WO2017055518A1

WO2017055518A1 - Means and methods for producing an amide compound

Info

Publication number: WO2017055518A1
Application number: PCT/EP2016/073372
Authority: WO
Inventors: Michael Guenter BRAUN; Juergen Daeuwel; Peter OEDMAN; Michael Kiefer; Matthias Kleiner; Hans-Juergen Lang; Diego GHISLIERI
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2015-09-30
Filing date: 2016-09-29
Publication date: 2017-04-06
Anticipated expiration: 2018-03-30

Abstract

The present disclosure relates to methods for producing an amide compound from a nitrile compound using a dried biocatalyst which has been pre-treated with a buffer, wherein the dried and pre-treated biocatalyst has improved properties as compared to a dried biocatalyst which has not been pre-treated with a buffer. In particular, the present disclosure provides means and methods to enhance the NHase activity of a dried biocatalyst for use in the production of amide compounds from nitrile compounds in aqueous solutions thereby improving the bioconversion process.

Description

Means and Methods for Producing an Amide Compound

The present invention relates to a method for producing an amide compound from a nitrile compound in aqueous mixture, the method comprising: (a) a pre-treatment step comprising mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and (b) converting the nitrile compound to the amide compound using the biocatalyst in a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. The present invention further relates to an aqueous amide compound mixture obtainable or being obtained by said method. The present invention still further relates to a method of pre-treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture. Moreover, the present invention provides a composition comprising an amide compound and a biocatalyst, wherein the biocatalyst has been pre-treated according to the method of the present invention. The present invention further relates to a method for increasing the reaction rate of converting a nitrile compound to an amide compound using a biocatalyst in aqueous mixture and a method for reducing the biocatalyst amount necessary for converting a nitrile compound to an amide compound in aqueous mixture. The present invention also provides for the use of an aqueous solution or mixture comprising a buffer for pre-treatment of a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture.

Acrylamide is used as a monomer to form polymers and copolymers of acrylamide. For these polymerization and copolymerization reactions aqueous acrylamide solutions prepared by bioconversion can be used. Since the discovery of nitrile hydratase (NHase), a microbial enzyme that converts nitriles to amides, microorganisms having NHase activity have been intensively used for the industrial production of amide compounds. Due to milder reaction conditions compared to the chemical synthesis of amides, the use of NHase producing microorganisms as biocatalysts is more and more on the rise. In fact, one of the most well- known commercial examples of nitrile bioconversion by NHase producing microorganisms is the manufacture of acrylamide from acrylonitrile. However, there is still a need to improve the processes for the production of amide compounds via a bioconversion. The technical problem underlying the present invention is therefore to provide an improved bioconversion process of a nitrile compound to an amide compound.

The technical problem is solved by providing the embodiments reflected in the claims, described in the description and illustrated in the examples and figures that follow.

The present inventors have found that a biocatalyst may be dried in order to improve its long- term stability and that such a dried biocatalyst may then be applied directly into the reaction mixture, or alternatively, the biocatalyst may be dissolved or re-suspended in an aqueous solution prior to the addition to the reaction mixture. The present inventors have further considered that bioconversion reactions, especially in industrial scale, should preferably be conducted in a rather simple way. In particular, in regard to the bio-conversion of a nitrile compound to an amide compound using a NHase comprising biocatalyst, the present inventors have considered that keeping the molar concentration of a buffer in the reaction mixture comparably low, preferably as low as possible, has several advantages for the bioconversion process. For example, if a comparably large amount of buffer is present in the reaction mixture, it would consequently be present in the wastewater in comparably large amounts. This means that the buffer will have to be removed from the wastewater again, which will result in additional technical efforts and costs. In addition, the presence of comparably large amounts of a buffer in the product, i.e. the amide solution, may negatively influence subsequent reaction steps, such as, for example, polymerization or copolymerization reactions. Hence, if a comparably large amount of buffer is added to the reaction mixture of the bioconversion, it will either have to be separated from the amide solution prior to further reaction steps, which will be accompanied by additional technical efforts and costs, or the buffer may result in a decrease of the quality of the product. Consequently, the bioconversion of a nitrile compound to an amide compound is preferably conducted in aqueous solution in the presence of comparably low amounts of buffer.

The present inventors have further discovered that keeping the molar buffer concentration in the reaction mixture comparably low has no influence on the bioconversion of a nitrile compound into an amide compound. Furthermore, the present inventors have found that a pH control is not necessary for keeping the pH within a certain range during the bioconversion of a nitrile compound to an amide compound. Typically, the bioconversion may start at a pH in a range of pH 7 to 8.5, preferably pH 7.5 to 8.0 and stays within this range until the end of the bioconversion without pH control and/or addition of further buffer.

For the preparation of a reaction mixture for the bioconversion of a nitrile compound to an amide compound, a dried biocatalyst, which has been obtained, for example, by spray drying or freeze drying, may in general be suspended in water, and said aqueous mixture containing the biocatalyst may be then transferred to the reactor in which the bioconversion is carried out and where the biocatalyst is contacted with an aqueous mixture and a nitrile compound that is to be converted into the corresponding amide. However, the present inventors have surprisingly found that, if a dried biocatalyst is mixed with a non-buffered aqueous solution, the pH of the aqueous mixture containing the biocatalyst will be in a weakly acidic range (e.g. pH 5 to 6.5). This is surprising since prior to drying, e.g. spray drying or freeze drying, the wet biocatalyst is in a medium that typically has a neutral pH (e.g. pH 6.7 to 7.5). Moreover, the reaction mixture during bioconversion is rather weakly basic. Without wishing to be bound by theory, it is believed that during the drying step, ammonia (NH3) strips from the medium which results in the weakly acidic pH of the dried biocatalyst when mixed with an aqueous solution prior to bioconversion.

In this regard, the present inventors have surprisingly discovered that the acidic pH of the aqueous mixture of the dried biocatalyst results in a diminished activity of the NHase and that this diminishment may be irreversible, which means that the NHase activity will remain diminished even if the bioconversion is conducted in a reaction mixture that has a neutral or slightly basic pH.

The present inventors have conducted various experiments and have found out that if the dried biocatalyst is pre-treated by suspending it in a buffered aqueous solution prior to bioconversion, wherein the solution has a neutral or slightly basic pH (e.g. pH 6.6 to 9), the biocatalyst will have a substantially increased NHase activity. This high NHase activity is maintained even if the pre-treatment mixture (i.e. the buffered aqueous mixture comprising the biocatalyst) is transferred to a non-buffered aqueous solution in order to give the reaction mixture. By this increased NHase activity the total reaction time of the bioconversion is crucially decreased as compared to the reaction time of the bioconversion where the same amount of biocatalyst has been suspended in water without buffer after spray drying (Fig. 1 , 2 and 5-13). Moreover, simply adding the buffer to the reaction mixture does not lead to the same effect as when the dried biocatalyst has been re-suspended in the buffer as pre- treatment before adding to the reaction mixture (Fig. 1 and 3). According to further experiments conducted by the present inventors, the biocatalyst also has a substantially increased NHase activity when the buffer is added to the cell suspension after fermentation and the biocatalyst is dried with the buffer and afterwards re-suspended in water (Fig. 14) or buffer (Fig. 15). Thus, drying the biocatalyst together with a buffer leads to a shorter reaction time as well, i.e. a higher reaction rate of the bioconversion when compared to a reference in which no pretreatment with the buffer has been performed (Fig. 12). Drying the biocatalyst with the buffer and subsequently re-suspending this biocatalyst again in a buffered aqueous solution has further slightly improved the observed effect (Fig. 14 and 15). As set out above, the pre-treatment of the biocatalyst may be performed by suspending the dried biocatalyst in an aqueous solution containing a buffer. Such pre-treatment can be performed on a small scale, i.e. the reaction volume required for the pre-treatment is comparably small. On the other hand, the reaction mixture in which the bioconversion of the nitrile compound to the amide compound is performed in general has a comparably large volume. Due to the low volume of the pre-treatment mixture compared to the volume of the reaction mixture, the buffer component is diluted in the reaction mixture when the pre- treatment mixture is transferred to the reactor for the bioconversion of the nitrile compound to the amide compound. Nevertheless, the beneficial effect of the buffer during the pre- treatment is preserved in the bioconversion. As said before, as a consequence of the enhanced NHase activity, the bioconversion of a nitrile compound to an amide compound using the biocatalyst exhibits a higher reaction rate if the same amount of biocatalyst is employed (Fig. 1 , 2 and 5-13). Further, the amount of biocatalyst can be reduced while achieving a reaction rate that is even higher than the reaction rate when using a non-reduced amount of un-pre-treated biocatalyst, i.e. a biocatalyst re-suspended in water after drying (Fig. 1 and 4). In this regard, different buffers, in particular neutral buffers, such as, for example, phosphate buffer, citrate buffer, Tris, TES, ACES, PIPES and others can be used to achieve the described effect of an increased NHase activity (Fig. 11 -13).

In sum, the present inventors have dissolved the discrepancy that a buffer is not desired in the reaction mixture, i.e. that the amount of the buffer in the reaction mixture should be kept low in the reaction mixture, but that a buffer is desired for the pre-treatment of a dried biocatalyst prior to bioconversion of a nitrile compound to an amide compound. Hence, the advantages of having a buffer in the pre-treatment mixture and having only low amounts of buffer in the reaction mixture can be combined according to the present invention. The present invention demonstrates that the amount of biocatalyst necessary for a full conversion of a nitrile compound to an amide compound can be strongly reduced by pre-treating the biocatalyst with a buffer, wherein the ratio of the molar concentration of the buffer in the pre- treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more.

This technical solution is surprising, as it has not been suggested in the prior art that the pre- treatment of a biocatalyst with a buffer prior to bioconversion, such as a pre-treatment by, for example, suspending a dried biocatalyst in a buffer prior to the bioconversion, will lead to an increased NHase activity of said biocatalyst. Moreover, there is no hint in the prior art to a pre-treatment comprising that a biocatalyst is dried in a buffer after fermentation in order to increase the NHase activity of said biocatalyst. For example, WO 02/18612 suggests that a dried biocatalyst may be suspended in the same buffer, in which the bio-conversion is carried out. However, WO 02/18612 does not give a hint that pre-treatment of a dried biocatalyst with a buffered aqueous solution will increase the NHase activity of the biocatalyst compared to re-suspending the dried biocatalyst in non-buffered aqueous solution.

Accordingly, on a general basis, the present invention relates to means and methods to improve the bioconversion of a nitrile compound to an amide compound by enhancing the NHase activity of a biocatalyst used in said bioconversion. In this regard, the present inventors suggest that the ratio of the molar concentration of the buffer used during pre- treatment of the biocatalyst, i.e. prior to the bioconversion, to the molar concentration of said buffer in the reaction mixture should be at least 2:1 or more. Thus, the amount of buffer during pre-treatment of the biocatalyst is diluted in the reaction mixture, i.e. during the bioconversion of a nitrile compound to an amide compound.

Thus, according to a first aspect, the present invention relates to a method for producing an amide compound from a nitrile compound in aqueous mixture, the method comprising: (a) a pre-treatment step comprising mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer; and (b) converting the nitrile compound to the amide compound using the biocatalyst in a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion. It is not required in any one of the methods disclosed herein that the ratio is maintained constant during the conversion. Rather the ratio may vary during the conversion, as long as the ratio is about 2:1 or more. For example, the ratio may increase during the conversion. This may be the case if reactants are added to the reaction mixture during the conversion which dilute the reaction mixture and thereby decrease the buffer concentration in the reaction mixture. For example, the nitrile compound and/or water may be fed as reactants to the reaction mixture during the conversion. This increases the volume of the reaction mixture, and, thus, decreases the molar concentration of the buffer in the reaction mixture. As a result of the decrease of the molar concentration of the buffer in the reaction mixture, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of the buffer in the reaction mixture increases. Thus, as can be seen from this example, the molar ratio can vary over the course of conversion reaction. It is further envisaged that the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture may be about 3:1 or more, preferably about 4:1 or more, more preferably about 5:1 or more, even more preferably about 7:1 or more, still more preferably about 10:1 or more, still more preferably about 20:1 or more, still more preferably about 50:1 or more, even more preferably about 75:1 or more, most preferably about 100:1 or more. In particular, these ratios are present before the end of the conversion. Regarding the molar concentration of the buffer in the pre-treatment mixture and the molar concentration of the buffer in the reaction mixture, these concentrations are both indicated in mol/L (moles per liter). When calculating the ratio of the molar concentration of the buffer in the pre-treatment mixture and the molar concentration of the buffer in the reaction mixture, both the molar concentration of the buffer in the pre-treatment mixture and the molar concentration of the buffer in the reaction mixture have to be taken in mol/L. It is also contemplated by the invention that the buffer of the pre-treatment mixture may be at least partially removed after the pre-treatment step and before the biocataiyst is contacted with a nitrile compound. As an illustrative example, this can be done by centrifugation of the pre-treatment mixture followed by discarding the supernatant, optionally followed by contacting the biocataiyst with another aqueous solution, or, as another illustrative example, by filtration. In such a case, the biocataiyst (suspension) will typically still contain residual buffer when the biocataiyst is contacted with the nitrile compound. It is understood by the skilled artisan, that the less residual buffer is present in the biocataiyst, the higher the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture may typically be. It is further envisaged that the pre-treatment may comprise the steps of: (i) contacting an aqueous solution with a buffer to give a buffered aqueous solution, and (ii) mixing the dried biocatalyst with the buffered aqueous solution to give a pre-treatment mixture. It is also envisaged that the pre-treatment comprises contacting a mixture of dried biocatalyst and buffer with an aqueous solution to give a pre-treatment mixture. Thus, the biocatalyst and the buffer can be mixed prior to or during the drying step. Also, the dried biocatalyst may be mixed with buffer prior to contacting the mixture with an aqueous solution.

The term "pre-treatment" or "pre-treating" as used herein in the context of a dried biocatalyst in general refers to mixing the dried biocatalyst with an aqueous solution to give an aqueous mixture comprising the biocatalyst and a buffer. Said mixture is also referred herein as "pre- treatment mixture". In accordance with any one of the methods described herein, the pre- treatment mixture may be prepared by mixing a buffer with an aqueous solution to give a buffered aqueous solution and subsequently dissolving or suspending the dried biocatalyst in the buffered aqueous solution. The pre-treatment mixture can also be prepared by mixing the dried biocatalyst with buffer components, in particular dry buffer components, and subsequently adding water to the mixture or adding the mixture to water, and dissolving the buffer components as well as dissolving or re-suspending the dried biocatalyst.

Thus, according to another aspect, the present invention also relates to a method of pre- treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, comprising: (a) mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer. Moreover, the present invention provides for a method of pre-treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, comprising: (a) mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and (b) contacting the biocatalyst with an aqueous mixture and/or a nitrile compound to form a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion. Also, the present invention relates to the use of an aqueous solution or mixture comprising a buffer for pre-treatment of a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, the pre-treatment comprising mixing the dried biocatalyst the aqueous solution or mixture to give a pre-treatment mixture, the use preferably further comprising contacting the pre-treated biocatalyst with an aqueous mixture and/or a nitrile compound to form a reaction mixture for the conversion of the nitrile compound to the amide compound, wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion.

It is also contemplated in the methods disclosed herein that the buffer can be added to a biocatalyst suspension or solution before the biocatalyst is subjected to a drying step to give a dried biocatalyst. The biocatalyst may also be washed before the buffer is added. By adding the buffer prior to the drying step, the dried biocatalyst comprises the dried buffer components that have been added prior to the drying step. Thus, when contacting the dried biocatalyst comprising a buffer with an aqueous solution, the buffer components dissolve, which, together with the biocatalyst, gives a pre-treatment mixture. Further, it is also contemplated in the methods disclosed herein that the biocatalyst treated with buffer before the biocatalyst is subject of a drying step to give a dried biocatalyst can subsequently be re- suspended or dissolved in a buffer solution to give a pre-treatment mixture. In a preferred embodiment the buffer is added to the dried biocatalyst.

When the biocatalyst is treated with a buffer according to the methods disclosed herein, said pre-treatment does not require a long period of time. Preferably, said pre-treatment of the biocatalyst is carried out for about 1 minute or more, more preferably for about 5 minutes or more, even more preferably from about 10 minutes to about 10 hours, still more preferably from about 20 minutes to about 5 hours, most preferably from about 30 minutes to about 2 hours. When a dried biocatalyst is treated with a buffered aqueous solution to give the pre- treatment mixture, said pre-treatment mixture is typically directly used for the bioconversion, i.e. directly mixed with an aqueous solution and a nitrile compound to give the reaction mixture. On the other hand, if the biocatalyst is pre-treated with a buffered solution or a buffer salt prior to the drying step, the dried biocatalyst can be subsequently stored for several months before bringing together said pre-treated biocatalyst with an aqueous solution to give a pre-treatment mixture and to further mix said pre-treatment mixture with an aqueous solution and a nitrile compound to give the reaction mixture. As found out by the present inventors, said biocatalyst does not significantly lose activity during the storage period. This can be seen as a further advantage of said pre-treatment variants. It is also envisaged by the methods disclosed herein, that, if no buffer is added to the biocatalyst prior to the drying step, the biocatalyst prior to the drying step may be suspended or dissolved in a medium that comprises fermentation broth. Said fermentation broth may comprise traces of buffer. However, as surprisingly found by the inventors, said traces of buffer in the fermentation broth are not sufficient to effectively pre-treat the dried biocatalyst. Hence, in accordance with the methods disclosed herein, a pre-treatment step (a) is required which comprises mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer. For example, pre-treatment step (a) can be performed by mixing the buffer with the biocatalyst either prior to the drying step or after drying in order to effectively pretreat the biocatalyst.

As disclosed herein, the biocatalyst used for the bioconversion of a nitrile compound to an amide compound has a substantially increased NHase activity when pre-treated with buffer after fermentation but before the drying step, or when treated with a buffered solution after drying, but before bringing together the dried biocatalyst with an aqueous solution and a nitrile compound to give the reaction mixture. Thus, according to another aspect, the present invention also relates to a method for reducing the biocatalyst amount necessary for converting a nitrile compound to an amide compound in aqueous mixture, the method comprising: (a) a pre-treatment step comprising mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and (b) converting the nitrile compound to the amide compound using the biocatalyst in a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more.

As described by the present inventors, an enhanced NHase activity of a biocatalyst used for the bioconversion of a nitrile compound to an amide compound can be conducted at a higher reaction rate if the same amount of biocatalyst is employed (Fig. 1 , 2 and 5-13). Accordingly, the present invention also provides for a method for increasing the reaction rate of converting a nitrile compound to an amide compound using a biocatalyst in aqueous mixture, the method comprising: (a) a pre-treatment step comprising mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and (b) converting the nitrile compound to the amide compound using the biocatalyst in a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion.

Any one of the methods disclosed herein contemplates that a reaction mixture can be created by mixing the pre-treatment mixture with an aqueous solution and a nitrile compound. Here the aqueous solution typically has a volume that is greater than the volume of the pre-treatment mixture. Hence, by mixing the three components, the buffer from the pre-treatment mixture is diluted and the molar concentration of the buffer in the reaction mixture is substantially lower than in the pre-treatment mixture.

In order to create the reaction mixture, the aqueous solution and a nitrile compound may first be mixed together in a reactor while the pre-treatment solution is added subsequently. This option is preferred. Alternatively, the pre-treatment mixture may also be added to a reactor comprising the aqueous solution, while the nitrile compound is subsequently added. Also, the pre-treatment solution may be comprised in a reactor and a mixture comprising the aqueous solution and a nitrile compound is added to the pre-treatment mixture. It is also possible, that the pre-treatment mixture is comprised in a reactor and the aqueous solution is added to it, followed by adding the nitrile compound.

Accordingly, it is envisaged that the reaction mixture is an aqueous mixture comprising a biocatalyst and a nitrile or amide compound. In this regard the pre-treatment mixture is added to an aqueous solution or mixture comprising a nitrile compound to give the reaction mixture. It is further envisaged that the pre-treatment mixture is added to an aqueous solution or mixture, wherein the nitrile compound is subsequently added to give the reaction mixture. Moreover, it is envisaged that an aqueous solution or mixture comprising a nitrile compound is added to the pre-treatment mixture to give the reaction mixture. It is also envisaged that an aqueous solution or mixture is added to the pre-treatment mixture, wherein the nitrile compound is subsequently added to give the reaction mixture.

The term "reaction mixture" as used herein refers to an aqueous mixture comprising a biocatalyst and a nitrile compound and/or an amide compound. Typically, the reaction mixture according to any one of the methods disclosed herein is created by combining a biocatalyst that has undergone a pre-treatment step according to the invention, an aqueous solution and a nitrile compound. Typically, the biocatalyst catalyzes the conversion of the nitrile compound to the amide compound in the reaction mixture. Thus, the term "reaction mixture" typically refers to a mixture including water, a biocatalyst, and a nitrile and/or an amide compound, at any time of a conversion process, including at the beginning of the reaction, when in an aqueous solution a biocatalyst is first contacted to a nitrile compound, as well as after the conversion has been stopped or ended but when the aqueous solution, the biocatalyst and an amide or nitrile compound are still present in the mixture. The term "before the end of the conversion" as used herein refers to any time while a conversion of nitrile to amide in the reaction mixture is still ongoing. Typically it refers to any time, in which a reaction mixture is present and in which the conversion has not yet ended or stopped. According to the methods disclosed herein, the ratio of the volume of the pre-treatment mixture to the volume of the reaction mixture is about 1 :2 or less, preferably about 1 :3 or less, more preferably about 1 :4 or less, even more preferably about 1 :5 or less, still more preferably from about 1 :5 to about 1 :1000, still more preferably from about 1 :10 to about 1 :500, still more preferably from about 1 :20 to about 1 :200, most preferably from about 1 :50 to about 1 :100. In particular, these ratios are present before the end of the conversion. Regarding the volume of the pre-treatment mixture and the volume of the reaction mixture, these volumes are both indicated in liters (L). When calculating the ratio of the volume of the pre-treatment mixture and the volume of the reaction mixture, both the volume of the pre- treatment mixture and the volume of the reaction mixture have to be taken in liters.

It is further envisaged that the pre-treatment mixture has a biocatalyst concentration of from about 0.1 to about 100 grams dry weight of the biocatalyst (gDw) per litre (L), preferably from about 0.2 to about 50 gDw L^-1, more preferably from about 0.5 to about 20 gDw L^-1, most preferably from about 1 to about 10 gDw L^_1. It is also envisaged that the pre-treatment of biocatalyst in the pre-treatment mixture is carried out for about 1 minute or more, preferably for about 5 minutes or more, more preferably from about 10 minutes to about 10 hours, even more preferably from about 20 minutes to about 5 hours, most preferably from about 30 minutes to about 2 hours. As used herein, the terms "dry weight" or "dry cell weight" refer to weight as determined in the relative absence of water. For example, reference to a component as comprising a specified percentage by dry weight means that the percentage is calculated based on the weight of the biomass after substantially all water has been removed.

It is also envisaged that the reaction mixture may comprise more than about 10 w/w % of the amide compound, preferably more than about 15 w/w % of the amide compound, more preferably more than about 20 w/w % of the amide compound, even more preferably more than about 25 w/w % of the amide compound, still more preferably more than about 30 w/w % of the amide compound, still more preferably more than about 35 w/w % of the amide compound, still more preferably more than about 40 w/w % of the amide compound, still more preferably more than about 45 w/w % of the amide compound, still more preferably more than about 50 w/w % of the amide compound, still more preferably more than about 55 w/w % of the amide compound, most preferably more than about 60 w/w % of the amide compound, each based on 100 w/w % of the reaction mixture. In particular, these contents of the amide compound are present before the end of the conversion.

It is also contemplated by the methods disclosed herein that the buffer comprised in the pre- treatment mixture has a pKa in a range of from about 6 to about 9, preferably from about 6.5 to about 8. Here, the buffer may comprise a single component, or can be a mixture of more than one buffer component. It is also understood that one single component can have more than one pKa values. A buffer has typically a pKa in a range from about 6 to about 9 if it comprises a buffer component that has a pKa in the range from about 6 to about 9. For example, phosphate has three pKa values, 2.1 , 7.2 and 12.7. Since one of the pKa values of phosphate is within the range of from about 6 to about 9, a buffer comprising phosphate may be understood as a buffer having a pKa in a range from about 6 to about 9.

It is also envisaged that the pre-treatment mixture has a pH value of from about 6.6 to about 9, preferably from about 6.6 to about 8.8, more preferably from about 6.7 to about 8.6, even more preferably from about 6.8 to about 8.4, still more preferably from about 6.9 to about 8.2, most preferably from about 7 to about 8. The buffer may have a pKa in a range of from about 6 to about 9, preferably from about 6.5 to about 8.

As shown by the present inventors, different buffers are well suited to be used in the methods disclosed herein, i.e. to increase the NHase activity of a biocatalyst (Fig. 11 -15). It is envisaged that the buffer comprises an inorganic buffer or an organic buffer. It is further envisaged that the buffer may comprise a non-sulfonic acid buffer or a carboxylic acid buffer. Suitable buffers which may be used in the present invention may comprise a compound selected from the group consisting of phosphate, citrate, carbonate, 2-[(2-hydroxy-1 ,1 - bis(hydroxymethyl)ethyl)amino] ethanesulfonic acid (TES), 1 ,4-piperazinediethanesulfonic acid (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), and tris(hydroxymethyl) aminomethane (TRIS), and any combination thereof. In particular, the buffer comprises a phosphate buffer or a citrate buffer or a combination thereof. Preferably, the buffer is a phosphate buffer.

It is further envisaged that the buffer is in a concentration in the pre-treatment mixture of more than 50 mM, preferably more than 60 mM, more preferably more than 80 mM and most preferably more than 90 mM, such as more than 100 mM, more than 120 mM, more than 150 mM or more than 200 mM. The buffer may be in a concentration in the pre-treatment mixture of about 50 mM to 1 M, preferably about 60 mM to 1 M, even more preferably about 70 mM to 1 M, still more preferably about 80 mM to 1 M, most preferably about 90 mM to about 1 M. The buffer may be in a concentration in the pre-treatment mixture of about 50 mM to about 500 mM, preferably about 60 mM to about 500 mM, even more preferably about 70 mM to about 500 mM, still more preferably about 80 mM to about 500 mM, most preferably about 90 mM to about 500 mM. Particularly, the buffer may be in a concentration of about 10 mM to about 1 M, preferably about 20 mM to about 500 mM, more preferably about 50 mM to about 200 mM, even more preferably about 70 mM to about 130 mM, most preferably about 80 mM to about 120 mM, such as about 100 mM. It is further envisaged that the buffer concentration in the reaction mixture is about 100 mM or less, preferably about 50 mM or less, more preferably about 20 mM or less, even more preferably about 10 mM or less, still more preferably from about 5 mM to about 1 pM, still more preferably from about 4 mM to about 1 pM, still more preferably from about 3 mM to about 1 pM, still more preferably from about 2 mM to about 1 pM, still more preferably from about 1 mM to about 1 pM, still more preferably from about 0.8 mM to about 1 pM, still more preferably from about 0.5 mM to about 1 pM, still more preferably from about 0.4 mM to about 1 pM, still more preferably from about 0.3 mM to about 1 pM, still more preferably from about 0.2 mM to about 1 pM, most preferably from about 0.1 mM to about 1 pM.

It is also envisaged that the temperature of the pre-treatment is in the range of from about 0 °C to about 50 °C, preferably from about 10° to about 40°C, more preferably from about 20°C to about 37°C. In the specific embodiment, the pH is not controlled during step (b), i.e. during the

bioconversion of a nitrile compound to an amide compound. By using the method of the invention it is not necessary to keep the pH within a certain range during the bioconversion, therefore the bioconversion can be simplified. The term "the pH is not controlled" in the context of the present invention means that the pH is not adjusted to be kept within a certain range, in particular in response to the measured pH value in the solution. In a specific embodiment, this means that the pH is also not measured during step (b).

It is envisaged that the amide compound is acrylamide, methacrylamide, acetamide or nicotinamide. Preferably, the amide compound is acrylamide. It is further envisaged that the nitrile compound is acrylonitrile, methacrylonitrile, acetonitrile or 3-cyanopyridine. Preferably, the nitrile compound is acrylonitrile.

The biocatalyst used in any one of the methods disclosed herein is typically a microorganism, preferably a nitrile hydratase (NHase) producing microorganism. In the context of the present disclosure, "Nitrile hydratase"("NHase") refers to a microbial enzyme that catalyzes the hydration of nitriles to their corresponding amides (IUBMB Enzyme Nomenclature EC 4.2.1 .84). However, the terms "Nitrile hydratase" and "NHase" as used herein also encompass modified or enhanced enzymes which are, e.g., capable of converting a nitrile compound (e.g. acrylonitrile) to an amide compound (e.g. acrylamide) more quickly, or which can be produced at a higher yield/time-ratio, or which are more stable, as long as they are capable to catalyze conversion (i.e. hydration) of a nitrile compound (e.g. acrylonitrile) to an amide compound (e.g. acrylamide). In the methods disclosed herein "NHase producing microorganisms" are used, or are for use, as a biocatalyst for converting a nitrile compound into the corresponding amide compound. A "nitrile compound" is converted by a microorganism in the methods disclosed herein into an amide compound by the action of NHase. A nitrile compound is any organic compound that has a -C≡N functional group. A preferred nitrile compound is acrylonitrile. An amide compound has the functional group R_nC(0)_xNR'2, wherein R and R' refer to H or organic groups. For organic amides n=1 , x=1. An example of an amide compound is acrylamide.

Regarding the methods disclosed herein, a "NHase producing microorganism" may be any microorganism which is able to produce the enzyme NHase. With this regard, it is not relevant whether the microorganism naturally encodes NHase or whether it has been genetically modified to encode said enzyme. Furthermore, the biocatalyst may be a microorganism which naturally encodes NHase and/or which is further genetically engineered, e.g., to increase production of NHase, or to increase stability and/or export of NHase or to decrease production of Amidase, or to increase stability and/or export of Amidase.

The term "biocatalyst" as used herein refers to any catalytic material of biological origin. The biocatalyst can be homogeneous or heterogeneous, and can be an enzyme or a cellular biocatalyst, in particular microorganisms (e.g., bacteria or protozoic eukaryotes), for example, and comprises intact cell, such as a bacterial cells, a fungal cells or a yeast cells, or a cell fragment. Typically, a biocatalyst referred to in the context of the methods disclosed herein comprises a nitrile hydratase (NHase) and is typically able to catalyze the conversion of a nitrile compound to an amide compound. Methods for determining the ability of a given biocatalyst (e.g., microorganism or enzyme) to convert a nitrile compound, e.g. acrylonitrile, to an amide compound, e.g. acrylamide, are well known in the art. As an example, in context with any one of the methods and uses disclosed herein, activity of a given biocatalyst to be capable of converting acrylonitrile to acrylamide in the methods disclosed herein may be determined as follows: First reacting 100 μΙ of a cell suspension, cell lysate, dissolved enzyme powder or any other preparation containing the supposed biocatalyst with 875 μΙ of an 50 mM potassium phosphate buffer and 25 μΙ of acrylonitrile at 25 °C on an Eppendorf tube shaker at 1 ,000 rpm for 10 minutes. After 10 minutes of reaction time, samples may be drawn and immediately quenched by adding the same volume of 1.4% hydrochloric acid. After mixing of the sample, cells may be removed by centrifugation for 1 minute at 10,000 rpm and the amount of acrylamide formed is determined by analyzing the clear supernatant by HPLC. For affirmation of a biocatalyst to be capable of converting acrylonitrile to acrylamide in context with the methods disclosed herein, the concentration of acrylamide shall be between 0.25 and 1.25 mmol/l - if necessary, the sample has to be diluted accordingly and the conversion has to be repeated. The activity may then be deduced from the concentration of acrylamide by dividing the acrylamide concentration derived from HPLC analysis by the reaction time, which has been 10 minutes and by multiplying this value with the dilution factor between HPLC sample and original sample. Activities >5 U/mg dry cell weight, preferably >25 U/mg dry cell weight, more preferably >50 U/mg dry cell weight, most preferably >100 U/mg dry cell weight indicate the presence of a functional biocatalyst and are considered as biocatalyst capable of converting acrylonitrile to acrylamide in context with the methods disclosed herein.

The term "bioconversion" when used in the context of the methods disclosed herein refers to the total or partially reaction of the nitrile compound to an amide compound in aqueous solution and in the presence of a biocatalyst. More precisely, "conversion" or "converting" means, that the nitrile compound is allowed to undergo hydration reaction by the use of a biocatalyst in aqueous solution in order to obtain an amide reaction solution having a desired concentration. According to the present invention, this hydration reaction may be carried out in any conventional manner. The concentration of the nitrile compound is not specifically restricted provided that the amide reaction solution of a desired concentration is obtained. Although the upper limit of the concentration of the nitrile compound is not specifically restricted, feed of excess nitrile needs a large catalytic amount for completion of the reaction, a reactor having an excess volume and an excess heat exchanger for removal of heat, so that the economic burden in the equipment aspect becomes heavy. Preferably, the term "bioconversion" denotes a reaction, wherein methacrylonitrile is converted to acrylamide in aqueous solution and in the presence of a biocatalyst. The acrylamide is dissolved in the water, such that by any one of the methods described and provided herein an aqueous acrylamide solution is formed.

In context with any one of the methods disclosed herein, the nitrile compound may be added to the reactor before the water is added, after water is added, or added together with water. According to any one of the methods described herein, the nitrile compound may be added continuously or intermittently. Addition of nitrile compound may be at constant or variable feed rate or batch-wise. The water may be added as such, be part of the pre-treatment mixture as described herein, be part of a nitrile solution as described herein, or otherwise be added. In case that the water is added as such, in general tap water or deionized water may be used. The microbial catalyst may be used in any amount provided that the amide reaction solution of a desired concentration is obtained, and the amount thereof is properly determined according to the reaction conditions, the type of the catalyst and the form thereof. However, the amount of the microbial catalyst is in the range of usually 10 to 50000 ppm by weight, preferably 50 to 30000 ppm by weight, in terms of weight of dry bacterial cell, based on the aqueous medium.

The reaction time of the conversion of the nitrile compound to the amide compound is not specifically restricted either provided that the amide reaction solution of a desired concentration is obtained. Although the reaction time depends upon the amount of the catalyst used and the conditions such as temperature, it is specifically in the range of usually 1 to 80 hours, preferably 2 to 40 hours, based on one reactor. Although the conversion of the nitrile compound to the amide compound is usually carried out at atmospheric pressure, it may be carried out under pressure in order to increase solubility of the nitrile compound in the aqueous medium. The reaction temperature is not specifically restricted provided that it is not lower than the ice point of the aqueous medium. However, it is desirable to carry out the conversion at a temperature of usually 0 to 50°C, preferably 10 to 40°C, more preferably 20 to 37°C. The conversion of the nitrile compound to the amide compound may be carried out by any of a batch process and a continuous process, and the conversion may be carried out by selecting its reaction system from reaction systems such as suspended bed, a fixed bed, a fluidized bed and the like or by combining different reaction systems according to the form of the catalyst.

In accordance with any one of the methods disclosed herein, the biocatalyst capable of converting a nitrile compound to an amide compound may be a microorganism which encodes the enzyme nitrile hydratase. With this regard, it is not relevant for the present invention whether the microorganism is naturally encoding nitrile hydratase, or whether it has been genetically modified to encode said enzyme, or whether a microorganism naturally encoding nitrile hydratase has been modified such as to be able to produce more and/or enhanced nitrile hydratase. As used herein, the expression "biocatalyst (e.g., microorganism) encoding (the enzyme) nitrile hydratase" or the like generally means that such a microorganism is generally also able to produce and stably maintain nitrile hydratase. That is, as used herein and as readily understood by the skilled person, a biocatalyst (e.g., a microorganism) to be employed in accordance with the methods disclosed herein which (naturally or non-naturally) encodes nitrile hydratase is generally also capable of producing and stably maintaining nitrile hydratase. However, in accordance with the methods disclosed herein, it is also possible that such microorganisms only produced nitrile hydratase during cultivation (or fermentation) of the microorganism - thus then containing nitrile hydratase - before being added to a reactor. In such a case, it is possible that the microorganisms do not produce nitrile hydratase during the methods described and provided herein any more, but they act only via the nitrile hydratase units which they have produced before and which they still contain. As readily understood by the person skilled in the art, it is also possible that some nitrile hydratase molecules may leave the microorganism (e.g., due to lysis of the microorganism) and act freely in the solution as biocatalyst. As such, it also possible that the term "biocatalyst" as used herein encompasses the enzyme nitrile hydratase per se, as long as it is able to convert acrylonitrile to acrylamide as described and exemplified herein. In context with the methods disclosed herein, it is also possible to directly employ nitrile hydratase as biocatalyst. In context with the methods disclosed herein, microorganisms naturally encoding nitrile hydratase, which can be used as biocatalyst in any one of the methods described herein, is a bacterium. It is further envisaged that the microorganism is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus. Preferably, the microorganism is selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus. It is further envisaged that the microorganism is selected from the group consisting of Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp. RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1 , Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG- Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1 , Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosteroni, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, and Nocardia sp. 163. It is also envisaged that the microorganism is Rhodococcus rhodochrous or Rhodococcus pyridinovorans. It is further envisaged that the microorganism is Rhodococcus rhodochrous (NCIMB 41 164), Rhodococcus rhodochrous (FERM BP-1478), or Rhodococcus rhodochrous M33.

In accordance with any one of the methods described herein, combinations of these microorganisms can be used as well. Further, the above microorganisms can be cultured by any method that is appropriate for a given microbial species. The microbial biocatalyst that is prepared from microorganisms refers to a culture solution obtained by culturing microorganisms, cells obtained by a harvesting process or the like, cell disrupted by ultrasonication or the like, or those prepared after cell disruption including a crude enzyme, a partially-purified enzyme or a purified enzyme. A mode to use the microbial catalyst may be appropriately selected depending on enzyme stability, production scale and the like.

In context with the present disclosure, nitrile hydratase encoding microorganisms which are not naturally encoding nitrile hydratase may be genetically engineered microorganisms which naturally do not contain a gene encoding a nitrile hydratase but which have been manipulated such as to contain a polynucleotide encoding a nitrile hydratase (e.g., via transformation, transduction, transfection, conjugation, or other methods suitable to transfer or insert a polynucleotide into a cell as known in the art; cf. Sambrook and Russell 2001 , Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), thus enabling the microorganisms to produce and stably maintain the nitrile hydratase enzyme. For this purpose, it may further be required to insert additional polynucleotides which may be necessary to allow transcription and translation of the nitrile hydratase gene or mRNA, respectively. Such additional polynucleotides may comprise, inter alia, promoter sequences, polyT- or polyU-tails, or replication origins or other plasmid-control sequences. In this context, such genetically engineered microorganisms which naturally do not contain a gene encoding a nitrile hydratase but which have been manipulated such as to contain a polynucleotides encoding a nitrile hydratase may be prokaryotic or eukaryotic microorganisms. Examples for such prokaryotic microorganisms include, e.g., representatives of the species Escherichia coli. Examples for such eukaryotic microorganisms include, e.g., yeast (e.g., Saccharomyces cerevisiae). In context of the present disclosure, the term "nitrile hydratase" (also referred to herein as NHase) generally means an enzyme which is capable of catalyzing the conversion (i.e. hydration) of acrylonitrile to acrylamide. Such an enzyme may be, e.g., the enzyme registered under IUBMB nomenclature as of April 1 , 2014: EC 4.2.1.84; CAS-No. 2391 -37-5. However, the term "nitrile hydratase" as used herein also encompasses modified or enhanced enzymes which are, e.g., capable of converting acrylonitrile to acrylamide more quickly, or which can be produced at a higher yield/time-ratio, or which are more stable, as long as they are capable to catalyze conversion (i.e. hydration) of acrylonitrile to acrylamide. Methods for determining the ability of a given biocatalyst (e.g., microorganism or enzyme) for catalyzing the conversion of acrylonitrile to acrylamide are known in the art. As an example, in context with the present disclosure, activity of a given biocatalyst to act as a nitrile hydratase in the sense of the present disclosure may be determined as follows: First reacting 100 μΙ of a cell suspension, cell lysate, dissolved enzyme powder or any other preparation containing the supposed nitrile hydratase with 875 μΙ of an 50 mM potassium phosphate buffer and 25 μΙ of acrylonitrile at 25 °C on an Eppendorf tube shaker at 1 ,000 rpm for 10 minutes. After 10 minutes of reaction time, samples may be drawn and immediately quenched by adding the same volume of 1 .4% hydrochloric acid. After mixing of the sample, cells may be removed by centrifugation for 1 minute at 10,000 rpm and the amount of acrylamide formed is determined by analyzing the clear supernatant by HPLC. For affirmation of an enzyme to be a nitrile hydratase in context with the present disclosure, the concentration of acrylamide shall be between 0.25 and 1.25 mmol/l - if necessary, the sample has to be diluted accordingly and the conversion has to be repeated. The enzyme activity may then be deduced from the concentration of acrylamide by dividing the acrylamide concentration derived from HPLC analysis by the reaction time, which has been 10 minutes and by multiplying this value with the dilution factor between HPLC sample and original sample. Activities >5 U/mg dry cell weight, preferably >25 U/mg dry cell weight, more preferably >50 U/mg dry cell weight, most preferably >100 U/mg dry cell weight indicate the presence of a functional biocatalyst and are considered as biocatalyst capable of converting acrylonitrile to acrylamide in context with the present disclosure. In context with the present disclosure, the nitrile hydratase may be a polypeptide encoded by a polynucleotide which comprises or consists of a nucleotide sequence which is at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99,5%, and most preferably 100% identical to the nucleotide sequence of SEQ ID NO: 1 (alpha-subunit of nitrile hydratase of Rhodococcus rhodochrous: GTGAGCGAGCACGTCAATAAGTACACGGAGTACGAGGCACGTACCAAGGCGATCGAAA CCTTGCTGTACGAGCGAGGGCTCATCACGCCCGCCGCGGTCGACCGAGTCGTTTCGTA CTACGAGAACGAGATCGGCCCGATGGGCGGTGCCAAGGTCGTGGCCAAGTCCTGGGT GGACCCTGAGTACCGCAAGTGGCTCGAAGAGGACGCGACGGCCGCGATGGCGTCATT GGGCTATGCCGGTGAGCAGGCACACCAAATTTCGGCGGTCTTCAACGACTCCCAAACG CATCACGTGGTGGTGTGCACTCTGTGTTCGTGCTATCCGTGGCCGGTGCTTGGTCTCC CGCCCGCCTGGTACAAGAGCATGGAGTACCGGTCCCGAGTGGTAGCGGACCCTCGTG GAGTGCTCAAGCGCGATTTCGGTTTCGACATCCCCGATGAGGTGGAGGTCAGGGTTTG GGACAGCAGCTCCGAAATCCGCTACATCGTCATCCCGGAACGGCCGGCCGGCACCGA CGGTTGGTCCGAGGAGGAGCTGACGAAGCTGGTGAGCCGGGACTCGATGATCGGTGT CAGTAATGCGCTCACACCGCAGGAAGTGATCGTATGA) and/or to the nucleotide sequence of SEQ ID NO: 3 (beta-subunit of nitrile hydratase of Rhodococcus rhodochrous: ATGGATGGTATCCACGACACAGGCGGCATGACCGGATACGGACCGGTCCCCTATCAGA AGGACGAGCCCTTCTTCCACTACGAGTGGGAGGGTCGGACCCTGTCAATTCTGACTTG GATGCATCTCAAGGGCATATCGTGGTGGGACAAGTCGCGGTTCTTCCGGGAGTCGATG GGGAACGAAAACTACGTCAACGAGATTCGCAACTCGTACTACACCCACTGGCTGAGTG CGGCAGAACGTATCCTCGTCGCCGACAAGATCATCACCGAAGAAGAGCGAAAGCACCG TGTGCAAGAGATCCTTGAGGGTCGGTACACGGACAGGAAGCCGTCGCGGAAGTTCGAT CCGGCCCAGATCGAGAAGGCGATCGAACGGCTTCACGAGCCCCACTCCCTAGCGCTTC CAGGAGCGGAGCCGAGTTTCTCTCTCGGTGACAAGATCAAAGTGAAGAGTATGAACCC GCTGGGACACACACGGTGCCCGAAATATGTGCGGAACAAGATCGGGGAAATCGTCGCC TACCACGGCTGCCAGATCTATCCCGAGAGCAGCTCCGCCGGCCTCGGCGACGATCCTC GCCCGCTCTACACGGTCGCGTTTTCCGCCCAGGAACTGTGGGGCGACGACGGAAACG GGAAAGACGTAGTGTGCGTCGATCTCTGGGAACCGTACCTGATCTCTGCGTGA), provide d that the polypeptide encoded by said polynucleotide is capable of catalyzing hydration of acrylonitrile to acrylamide (i.e. has nitrile hydratase activity) as described and exemplified herein. Also in the context with the present disclosure, the nitrile hydratase may be a polypeptide which comprises or consists of an amino acid sequence which is at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99,5%, and most preferably 100% identical to the amino acid sequence of SEQ ID NO: 2 (alpha-subunit of nitrile hydratase of Rhodococcus rhodochrous: VSEHVN KYTE YEARTKAI ET LLYERGLITP AAVDRVVSYY EN EIGPMGGA KVVAKSWVDP EYRKWLEEDA TAAMASLGYA GEQAHQISAV FN DSQTH HVV VCTLCSCYPW PVLGLPPAWY KSMEYRSRVV ADPRGVLKRD FGFDI PDEVE VRVWDSSSEI RYIVI PERPA GTDGWSEEEL TKLVSRDSMI GVSNALTPQE VIV) and/or to the amino acid sequence of SEQ ID NO: 4 (beta-subunit of nitrile hydratase of R. rhodochrous: MDGIHDTGGM TGYGPVPYQK DEPFFHYEWE GRTLSILTWM HLKGISWWDK SRFFRESMGN ENYVNEIRNSY YTHWLSAAE RILVADKIIT EEERKHRVQE ILEGRYTDRK PSRKFDPAQI EKAIERLHEP HSLALPGAEP SFSLGDKIKV KSMNPLGHTR CPKYVRNKIG EIVAYHGCQI YPESSSAGLG DDPRPLYTVA FSAQELWGDD GNGKDVVCVD LWEPYLISA), provided that said polypeptide is capable of catalyzing hydration of acrylonitrile to acrylamide as described and exemplified herein.

The level of identity between two or more sequences (e.g., nucleic acid sequences or amino acid sequences) can be easily determined by methods known in the art, e.g., by BLAST analysis. Generally, in context with the present disclosure, if two sequences (e.g., polynucleotide sequences or amino acid sequences) to be compared by, e.g., sequence comparisons differ in identity, then the term "identity" may refer to the shorter sequence and that part of the longer sequence that matches said shorter sequence. Therefore, when the sequences which are compared do not have the same length, the degree of identity may preferably either refer to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that matches the shorter sequence. Furthermore, as used herein, identity levels of nucleic acid sequences or amino acid sequences may refer to the entire length of the respective sequence and is preferably assessed pair-wise, wherein each gap is to be counted as one mismatch. These definitions for sequence comparisons (e.g., establishment of "identity" values) are to be applied for all sequences described and disclosed herein.

Moreover, the term "identity" as used herein means that there is a functional and/or structural equivalence between the corresponding sequences. Nucleic acid/amino acid sequences having the given identity levels to the herein-described particular nucleic acid/amino acid sequences may represent derivatives/variants of these sequences which, preferably, have the same biological function. They may be either naturally occurring variations, for instance sequences from other varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described nucleic acid sequences may have been produced, e.g., by deletion, substitution, addition, insertion and/or recombination. The term "addition^" refers to adding at least one nucleic acid residue/amino acid to the end of the given sequence, whereas "insertion" refers to inserting at least one nucleic acid residue/amino acid within a given sequence. The term "deletion" refers to deleting or removal of at least one nucleic acid residue or amino acid residue in a given sequence. The term "substitution" refers to the replacement of at least one nucleic acid residue/amino acid residue in a given sequence. Again, these definitions as used here apply, mutatis mutandis, for all sequences provided and described herein.

Generally, as used herein, the terms ..polynucleotide" and ..nucleic acid" or ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 552571 1 , US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339). Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present disclosure. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers. As a "reference microorganism" when referred to herein, a dried biocatalyst (i.e. microorganism) not pre-treated with buffer after fermentation and prior to bioconversion may be used. As used herein, a reference microorganism is one which was dried after fermentation, but not pre-treated before or after drying with any kind of buffer, and which is then re-suspended in water before bioconversion. However, also the reference microorganism can be contacted with a nitrile compound in aqueous solution and converts the nitrile compound to an amide compound. However, said reference microorganism has a lower NHase activity as compared with a microorganism pre-treated with buffer prior to bioconversion. Thus, the use of said reference microorganism will be conducted at a lower reaction rate as compared to the reaction rate of a microorganism pre-treated with buffer, if the same amount of biocatalyst is employed during bioconversion.

According to the present invention, the microorganism used to convert a nitrile compound to an amide compound is necessarily a dried microorganism. In this regard it is particularly envisaged that the dried biocatalyst is a biocatalyst that has been subjected to a drying step before mixing the biocatalyst with an aqueous solution. The drying can be mediated by spray drying, freeze-drying, heat drying, air drying, vacuum drying, fluidized-bed drying and/or spray granulation, wherein spray drying and freeze drying are preferred, wherein spray drying is most preferred.

Within the methods disclosed herein, is envisaged that the dried biocatalyst is in the form of a powder, granule, suspension, and/or matrix bound microorganism. Regarding the drying method, in any one of the methods described and provided herein, independently of whether the biocatalyst is dried before being contacted with buffer, or whether the biocatalyst is dried after being contacted with buffer, the biocatalyst may be dried using the drying methods described herein.

The term "dried biocatalyst" as used herein refers to a biocatalyst that has been subjected to a drying step. A dried biocatalyst typically has a moisture content of less than about 20 w/w %, more preferably less than about 15 w/w %, even more preferably less than about 14 w/w %, most preferably from about 5 to about 10 w/w % based on the total weight of the biocatalyst sample. Methods of determining the moisture content are familiar to the skilled person. For example, in the context of the present invention the moisture content of a sample of the dried biocatalyst may be determined via thermogravimetric analysis. At the beginning of the thermogravimetric analysis the initial weight of the sample is determined. The sample is then heated and the moisture vaporizes. Heating is continued until the sample weight remains constant. The difference between the constant weight at the end of the analysis and the initial weight represents the amount of water vaporized during the analysis, which allows for calculation of the moisture content of the sample. For determination of the moisture content via thermogravimetric anaylsis, the biocatalyst sample may be, for example, analyzed on a 'Mettler Toledo HB43-S Halogen moisture analyzer', operated at 130 °C until the sample weight remains constant for at least 30 seconds. By performing any one of the methods described herein, the aqueous acrylamide solution may be obtained along with the biocatalyst. Accordingly, the biocatalyst may be separated from the obtained aqueous acrylamide solution. Such a separation of the biocatalyst may be performed with regard to the desired applications, which may, for example, include the homopolymerization or copolymerization of the acrylamide. Suitable methods for separation of the biocatalyst are known in the art and include, for example, centrifugation, sedimentation (e.g., with flocculation), membrane separation and filtration.

In any one of the methods described and provided herein, the separation of the biocatalyst is started after completion of the conversion of acrylonitrile to acrylamide using a biocatalyst. The term "after completion" when used herein can be understood as the point of time when the desired amide concentration in the aqueous solution is achieved. The desired amide concentration is disclosed elsewhere herein. The separation may be started immediately after completion of the conversion of acrylonitrile to acrylamide, or within a specific time interval. In a preferred embodiment of the present invention, the separation is started within 30 minutes after completion of the conversation of acrylonitrile to acrylamide. In a more preferred embodiment the separation is started within 20 minutes after completion of the conversation of acrylonitrile to acrylamide. In a most preferred embodiment the separation is started within 10 minutes after completion of the conversation of acrylonitrile to acrylamide. The present invention further relates to an aqueous amide compound solution obtainable or being obtained by any one of the methods described and provided herein.

An aqueous amide compound solution, in particular an aqueous amide compound solution obtainable or being obtained by any one of the methods described herein, may contain 20 to 65 w/w % of the amide compound, wherein indications of w/w % are each referred to the total weight of the solution. Preferably, the aqueous amide compound solution contains 25 to 60 w/w % of the amide compound, wherein indications of w/w % are each referred to the total weight of the solution. More preferably, the aqueous amide compound solution contains 30 to 58 w/w % of the amide compound, wherein indications of w/w % are each referred to the total weight of the solution. Most preferably, the aqueous amide compound solution contains 35 to 55 w/w % of the amide compound, wherein indications of w/w % are each referred to the total weight of the solution.

In any one of the aqueous acrylamide solutions disclosed herein, the acrylamide content, the acrylonitrile and/or the acrylic acid concentration may be determined using HPLC. Preferably, an HPLC method is used as set forth below under the Examples.

The present invention further relates to a composition comprising an amide compound and a biocatalyst, wherein the biocatalyst has been pre-treated by a method of pre-treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, comprising: (a) mixing a dried biocatalyst with an aqueous solution to give a pre- treatment mixture, wherein the pre-treatment mixture comprises a buffer, and (b) contacting the biocatalyst with an aqueous mixture and/or a nitrile compound to form a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion. Preferably, the amide compound is acrylamide. Accordingly, said composition preferably comprises acrylamide and a biocatalyst. The term "composition" includes but is not limited to solutions and/or suspensions, dispersions, concentrations, ready mix, powders, and granules comprising an amide compound, preferably acrylamide and a biocatalyst.

The present invention also relates to the use of a composition comprising a biocatalyst pre- treated according to the method of pre-treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, comprising: (a) mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre- treatment mixture comprises a buffer, and (b) contacting the biocatalyst with an aqueous mixture and/or a nitrile compound to form a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), and wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more for converting a nitrile compound to an amide compound, wherein the conversion is conducted in a reaction mixture, wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more. In particular, the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture is about 2:1 or more before the end of the conversion.

The embodiments and definitions described herein in the context of the methods of the present invention are equally applicable to the uses of the present invention, mutatis mutandis.

Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below. Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and uses described herein. Such equivalents are intended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "consisting", "consisting of" and "consisting essentially of" may be replaced with either of the other two terms. As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein.

As described herein, "preferred embodiment" means "preferred embodiment of the present invention". Likewise, as described herein, "various embodiments" and "another embodiment" means "various embodiments of the present invention" and "another embodiment of the present invention".

The word "about" as used herein refers to a value being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. The term "about" is also used to indicate that the amount or value in question may be the value designated or some other value that is approximately the same. The phrase is intended to convey that similar values promote equivalent results or effects according to the invention. In this context "about" may refer to a range above and/or below of up to 10%. The word "about" refers in some embodiments to a range above and below a certain value that is up to 5%, such as up to up to 2%, up to 1 %, or up to 0.5 % above or below that value. In one embodiment "about" refers to a range up to 0.1 % above and below a given value.

Several documents are cited throughout the text of this disclosure. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

This description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 1 16 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re- suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 13.78 h.

Figure 2: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 33 ml. of 100 mM phosphate buffer (pH 7.0), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 1 h. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 1 16 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from O h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 % w/w, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 2.31 h. Figure 3: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 1 16 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. Directly prior to biocatalyst addition, 33 ml. of 100 mM phosphate buffer (pH 7.0) was added to the reactor, which corresponds to the amount of buffer that was used in the pre-treatment step of the experiment depicted in Figure 2. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from O h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 1 1.98 h.

Figure 4: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 7.0), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. 1.8 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 1 16 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 23 °C. At the beginning of the reaction, re- suspended biocatalyst corresponding to 1 .8 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration after beginning of the bioconversion was maintained at 1 w/w % by feeding acrylonitrile to the reactor, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 7.13 h.

Figure 5: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1.73 g of the dried biocatalyst (batch Ch32) was employed, which had an NHase activity of 137 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1 .24 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re- suspended biocatalyst corresponding to 0.49 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. No full conversion (< 100 ppm residual acrylonitrile) was reached after 19 h.

Figure 6: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 7.5), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. A total amount of 1 .73 g of the dried biocatalyst (batch Ch32) was employed, which had an NHase activity of 137 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1.24 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.49 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 4.6 h.

Figure 7: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1 .29 g of the dried biocatalyst (batch Ch30) was employed, which had an NHase activity of 203 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re- suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. No full conversion (< 100 ppm residual acrylonitrile) was reached after 19 h.

Figure 8: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 7.5), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. A total amount of 1.29 g of the dried biocatalyst (batch Ch30) was employed, which had an NHase activity of 203 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 4.3 h.

Figure 9: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity of 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. No full conversion (< 100 ppm residual acrylonitrile) was reached after 20 h.

Figure 10: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 8.0), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity of 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 4.4 h.

Figure 11 : Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM citrate buffer (pH 7.0), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity of 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 7.25 h. Figure 12: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1.84 g of the dried biocatalyst (batch Ch08) was employed, which had an NHase activity of 136 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1 .31 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re- suspended biocatalyst corresponding to 0.53 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. No full conversion (< 100 ppm residual acrylonitrile) was reached after 18 h.

Figure 13: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 7.0), which corresponds to the pre-treatment step of the invention. Pre-treatment was conducted for 0.5 h. A total amount of 1 .84 g of the dried biocatalyst (batch Ch08) was employed, which had an NHase activity of 136 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1.31 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.53 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 8.0 h.

Figure 14: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1.94 g of the dried biocatalyst (batch Ch13-PP/200) was employed, which had an NHase activity of 129 kU/g as measured before the beginning of the bioconversion. Batch Ch13-PP/200 of the biocatalyst Rhodococcus rhodochrous used in the experiments of Fig. 14 and 15 differs from batch Ch08 used in the experiments of Fig. 12 and 13 only in that Ch13-PP/200 has been spray dried together with phosphate buffer according to Example 2, while Ch08 has been spray dried without addition of buffer. Both batches Ch08 and Ch13-PP/200 have been obtained from the same concentrated cell suspension. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1.39 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.55 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 8.9 h.

Figure 15: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41 164 as biocatalyst.

The dried biocatalyst has been re-suspended in 30 ml. of 100 mM phosphate buffer (pH 7.0).

Treatment with the phosphate buffer was conducted for 0.5 h. A total amount of 1 .94 g of the dried biocatalyst (batch Ch13-PP/200, wherein the biocatalyst has been spray dried together with phosphate buffer according to Example 2) was employed, which had an NHase activity of 129 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L = liter) at 26 °C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1.39 g dried biocatalyst was added to the reactor. The acrylonitrile (ACN) concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding acrylonitrile to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.55 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the acrylonitrile concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (< 100 ppm residual acrylonitrile) was 6.63 h.

EXAMPLES

The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration, and the present invention is limited only by the claims. It will be clear to a person skilled in the art that the invention may be practiced in other ways than as particularly described in the present description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

Example 1 : Pretreatment of a dried biocatalyst with buffer

Spray dried biocatalyst is weighed out in a centrifuge tube (Falcon®) and suspended in 30 ml. buffer for the pre-treatment step according to the invention. Unless indicated otherwise, said buffer was 100 mM phosphate buffer, pH 7.0. The biocatalyst is buffer-treated for 0.5 h at room temperature. Then the biomass (biocatalyst) suspension is transferred to the reactor and further incubated for 1 h. After addition of the biomass suspension to the reactor, the centrifuge tube is rinsed with water and the solvent is transferred as well to the reactor. This amount of water is considered for the water weighing into the reactor. Example 2: Pre-treatment of a biocatalyst with buffer before spray drying

After fermentation, the cell suspension is concentrated by mechanical means, e.g. by centrifugation, filtration or membrane processes. The concentrate has a molar phosphate concentration of about 10 mM. The concentrate is the form of the biocatalyst prior to its drying step. Concentrate means that the fermentation broth is concentrated by reducing liquid fermentation broth, e.g. by centrifugation and/or filtration. Thus, the fermentation broth, the concentrate and the dried powder as used in this Example contain the same biocatalyst. Then, a concentrated buffer solution is added to the cell suspension, thereby increasing the phosphate amount up to 200 mM. Shortly afterwards the cell suspension is spray dried. For preparation of the biocatalyst lot Ch13PP/200 (used in the experiments depicted in Figure 14 and 15), 1 .3 kg of a 6 M potassium phosphate buffer, pH 8.0, was added to 40 kg of a fermentation broth, which had been concentrated to 15.3 w/w % total dry mass by centrifugation. The mixture was kept at 4-8 °C. The mixture was used as the feed to the spray dryer. Spray drying was operated with N2 as drying gas, at 1 15 °C gas inlet temperature and 65°C gas outlet temperature. The same concentrated fermentation broth, before phosphate buffer addition, had been used to produce the biocatalyst lot Ch08 (used in the experiments depicted in Figure 13 and 14), under identical drying conditions.

Example 3: General protocol for bioconversion

The hydration of acrylonitrile is generally carried out in a stirred tank reactor (rpm = 250, volume V = 4 L) with an external circulating loop for cooling. For this purpose 2.4 L of water is filled in the reactor as well as the biocatalyst. Biomass is added as spray dried cells of Rhodococcus rhodochrous, which has been previously suspended into water. As described herein, the spray dried cells can also be suspended in buffer as pre-treatment. In order to start the reaction acrylonitrile is dosed into the stirred tank reactor employing a process control system. A constant concentration of acrylonitrile of 0.5 to 5 w/w % is adjusted by the use of an online Fourier Transform Infrared (FTIR) analysis, which directly communicates with the process control unit (Labview). The reaction temperature is constantly kept at 20 to 29°C. The dosage of acrylonitrile is stopped after the addition of 1553 g acrylonitrile. After the complete conversion of residual acrylonitrile, i.e. when a residual acrylonitrile (ACN) concentration of < 100 ppm is reached, and obtaining 52 w/w % acrylamide, the reaction is finished. Such concentrations of acrylamide and acrylonitrile are determined via HPLC using the method set forth in Example 4. The acrylamide concentration determined via HPLC may slightly deviate from the acrylamide concentration shown in the Figures, which have been monitored via FTIR.

Example 4: Determination of the concentration of acrylic acid, acrylamide, acrylic acid and acrylonitrile in the obtained aqueous acrylamide solutions by HPLC

The following conditions were applied in order to determine the contents of acrylamide, acrylic acid and acrylonitrile: Column: Aqua C18, 250^*4.6 mm (Phenomenex) Guard column: C18 Aqua

Temperature: 40 °C

Flow rate: 1 .00 ml/min

Injection volume: 1 .0 μΙ

Detection: UV detector, wavelength 210 nm

Stop time: 8.0 minutes

Post time: 0.0 minutes

Maximum pressure: 250 bar

Eluent A: 10 mM KH₂P0₄, pH 2.5

Eluent B: Acetonitrile

Gradient:

Fermentation broths, bioconversion mixtures Sample is filtered through 0.22 μηη

Analytes:

Example 5:

Spray dried Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch 10 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Example 4.

In Run 1 (depicted in Fig. 1 ), 3.36 g biocatalyst that has been re-suspended in water was used (2.4 g was added at the beginning of the bioconversion, 0.96 g was added after 1 h). In Run 2 (depicted in Fig. 2), 3.36 g biocatalyst that has been pre-treated with 100 mM phosphate buffer (pH 7.0) was used (2.4 g was added at the beginning of the bioconversion, 0.96 g was added after 1 h). In Run 3 (depicted in Fig. 3), 3.36 g biocatalyst that has been re-suspended in water was used (2.4 g biocatalyst was added at the beginning of the bioconversion, 0.96 g biocatalyst was added after 1 h), but the same amount of phosphate buffer that was used in the pre-treatment step of Run 2 was directly added to the reactor prior to the addition of biocatalyst. In Run 4 (depicted in Fig. 4), 1 .8 g biocatalyst that has been pre-treated with 100 mM phosphate buffer (pH 7.0) was used (1 .8 g biocatalyst was added at the beginning of the bioconversion). The results are outlined in the table below.

(100 mM, pH 7.0) to

the reactor

4 1 .8 g Rhodococcus 100 mM phosphate 7.13 h

rhodochrous (NCI MB buffer (pH 7.0)

41 164) of batch

Ch10

As can be seen from Run 2, pre-treatment of the dried biocatalyst with phosphate buffer reduces the total reaction time from 13.78 h to 2.31 h. Run 3 shows that addition of phosphate buffer to the reaction mixture without pre-treating the dried biocatalyst with buffer has almost no influence on the reaction time. Run 4 demonstrates that if the dried biocatalyst is pre-treated with phosphate buffer, the amount of the biocatalyst can be reduced from 3.36 g to 1 .8 g while the total reaction time is still less than using 3.36 g of non-pre-treated biocatalyst. Example 6:

Spray dried Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch32 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Example 3. In Run 5 (depicted in Fig. 5), 1 .73 g biocatalyst that has been re-suspended in water was used (1 .24 g biocatalyst was added at the beginning of the bioconversion, 0.49 g biocatalyst was added after 1 h). In Run 6 (depicted in Fig. 6), 1 .73 g biocatalyst that has been pre- treated with 100 mM phosphate buffer (pH 7.5) was used (1 .24 g biocatalyst was added at the beginning of the bioconversion, 0.49 g biocatalyst was added after 1 h). The results are outlined in the table below. Run # Biocatalyst Pre-treatment Total reaction time

5 1 .73 g Rhodococcus no / resuspension in Incomplete

rhodochrous (NCI MB water conversion after 19 h 41 164) of batch

Ch32

6 1 .73 g Rhodococcus 100 mM phosphate 4.6 h

rhodochrous (NCIMB buffer (pH 7.5)

41 164) of batch

Ch32

Similar to the results of Example 5, pre-treatment of Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch32 with phosphate buffer leads to a dramatic reduction of total reaction time from an incomplete conversion after 19 h to a complete conversion after 4.6 h.

Example 7

Spray dried Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch30 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Example 3.

In Run 7 (depicted in Fig. 7), 1.29 g biocatalyst that has been re-suspended in water was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). In Run 8 (depicted in Fig. 8), 1.29 g biocatalyst that has been pre- treated with 100 mM phosphate buffer (pH 7.5) was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). The results are outlined in the table below. Run # Biocatalyst Pre-treatment Total reaction time

7 1 .29 g Rhodococcus no / resuspension in Incomplete

rhodochrous (NCIMB water conversion after 19 h 41 164) of batch

Ch30

8 1 .29 g Rhodococcus 100 mM phosphate 4.3 h

rhodochrous (NCIMB buffer (pH 7.5)

41 164) of batch

Ch30

Similar to the results of Examples 6 and 7, pre-treatment of Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch30 with phosphate buffer leads to a dramatic reduction of total reaction time from an incomplete conversion after 19 h to a complete conversion after 4.32 h.

Example 8

Spray dried Rhodococcus rhodochrous (NCIMB 41 164) of batch V3 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Example 3.

In Run 9 (depicted in Fig. 9), 1.29 g biocatalyst that has been re-suspended in water was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). In Run 10 (depicted in Fig. 10), 1.29 g biocatalyst that has been pre- treated with 100 mM phosphate buffer (pH 8.0) was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). In Run 1 1 (depicted in Fig. 1 1 ), 1 .29 g biocatalyst that has been pre-treated with 100 mM citrate buffer (pH 7.0) was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). The results are outlined in the table below.

As can be seen from Runs 10 and 1 1 , both the pre-treatment of Rhodococcus rhodochrous (NCIMB 41 164) of batch V3 with phosphate buffer (100 mM, pH 8.0) and citrate buffer (100 mM, pH 7.0) leads to a dramatic reduction of total reaction time from an incomplete conversion after 20 h to a complete conversion after 4.39 h and 7.25 h, respectively. Example 9

Spray dried Rhodococcus rhodochrous (NCIMB 41 164) of batch Ch08 and Ch13-PP/200 were used for bioconversion reactions of acrylonitrile to acrylamide. Batch Ch13-PP/200 differs from batch Ch08 only in that batch Ch13-PP/200 has been spray dried together with phosphate buffer according to Example 2, while Ch08 has been spray dried without addition of buffer. Both batches Ch08 and Ch13-PP/200 have been obtained from the same concentrated cell suspension. The bioconversion reactions were carried out according to the protocol of Example 3. In Run 12 (depicted in Fig. 12), 1 .84 g biocatalyst of batch Ch08 that has been re-suspended in water was used (1 .31 g biocatalyst was added at the beginning of the bioconversion, 0.53 g biocatalyst was added after 1 h). In Run 13 (depicted in Fig. 13), 1 .84 g biocatalyst of batch Ch08 that has been pre-treated with 100 mM phosphate buffer (pH 7.0) was used (1.31 g biocatalyst was added at the beginning of the bioconversion, 0.53 g biocatalyst was added after 1 h). In Run 14 (depicted in Fig. 14), 1.94 g biocatalyst of batch Ch13-PP/200 that has been re-suspended in water was used (1 .39 g biocatalyst was added at the beginning of the bioconversion, 0.55 g biocatalyst was added after 1 h). In Run 15 (depicted in Fig. 15), 1.94 g biocatalyst of batch Ch13-PP/200 that has been further treated with 100 mM phosphate buffer (pH 7.0) was used (1 .39 g biocatalyst was added at the beginning of the bioconversion, 0.55 g biocatalyst was added after 1 h). The results are outlined in the table below.

Ch08

14 1 .94 g Rhodococcus addition of phosphate 8.9 h

rhodochrous (NCIMB buffer prior to spray

41 164) of batch drying

Ch13-PP/200 resuspension in

water

15 1 .94 g Rhodococcus addition of phosphate 6.63 h

rhodochrous (NCIMB buffer prior to spray

41 164) of batch drying

Ch13-PP/200 further treatment

(resuspension) In

100 mM phosphate

buffer (pH 7.0)

As can be seen from Run 14, addition of phosphate buffer prior to spray drying and re- suspending the dried biocatalyst in water achieves a similar reduction of total reaction time as re-suspending a dried biocatalyst (without prior addition of buffer) in phosphate buffer, as can be seen in Run 13. If buffer is added prior to spray drying, the buffer components (e.g. salts) will be contained in the dried biocatalyst. When said dried biocatalyst is re-suspended in water, the buffer components will dissolve as well and thus give a buffered aqueous mixture. However, re-suspending said biocatalyst in buffer will further reduce the total reaction time, as can be seen from Run 15.

Example 10

Spray dried biocatalyst powder with a specific nitrile hydratase activity of 90 kU/g powder was suspended in deionized water and several different buffers before being used in a bioconversion reaction. 70,7 mg biocatalyst powder was suspended in 10 g suspension medium (i.e. either deionized water or a buffer solution) and gently mixed at room

temperature for 10-20 minutes. The biocatalyst suspension was then transferred to a stirred reactor containing 50,5 g deionized water at room temperature. The temperature was adjusted to 20 °C, and this temperature was controlled throughout the complete

bioconversion reaction. 39,5 g acrylonitrile was added to the reactor at a constant rate of 9,9 g/h, so that the complete acrylonitrile amount was added within 4 hours. Two hours after the feed had stopped (i.e. after 6 hours total reaction time), a sample was taken from the reaction solution and the acrylamide concentration was analyzed by HPLC. Another sample was taken after 24 hours total reaction time. The results are shown in the table below.

TRIS-HCI: 2-Amino-2-(hydroxymethyl)-1 ,3-propanediol hydrochloride ACES: N-(2-Acetamido)-2-aminoethanesulfonic acid

TES: 2-[(2-Hydroxy-1 ,1 -bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid

As shown in the table above, conversion of acrylonitrile to acrylamide was significantly improved when the biocatalyst was suspended in a buffer solution before being added to the reaction solution (Exp.# 2-5) in comparison to suspension in deionized water (Exp.# 1 ). In Exp.# 2 and 5, no residual acrylonitrile was detected by the HPLC analysis after 6 h. In Exp# 3, no acrylonitrile was detected after 24 h. In Exp.# 4 approximately 1 % residual acrylonitrile was still present after 24 h, and in Exp.# 1 the residual acrylonitrile after 24 h was approximately 14 wt%.

Example 1 1 2410 g deionized water and 25 g ACN were placed in a reactor. 1 ,92 g biocatalyst powder containing spray dried cells of Rhodococcus rhodochrous NCIMB 41 164, with a specific nitrile hydratase activity of 153 kU/g dry powder and 1528 g additional ACN was then added to the reactor in the following manner:

Run #1 (reference, without buffer): 1 ,37 g biocatalyst powder was suspended in 20 ml deionized water at room temperature and added to the reactor, whereby the bioconversion reaction started. The mixing tube, with a total volume of 50 ml, was rinsed with 5 g deionized water, which were also added to the reactor. After 1 h reaction time, an additional 0,55 g biocatalyst powder was suspended in 20 g deionized water at room temperature and added to the reactor. Again, the mixing tube was rinsed with 5 g deionized water, which were also added to the reactor. 1528 g ACN was added continuously to the reactor. The ACN concentration was measured by on-line FTIR, and the rate of addition of ACN was adjusted so that the ACN concentration in the reaction mixture was kept constant at 1.0 ± 0.1 % (w / w) until the entire ACN had been added to the reaction mixture. The reaction was stopped after ACN concentration had decreased to <100 ppm due to conversion. Runs #2 and #3: 1 ,92 g biocatalyst powder was suspended in 30 ml buffer solution at room temperature and mixed for 15 minutes. From this suspension, 22 ml was added to the reactor, whereby the bioconversion reaction started. After 1 h reaction time, the rest of the biocatalyst suspension (9 ml) was added to the reactor. Acrylonitrile addition was performed as described for Run# 1 above. In all runs, the reaction temperature was controlled at 23 °C throughout the complete reaction time. At the end of the reaction, the ACM concentration in every run was≥ 51 % (w / w), determined using HPLC according to the method provided below. The results are shown in the table below. Reaction time

Run Pre-treatment mixture

[h]

#1 Deionized water 7,8

#2 50 mM potassium phosphate buffer, pH 7,0 5,1

#3 100 mM potassium phosphate buffer, pH 7,0 5,0

As shown in the table above, the conversion of acrylonitrile to acrylamide was significantly faster when the biocatalyst had been suspended in a small volume of buffer solution before being added to the reaction solution. The use of a 100 mM buffer had a slightly better effect than a 50 mM buffer.

Claims

A method for producing an amide compound from a nitrile compound in aqueous mixture, the method comprising:

(a) a pre-treatment step comprising mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and

(b) converting the nitrile compound to the amide compound using the biocatalyst in a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture, optionally before the end of the conversion, is about 2:1 or more.

The method of claim 1 , wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture, optionally before the end of the conversion, is about 3:1 or more, preferably about 4:1 or more, more preferably about 5:1 or more, even more preferably about 7:1 or more, still more preferably about 10:1 or more, still more preferably about 20:1 or more, still more preferably about 50:1 or more, still more preferably about 75: 1 or more, most preferably about 100:1 or more.

The method of claim 1 or 2, wherein the pre-treatment mixture has a pH value from about 6.6 to about 9, preferably from about 6.6 to about 8.8, more preferably from about 6.7 to about 8.6, even more preferably from about 6.8 to about 8.

4, still more preferably from about 6.9 to about 8.2, most preferably from about 7 to about 8.

The method of any one of the preceding claims, wherein the buffer has a pKa in a range of from about 6 to about 9, preferably from about 6.

5 to about 8.

The method of any one of the preceding claims, wherein the buffer concentration in the pretreatment mixture is selected from the group consisting of more than 50 mM, more than 60 mM, more than 80 mM or more than 90 mM.

6. The method of any one of the preceding claims, wherein the buffer comprises a compound selected from the group consisting of phosphate, citrate, 2-[(2-hydroxy-1 ,1 - bis(hydroxymethyl)ethyl)amino] ethanesulfonic acid (TES), 1 ,4- piperazinediethanesulfonic acid (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), tris(hydroxymethyl) aminomethane (TRIS) and any combination thereof.

7. The method of any one of the preceding claims, wherein the amide compound is acrylamide, methacrylamide, acetamide or nicotinamide, preferably acrylamide; and/or wherein the nitrile compound is acrylonitrile, methacrylonitrile, acetonitrile or 3- cyanopyridine, preferably acrylonitrile.

8. The method of any one of the preceding claims, wherein the biocatalyst is a microorganism, preferably a nitrile hydratase (NHase) producing microorganism.

9. The method of claim 8, wherein the microorganism is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium,

Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.

10. The method of claim 9, wherein the microorganism is selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, wherein preferably the microorganism is Rhodococcus rhodochrous or Rhodococcus pyridinovorans.

1 1 . The method of any one of the preceding claims, wherein the dried biocatalyst is a biocatalyst that has been subjected to a drying step before mixing the biocatalyst with an aqueous solution, wherein preferably said drying is mediated by spray drying, freeze-drying, heat drying, air drying, vacuum drying, fluidized-bed drying and/or spray granulation, wherein spray drying and freeze-drying are more preferred.

12. The method of any one of the preceding claims, wherein the pre-treatment step comprises the steps of:

(i) contacting an aqueous solution with a buffer to give a buffered aqueous solution, and

(ii) mixing the dried biocatalyst with the buffered aqueous solution to give a pre- treatment mixture.

The method of any one of claims 1 to 1 1 , wherein the pre-treatment step comprises contacting a mixture of the dried biocatalyst and the buffer with an aqueous solution to give a pre-treatment mixture, wherein preferably the biocatalyst and the buffer are mixed prior to or during the drying step.

14. The method of any one of the preceding claims, wherein the pH is not controlled during step (b).

15. The method of any one of the preceding claims, wherein the ratio of the volume of the pre-treatment mixture to the volume of the reaction mixture, optionally before the end of the conversion, is about 1 :2 or less, preferably about 1 :3 or less, more preferably about 1 :4 or less, even more preferably about 1 :5 or less, still more preferably from about 1 :5 to about 1 :1000, still more preferably from about 1 :10 to about 1 :500, still more preferably from about 1 :20 to about 1 :200, most preferably from about 1 :50 to about 1 :100.

16. An aqueous amide compound solution obtainable or being obtained by the method of any one of the preceding claims.

17. A composition comprising an amide compound and a biocatalyst, wherein the biocatalyst has been pre-treated according to a method of pre-treating a dried biocatalyst for the production of an amide compound from a nitrile compound in aqueous mixture, comprising:

(a) mixing a dried biocatalyst with an aqueous solution to give a pre-treatment mixture, wherein the pre-treatment mixture comprises a buffer, and

(b) contacting the biocatalyst with an aqueous mixture and/or a nitrile compound to form a reaction mixture, wherein the reaction mixture comprises said buffer of step (a), wherein the ratio of the molar concentration of the buffer in the pre-treatment mixture to the molar concentration of said buffer in the reaction mixture, optionally before the end of conversion, is about 2:1 or more.