WO1998026058A1 - Thermostable alpha-glucosidase et pullulanase and their uses - Google Patents

Abstract

The invention concerns a novel alpha -glucosidase and a novel pullulanase obtained from a strain of the genus Thermococcus and the use for transforming a starch into a sugar syrup.

Description

THERMOSTABLE ALPHA-GLUCOSIDASE AND PULLULANASE AND USES THEREOF

The present invention relates to a new α-glucosidase and a new thermostable pullulanase and to their industrial uses.

In the starch industry, the transformation of starch into glucose syrup requires two enzymatic steps. The first step is currently carried out by the action of a thermostable α-amylase and the second by the action of a glucoamylase or (of an α-giucosidase) and of a thermosensitive pullulanase.

Strains of the genus Thermococcus allowing the production of enzymes are already known: for example the document WO-A-95/23852 relates to the strain Thermococcus celer capable of producing an amylase and a pullulanase. One of the aims of the present invention is therefore to provide an α-glucosidase which acts under the same conditions as the α-amylase currently used: thus the transformation of starch into glucose syrup could be done in one step.

This object, as well as others which will appear subsequently, is achieved by an α-glucosidase which is characterized, according to the present invention, by the fact that it is obtained by culture of the strain Thermococcus hydrothermalis, strain CNCM 1 1319 .

As stated above, the present invention also relates to a pullulanase which is characterized in that it is produced simultaneously with the above α-glucosidase. A copy of the strain of the genus Thermococcus hydrothermalis was deposited on June 16, 1993 at the National Collection of Cultures of Microorganisms under the number CNCM I 1319: it corresponds to the strain referenced AL 662 in the collection held by IFREMER.

The two enzymes from Thermococcus hydrothermalis can be used simultaneously with a commercial α-amylase to produce a sugar syrup from starch.

The present invention is described with reference to the appended figures, among which:

- Figure 1 shows the influence of temperature on the activity of an α-glucosidase according to the present invention at pH 5.5; - Figure 2 shows the influence of pH on the activity of an α-glucosidase according to the present invention at a temperature of 80 ° C;

- Figure 3 shows the effect of pH on the activity of a purified pullulanase according to the invention; - Figure 4 shows the effect of temperature on the activity of a purified pullulanase according to the invention as well as the protective effect of calcium at a concentration of 1 mM; and,

- Figure 5 shows the influence of pH on the activity of an α-glucosidase according to the invention and another produced by another strain of Thermococcus. According to the present invention, an α-glucosidase is obtained from a strain of the genus Thermococcus, in particular, Thermococcus hydrothermalis, and a pullulanase from a strain of the genus Thermococcus, in particular Thermococcus hydrothermalis.

A strain of Thermococcus hydrothermalis was made public under the number CNCM I 1319: it was deposited on June 16, 1993.

The α-glucosidase is a constitutive enzyme in Thermococcus hydrothermalis. The development of the production of this enzyme in this bacteria has shown that certain carbon sources (such as soluble starch, glycogen and dextrins) can induce the production of the enzyme by three. The addition of 4 g / l of soluble starch to the induction medium was chosen for the rest of the study.

The relationship between strain growth and enzyme production has been discussed. Thus, Thermococcus hydrothermalis, cultivated in a fermenter of a liter and a half on BHI medium + sulfur + soluble starch, has a generation time of 34 min. and reaches its stationary phase in about 8 hours. The maximum activity is reached in 6-7 hours at the level of the intracellular medium, then it is noted that the intracellular activity gradually decreases. The extracellular activity, which is weak in the first hours of culture, gradually increases up to 24 hours. This phenomenon can be explained by cell lysis of the strain: in fact, the α-glucosidase which is undoubtedly an intracellular enzyme, is released into the culture medium during the stationary phase and the decline phase, causing this increase in extracellular activity.

The study of the physicochemical properties of α-glucosidase in the intracellular medium has shown that this enzyme is optimally active at pH 5.0-5.5 and at approximately 1 10 ° C. It still exhibits 50% of the maximum activity between 96 ° C and 116 ° C. At 120 ° C, the enzyme is denatured. The α-glucosidase of the strain according to the invention is stable for 24 h at 80 ° C, while at 96 ° C, only 50% of the initial activity remains after 2 h 30. The thermal stability of the enzyme is increased in presence of starch. The higher the percentage of starch, the greater the protection of the enzyme against heat inactivation. Thus, at 106 ° C, in the presence of 10% starch, the half-life of the enzyme is 2 h, while without starch, the half-life is only 8 min.

The purification of the enzyme was discussed in order to be able to demonstrate these exact characteristics and so that the enzyme can be compared with the other α-glucosidases already described. For this, Thermococcus hydrothermalis is cultivated in a 200 liter fermenter on BHI medium + sulfur + starch, anaerobically, at 80 ° C and pH 6.0. After 6 hours of growth, the bacterial cells are recovered by centrifugation of the culture medium and broken by passing through a French press. The intracellular medium containing the α-glucosidase is dialyzed with an ultrafiltration cell, the cutoff threshold of which is 30 kDa and concentrated by precipitation of the proteins with ammonium sulphate (70% saturation). After centrifugation, the protein pellet is taken up in a small volume of buffer and dialyzed in order to remove the ammonium sulfate. The protein sample is deposited several times on an ion exchange column (Hitrap Q Sepharose High Performance). The proteins are eluted by means of a discontinuous gradient of Tris-HCL buffer containing 1M NaCl. The fractions having the α-pNPGase activity are pooled and concentrated on UF cell. The third purification step uses affinity chromatography on sepharose gel on which glucose residues are grafted. The enzyme is eluted from the column using a continuous gradient of phosphate buffer containing 1M NaCl. The last step is to pass the protein sample through a molecular sieving column (Sephacryl S200 High performance). The purified enzyme is obtained after all these steps. The protein is monomeric and has a molar mass of around 118,000 daltons.

For the demonstration of the production of a pullulanase, a culture medium was used, called BHIS, composed of infusion of heart-brain (9 g / l), NaCl (23 g / l), sulfur. elemental (5 g / l), PIPES buffer (6.05 g / l), resazurin (1 mg / l). After tyndallisation (40 min at 100 ° C two days successively), the medium is reduced by Na ₂ S (0.5 g / l) in an anaerobic enclosure where the gas composition is N ₂ / H ₂ / CO ₂ in the proportions 90% / 5% / 5%. Incubation is carried out at 80 ° C. After growth, the media are degassed with nitrogen to remove the H ₂ S formed by the isolate, filtered to retain elemental sulfur, then cells and supernatants are separated by centrifugation (10,000 g for 40 min).

The pullulanase activity is determined by measuring the reducing sugars released, by the dinitrosalicylic acid method (Miller 1959), when the enzymatic extract is incubated at 80 ° C in the presence of pullulanase (0.75%) in buffer sodium phosphate (200 mM, pH 6.0).

For the pH study, the conditions are identical to those mentioned above with a 200 mM citrate-phosphate buffer for pH 3.0-7.0 and a 200 mM sodium phosphate buffer for pH 6.0-8.0.

For the study of the induction of pullulanase activity by sugars, another technique was used. This involves monitoring the hydrolysis of the colored pullulan (shiny blue of remazol-pullulan) by the release of the colored group (shiny blue of remazol). The purification of the enzyme was discussed in order to be able to demonstrate these exact characteristics and so that the enzyme can be compared with the other pullulanases already described. For this Thermococcus Hydrothermalis was cultivated under the conditions described above, with the following differences: maltrose is used in place of starch and the culture lasts 23 hours the other conditions are unchanged.

Proteins precipitated with ammonium sulphate were redissolved in sodium phosphate buffer pH 6.0 and placed on a hydrophobic interaction chromatography column. Elution was carried out with a decreasing gradient of ammonium sulfate (from 1 to 0 molar) in sodium phosphate buffer pH 6.0. After this first step, the recovery yield of the pullulanase activity was 31% and the purification rate was 10.

Ion exchange chromatography was then carried out at pH 8.5 after concentration and diafliteration of the fractions containing the pullulanase activity. The proteins were eluted with a sodium chloride gradient (from 0 to 1 molar) in Tris-HCl buffer, pH 8.5. Compared to the starting crude extract, the recovery yield was 15% and the purification rate 30.

The last step was affinity chromatography after concentration and diafiltration of the fractions previously recovered. Malotriose was used as the affinity ligand, and was coupled to activated sepharose. The enzyme was eluted with a sodium chloride gradient (0 to 0.6 molar) in sodium acetate buffer pH 5.5. The recovery yield of this last step was 8% and the purification rate of 97 compared to the crude extract.

The purified enzyme is obtained after all these stages and has a molar mass of approximately 128,000 daltons. The properties of the purified enzyme are illustrated in Figures 3 and 4.

The enzymes according to the present invention are of great utility, especially in starch manufacture: the conversion of starch into various sugar syrups takes place at high temperatures due to the very low solubility of starch.

The use of an α-glucosidase compatible with the α-amylase used during the first stage of transformation makes it possible to reduce the transformation process in a single step. Likewise, the use of a pullulanase which exhibits maximum activity in this same temperature zone, makes it possible to contribute to the conversion of starch into various sugar syrups in a single and same step. The use of these enzymes is illustrated by the following two examples:

Example 1:

Action of a crude extract of the Thermococcus Hydrothermalis strain rich in α-glucosidase.

2 tests are carried out in parallel with native wheat starch as substrate

test 1: Native starch 90 g

CaCI2 60ppm Termamyl 1201 150 micro-liters

phosphate buffer pH 6.0 200 mM in sufficient quantity to have a reaction volume of 300 ml.

test 2 identical to test 1 but with 30 α-giucosidase unit derived from Thermococcus hydrothermalis For the two tests, the incubation temperature is 90 ° C. The results are presented in the following table I:

Table I

The tests were not continued beyond 7 hours, because as we used a crude extract as an enzyme source, very rich in protein, this led to the formation of Maillard compound in the reaction medium making it difficult to analysis of the reaction products.

However, this preliminary example shows that it is possible to carry out the liquefaction reaction and the saccharification reaction in a single step.

Example 2

Action of a crude extract of the Thermococcus hydrothermalis strain rich in pullulanase 2 tests are carried out in parallel with native wheat starch as a substrate test 1: Liquefaction

Starch 160 g / l

Termamyl 120 I 150 micro-liter

CaCl2 60 ppm phosphate buffer pH 6.2 200 mM in sufficient quantity to have a reaction volume of 300 ml.

Incubation for 2 hours at 90 ° C.

Saccharification addition of 55 amyloglucosidase unit AMG 300 L sold by the company Novo Nordisk.

The reaction is followed for 22 hours at 60 ° C. test 2 identical to test 1 but during the liquefaction step, various amounts of Thermococcus hydrothermalis pullulanase were added, namely: 34 Units, 68 Units, 136 Units. The results are shown in Figure 6 attached.

They clearly show us that the addition of thermostable pullulanase during the liquefaction stage makes it possible to significantly reduce the saccharification time.

As another strain of Thermococcus called Thermococcus AN1 is known as being able to produce enzymes with the same function as the strain Thermococcus hydrothermalis, the activity of these two α-giucosidase has been compared.

Thus, FIG. 5 represents the activity of the two enzymes as a function of the pH: it will be noted that the maximum of activity is different, as is the shape of the curve. The maximum activity of an α-glucosidase according to the invention is between 5.5 and 7.5 and is beyond 7 for that derived from Thermococcus NA1 currently called Thermococcus zilligii.

In Table II below, the residual activity rate of these two α-glucosidases has been grouped together during incubation with effectors at room temperature for 1 hour. Table II

SDS: sodium dodecylsulfate

DTT: dithiothreitol

BSA: bovine sero-albumin From these comparative tests, it appears that the α-glucosidase produced by Thermococcus hydrothermalis has its own properties.

Claims

1. α-glucosidase characterized in that it is obtained by culture of the Thermococcus hydrothermalis strain. 2. α-glucosidase, according to claim 1, characterized in that the strain producing it is Thermococcus hydrothermalis, CNCM I 1319. 3. α-glucosidase, according to claim 1 or 2, characterized in that: a) it has a maximum activity at a pH between 5.0-5.6 at a temperature of 96 and 116 ° C, the substrate being starch b) it exhibits a maximum inventive activity at a temperature of 110 ° C. at a pH of 5.5, the substrate being starch. 4. Pullulanase, characterized in that it is obtained by culture of the Thermococcus hydrothermalis strain. 5. Pullulanase, according to claim 4, characterized in that the strain producing it is Thermococcus hydrothermalis, CNCM I 1319. 6. Pullulanase, according to claim 4 or 5, characterized in that: a) it has a maximum activity at a pH between 5.0-6.2 for a temperature of 85 to 115 ° C, the substrate being l starch or pullulan b) it has a maximum activity at a temperature of 110 ° C. at a pH of 5.5, the substrate being pullulan or starch. 7. Process for producing a sugar syrup from starch, characterized by the fact that a commercial α-amylase, an α-glucosidase and a pullulanase derived from Thermococcus hydrothermalis are used simultaneously.