WO1991008288A1

WO1991008288A1 - An immobilized enzyme preparation whereby a basic amino acid has been incorporated and a process for the isomerization of glucose or xylose

Info

Publication number: WO1991008288A1
Application number: PCT/DK1990/000306
Authority: WO
Inventors: Sven Pedersen; Niels Bindesbøll NIELSEN
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1989-11-23
Filing date: 1990-11-23
Publication date: 1991-06-13
Anticipated expiration: 1992-05-23

Abstract

The activity of an immobilized enzyme at low pH (e.g. glucose isomerase at pH 5.6) can be increased by incorporating a basic amino acid such as ornithine. Addition of a C1-C3 carboxylate such as acetate improves the stability of immobilized glucose isomerase at low pH; the stability can be further increased by the presence of an iron salt.

Description

AN IMMOBILIZED ENZYME PREPARATION WHEREBY A BASIC AMINO ACID HAS BEEN INCORPORATED AND A PROCESS FOR THE ISOMERIZATION OF GLUCOSE OR XYLOSE.

TECHNICAL FIELD

This invention relates to a particulate immobilized enzyme preparation (particularly immobilized glucose isomerase), and to its production and use. Specifically, the invention relates to the improvement of activity and/or stability at low pH (i.e. below optimum pH) of the immobilized enzyme preparation.

BACKGROUND ART

Immobilized glucose isomerase has been used for many years in large- scale isomerization of glucose to produce fructose-containing syrups. Immobilized glucose isomerases have optimum activity and stability around pH 8, but during more than 15 years of industrial use of this immobilized enzyme there has been a wish to operate at lower pH, e.g. around pH 5-6, in order to reduce costs for pH adjustment and subsequent purification and to reduce the risk of microbial infection. It has also been suggested to use immobilized glucose isomerase

(which is also called xylose isomerase) for the simultaneous fermentation and isomerization of xylose (S.M. Lastick, Appl.Microbiol.Biotechnol (1989) 30:574-579). As fermentation by yeast occurs most efficiently at about pH 5.0, it is desirable to use the glucose isomerase (xylose isomerase) near this pH. Many methods for immobilization of enzymes such as glucose isomer¬ ase are known. Thus, cross-linking with a cross-linking agent such as giutaral- dehyde without the need for a solid carrier has been widely studied and also used commercially (e.g. US 3,980,521 , US 4,025,389, US 4,572,897). Such methods can be used for producing physically strong particles, suited for continuous operation in fixed-bed columns.

It is the object of the invention to provide an immobilized enzyme preparation, particularly immobilized glucose isomerase, having increased activity and/or stability at low pH.

There have been some reports of immobilized enzymes with increased activity at low pH. Thus, N. Kamath et al., Appl. Biochem. Biotech., 19, 251-258 (1988) describes urease immobilized on polyethyleneimine-coated cloth. L Goldstein, Methods in Enzymology, vol. 44, describes a polycationic, polyomithyl derivative of a protease. There is no report in the prior art of chemically modifying immobilized glucose isomerase to increase the activity or stability at low pH. And there is no report of such a treatment applied an immobilized enzyme produced by cross-linking with cross-linking agent.

SUMMARY OF THE INVENTION

We have found that, surprisingly, the low-pH activity of immobilized glucose isomerase or of an enzyme immobilized by cross-linking can be increased by incorporating a basic amino acid. We have also found that addition of a C,- C₃ carboxylate to the syrup improves the stability of immobilized glucose isomerase at low pH and that the stability can be further increased by the presence of an iron salt.

Accordingly, the invention provides a particulate immobilized enzyme preparation obtainable by cross-linking of enzymatically active biological material in aqueous phase with a cross-linking agent for amino groups, characterized in that a basic amino acid is incorporated before or during the cross-linking. The invention also provides a method of preparing this and use thereof in an enzyme- catalyzed process at a pH below optimum for the enzyme activity, preferably at least 1 pH unit below said optimum. In another aspect, the invention provides a particulate immobilized glucose isomerase preparation, characterized in that a basic amino acid is incorporated. The invention also provides a method of preparing this and use thereof for isomerization of glucose or xylose in an aqueous solution at a pH below 7, preferably below 6, e.g. 5-7 or 5-6.

Finally, the invention provides a process for the isomerization of glucose or xylose in aqueous solution in the presence of immobilized glucose isomerase below pH 6 (especially 5-6), characterized by the presence in the reaction system of 1-10 m-mole/l of

carboxylate.

DETAILED DESCRIPTION OF THE INVENTION

Enzvmaticallv active material

The enzymatically active material to be immobilized according to the invention may be a crude or purified solution of enzyme; it may comprise whole cells or cell mass from fully or partially disrupted cells; it is generally of microbial origin.

A purified enzyme solution may be preferred to obtain immobilized enzyme with high activity. On the other hand, in the case of immobilization by cross-linking without a carrier, use of cell mass may lead to physically stronger particles. An advantageous compromise is the use of crude enzymatically active cell mass fortified with purified enzyme.

Glucose isomerase

The enzymatically active biological material may particularly comprise glucose isomerase. The glucose isomerase to be used in the invention is preferably derived from a strain of Streptomyces (e.g. S. murinus), Bacillus (e.g. S. coagulans) or Actinoplanes (e.g. A. missouriensis). In the examples that follow, glucose isomerase activity is determined from isomerization of a 40% aqueous solution of pure fructose at 60 °C, pH 7.4 for

1 hour in a batch system. One New Glucose Isomerase Unit (1 NGIU) is defined as the amount of immobilized enzyme that isomerises fructose at an initial rate of 1 μmole/min at these conditions.

Basic amino acid

The basic amino acid used in the invention is one that has an excess of basic groups over acid groups and is therefore cationic at neutral pH. Examples are lysine, hydroxylysine, arginine, histidine and ornithine. The amount of amino acid is preferably 2-50% by weight of the dry matter in the biological material, especially 5-30%.

Cross-linking process

In the cross-linking process of the invention, the biological material and the basic amino acid are mixed in aqueous phase. The quantity of water present in the reaction mixture is not critical. Excess water will be removed during dewatering without any serious loss of active material. Thus, water may be added to obtain a convenient consistency during the process. Conveniently, the biological material is added in the form of an aqueous dispersion or solution typically with 1-25% (w/w) of dry substance, particularly 1-10% in the case of culture broth or 10-25% in the case of a purified enzyme.

Optionally, a polyamine, such as polyethyleneimine (abbreviated as PEI) or chitosan, may be incorporated before addition of cross-linking agent, e.g. in an amount of 2-15%, in order to improve the physical strength of the particles.

The cross-linking agent used in the invention is one that reacts with amino groups. Examples are glutaraldehyde, diisocyanates (e.g. toluylene or hexamethylene diisocyanate) and polyazetidine (as described in EP 297,912). The amount of cross-linking agent is preferably 10-20% by weight of the dry matter in the biological material. After adding this, the mixture is left for cross-linking to occur, e.g. 0.5-2 hours.

The temperature throughout the process is generally in the range 0- 60 °C. Temperature near ambient is often convenient, but lower temperature may be needed due to enzyme instability. pH throughout the process is generally around neutral, mostly between about 5 and about 9. Higher or lower pH may be preferred depending on the enzyme stability. A buffer may be included to stabilize pH during the reaction.

After the cross-linking, the immobilized preparation may be dewatered (e.g. by filtration), sub-divided into particles (e.g. by extrusion), optionally rounded (e.g. in a Marumerizer*) and dried by conventional means.

Isomerization process

Isomerization of glucose or xylose according to the invention can is advantageously done at pH 5-7, especially 5-6, where the invention provides improved enzyme stability and/or activity.

The isomerization process may be isomerization of glucose to produce fructose-containing syrup, typically at 40-50% dry solids, 50-70° C, with addition of 0.2-2 mM of a magnesium salt, optionally with addition of a sulphite in amount corresponding to 50-500 ppm S0₂. Alternatively, the process may be simultaneous fermentation and isomerization of xylose.

C,-C. carboxylate

The carboxylate salt used in the invention is preferably acetate. The carboxylate may be dissolved to the glucose (xylose) solution in the form of a soluble salt such as sodium acetate. Alternatively, the carboxylated may be added during production of the immobilized glucose isomerase, e.g. by immersing the immobilized preparation in a solution of a soluble salt, e.g. 15-30% sodium acetate at pH 5-9 for 2-6 hours. Iron salt

In the isomerization process at low pH with carboxylate according to the invention, the presence of an iron salt is preferred as this further improves the enzyme stability. This may be incorporated as 0.2-20 mM of a soluble non-toxic salt such as ferrous sulphate, ferric chloride or ferrous acetate.

Alternatively, 0.2-5% (as Fe relatively to dry matter) may be incorporated in the immobilized glucose isomerase. This may be incorporated incorporated as a soluble salt or as a cation exchange resin in Fe(ll) form, especially a complex binding resin, e.g. in an amount of 10-50% of total dry substance.

EXAMPLES

EXAMPLE 1

Immobilized glucose isomerase with ornithine

100 ml of a 15% (by weight) solution of glucose isomerase from Streptomyces murinus (prepared according to US 4,687,742) was mixed with 5 g of ornithine dissolved in 200 ml of water and 3 g of polyethyleneimine dissolved in 12.4 ml of water. pH was adjusted to 7.5, and 8.84 ml of a 50% (by weight) glutaraldehyde solution was added. pH was kept at 7.5 for 2 hours, whereafter the mixture was flocculated with a cationic flocculant (Cyanamid C-521). The cross- linked enzyme was then recovered by filtration, formed into particles by extrusion through a 0.8 mm screen and dried at room temperature.

A reference sample was prepared in the same way, but without ornithine.

The activity was determined at various pH, and the results, shown below, are expressed as activity in percent of that at pH 7.4. Activity

PH Invention Reference

(with ornithine) (without ornithine)

It is seen that ornithine changes the activity-pH curve of immobilized glucose isomerase to relatively higher activity at low pH, i.e. pH 5-6.

EXAMPLE 2

Preparations with Varying Ornithine Amount

A number of immobilized glucose isomerase preparations were made by the same procedure as in Example 1 , but with varying amounts of ornithine.

The activity of each preparation was measured by the NGIU method at several pH.

Results are shown below; the amount of ornithine is expressed in percent of enzyme solution dry matter.

PH

The activity at low pH (5.3-5.8) is significantly increased by incorporation of ornithine especially above 20-30%. Without ornithine, the activity optimum is about pH 7.2; with ornithine, it changes to about 6.0-6.5. A preparation made with 60% ornithine was judged to be physically too weak for practical use.

EXAMPLE 3

Isomerization in Presence of acetate and Fe Four experiments were made as described below, using the same batch of glucose isomerase-containing fermentation broth.

Experiment 1: Ornithine and Fe in immobilized preparation. 1 I of glucose isomerase containing fermentation broth from Streptomyces murinus (prepared according to US 4,687,742, dry substance content 4%) was mixed with 10 g MgSOψ 7H₂0 and 4 g L-ornithine hydrochloride and pH adjusted to 7.5. 5 ml 50% glutaraldehyde was added and the cell sludge was stirred for 10 minutes under pH adjustment to pH 7.5. Then 5.0% (of cell sludge dry matter) of polyethylene imine (Sedipur, product of BASF, West Germany) was added and after thorough mixing 11 ml glutaraldehyde (total glutaraldehyde 17.4% based on cell sludge plus L-ornithine hydrochloride plus polyethylene imine dry substance) foe crosslinking of the mixture. pH was constantly adjusted to 7.5. After 1 hour the crosslinked mixture was flocculated by addition of a cationic flocculant (Superfloc C 521, Cyanamid Int.). The crosslinked enzyme was recovered by filtration. 1 g FeS0₄, 7H₂0 was mixed thoroughly with the filter cake and the mixture formed into particles by extrusion through a 0.8 mm screen and dried at room temperature.

Experiment 2: Ornithine. Fe and ion exchange resin in preparation. The method of preparation was identical to preparation 1 up to and including the filtration step. The filter cake was then mixed with 0.5 g FeSOψ 7H₂0 and 5 g (dry weight) cation exchange resin (Amberlite CG-120) in Fe(ll) form. The mixture was formed into particles by extrusion through a 0.8 mm screen and dried at room temperature. The resin was pretreated as follows: The commercial resin (ⁱn Na+ f₀rm) was stirred for 40 minutes with 5 volumes 4% HCI, the resin was then washed with 10 volumes water (ion exchanged) and stirred for 60 minutes with 5 volumes 3% FeSOψ 7H₂0 solution (pH kept constant at 7.5 with NaOH). After washing with 10 volumes ion exchanged water the resin was ready for use. Experiment 3: ^• Ornithine. Fe and complex binding resin in preparation. An immobilized preparation was made as in example 2, except that a complexing cation exchange resin was used (Dowex™ Chelating Resin A-1). The resin was pretreated as described above.

Feed syrup for exps. 1-3. A 45% dry substance solution of crystalline dextrose in demineralized water was sterile filtered on a 0.2 micron filter. MgSOψ 7H 0 and sodium metabisulfite were added to obtain 45 ppm Mg ion and 100 mg/l S0₂, before adding 0.3 ml/I acetic acid (cone.) and adjusting to pH 5.5 with NaOH.

Experiment 4: Ornithine in preparation, Fe in syrup. An immobilized preparation was made as in experiment 1 , but without addition of iron salt to the preparation. Instead, FeS0_4> 7H₂0 was added to the feed syrup to obtain 2 mM Fe(ll).

Isomerization. 9 g of each enzyme preparation was soaked in 50 ml feed syrup for 90 min at ambient temperature, before being loaded into a 15 mm ID water jacketed glass column. A continuous flow of feed syrup was adjusted to maintain a dextrose conversion of 0.40-0.44 at 60" C. As reference, a different batch of the same enzyme was immobilized without ornithine and Fe by a prior-art method (according to US 4,687,742) and was tested without Fe.

Results. The isomerization activity was calculated each day. The results are summarized below (half-life calculated by regression analysis of activity decay). Data for the first 4-5 days were not used as operation was unsteady with pH increase over the columns. Experiment

Reference, without ornithine, Fe and acetate 1. Ornithine and Fe in immobilized preparation 2. Ornithine, Fe and ion exchange resin in preparation

3: Ornithine, Fe and complex binding resin in preparation 4: Ornithine in preparation, Fe in syrup

It is seen that addition of acetate in the preparation together with Fe in the preparation or in the syrup improves the stability (half-life) at low pH. Addition of cation exchange resin together with Fe further improves the stability.

EXAMPLE 4

Isomerization with acetate

A series of preparations were made as described in Example 3, Experiment 1 , except that ornithine was not used; the samples were made with and without Fe (1 g FeS0₄, 7H₂0) or cation exchange resin (5 g Duolite^® C464 in Fe(ll) form).

The preparations were tested in column experiments where half-life was determined. In one set of experiments the syrup consisted of a 45% dry substance solution of crystalline dextrose in demineraiized water, sterile filtered on a 0.2 micron filter. MgS0₄, 7H₂0 and sodium metabisulfite were added to obtain 45 ppm Mg⁺⁺ and 100 mg/l S0₂. pH was adjusted to 5.5 with NaOH. In another set of experiments, 0.3 ml/I of acetic acid (concentrated) was added before the pH adjustment.

The following results show average outlet pH and half-life in days:

It is seen that addition of acetate to the syrup improves the enzyme stability. Addition of Fe together with the acetate improves this further.

EXAMPLE 5

A series of preparations were made as in Example 4, but with and without ornithine hydrochloride (4 g). The preparations were tested in column isomerization with acetate as in Example 4, and initial activity was determined.

The following table shows initial activity (IGIU/g). Average outlet pH was 5.5 in all experiments.

With FeS0₄

With Fe-resin

With FeS0₄ + Fe-resin

It is seen that incorporation of ornithine significantly improves the activity at pH 5.5 in each case. EXAMPLE 6

Isomerization with ornithine. acetate and Fe-complex binding resin

The preparation without iron of Example 4 was used together with a similar preparation made with ornithine hydrochloride (4 g) and Fe-complex binding resin (5 g Dowex^R Chelating Resin A-1 in Fe(ll) form). The preparations were tested in column isomerization with acetate as in Example 4.

The initial activities (IGIU/g) and half-lives (days) obtained were as follows:

Activity Half-life Without ornithine and Fe 110 21

With ornithine and Fe-resin 140 40

The combined use of ornithine and Fe-complex binding resin in the perparation and acetate in the syrup is seen to be particularly advantageous as it provides good acitivity and stability at low pH.

EXAMPLE 7

Immobilization of fortified glucose isomerase

A preparation was made as in Experiment 1 of Example 3 except for: 75 ml of a partly purified glucose isomerase solution from Streptomyces murinus (activity 1000 FINU/g) was added to the fermentation broth (activity 90 FINU/g) before cross-linking. 10 ml 50% glutaraldehyde was used in the first crosslinking step. 2 g FeS0_4I 7H₂0 (instead of 1 g) was used.

A second preparation was made in the same way, but without any FeS0₄. Instead, 5 g Dowex A1 ion exchange resin in Fe(ll) form was added to the filter cake. The two preparations were tested in column isomerization with acetate as in Example 4, and average outlet pH, activity (IGIU/g) and half-life (days) were determined. Results:

Ave. PH Activity Half-life With FeS0₄ 5.5 160 50

With Fe-resin 5.5 160 65

This example illustrates the benefits of using of fortifying cell mass with purified glucose isomerase to obtain high activity and physically strong particles. The use of ornithine, Fe and acetate ensure good activity and stability at low pH.

Claims

1. A particulate immobilized enzyme preparation obtainable by cross- linking of enzymatically active biological material in aqueous phase with a cross- linking agent for amino groups, characterized in that a basic amino acid is incorporated before or during the cross-linking.

2. A preparation according to Claim 1 , whereby the basic amino acid is ornithine, lysine or arginine.

3. A preparation according to Claim 1 or 2, whereby the amount of basic amino acid is 2-50% by weight of the dry matter in the preparation, preferably 5-30%.

4. A preparation according to any of claims 1 - 3, whereby said cross-linking agent is glutaraldehyde, polyazetidine or a diisocyanate.

5. A preparation according to any of claims 1 - 4, whereby the amount of cross-linking agent used is 2-40% by weight of the dry matter in the biological material.

6. A preparation according to any of claims 1 - 5, whereby a polyamine (preferably polyethyleneimine or chitosan) is incorporated before or during the cross-linking, preferably in an amount of 2-15% by weight of the dry matter in the biological material.

7. A method for preparing a particulate immobilized enzyme prepara¬ tion comprising cross-linking of enzymatically active biological material in aqueous phase with a cross-linking agent for amino groups, characterized in that a basic amino acid is incorporated before or during the cross-linking.

5 8. ^' Use of an immobilized enzyme preparation according to any of claims 1 - 6 in an enzyme-catalyzed process at a pH below optimum for the enzyme activity, preferably at least 1 pH unit below said optimum.

9. A particulate immobilized glucose isomerase preparation, characterized in that a basic amino acid is incorporated.

10 10. A preparation according to Claim 9, whereby the basic amino acid is ornithine, lysine or arginine.

11. A preparation according to Claim 9 or 10, whereby the amount of basic amino acid is 2-50% by weight of the dry matter in the preparation, preferably 5-30%.

15 12. A preparation according to any of claims 9 - 11 , obtainable as indicated in any of claims 1 - 6.

13. A preparation according to any of claims 9 - 12, wherein the glucose isomerase is derived from a strain of Streptomyces, Bacillus or Actino- planes.

0 14. A preparation according to any of claims 9 - 13, wherein an iron salt is incorporated, preferably in an amount of 0.2-5% (as Fe) of total dry substance.

15. A preparation according to Claim 14, wherein a cation exchange resin in the Fe(ll) form is incorporated, preferably a complex binding resin.

16. A preparation according to Claim 15, wherein the amount of said resin is 10-50% of total dry substance.

17. A method for preparing a particulate immobilized glucose isomerase preparation, characterized by comprising the incorporation of a basic amino acid.

18. Use of an immobilized glucose isomerase preparation according to any of claims 9 - 16 for isomerization of glucose or xylose in an aqueous solution at a pH below 7, preferably below 6, e.g. 5-7 or 5-6.

19. A process for the isomerization of glucose or xylose in aqueous solution in the presence of immobilized glucose isomerase below pH 6 (especially 5-6), characterized by the presence in the reaction system of 1-10 m-mole/l of C,- C₃ carboxylate.

20. Use according to Claim 19 in the presence of an iron salt, preferably in the form of 0.2-10 mM of a dissolved salt and/or 0.2-5% (as Fe relative to total dry substance) in the immobilized preparation.

21. Use according to any of Claims 18 - 20 in a continuous process with the immobilized enzyme in a fixed bed for production of a fructose-containing syrup.

22. A process according to any of Claims 18 - 20 for simultaneous fermentation and isomerization of xylose.