JP4729408B2 - Method for producing immobilized enzyme - Google Patents

Description

  The present invention relates to an immobilized enzyme having a significantly improved enzyme life, a method for producing the same, and a method for producing an optically active cyanohydrin using the immobilized enzyme. More specifically, the present invention relates to an (S) -hydroxynitrile lyase-immobilized enzyme that is produced by adding a specific polymer and has a significantly improved enzyme life, and an industrial process for producing an optically active cyanohydrin using the immobilized enzyme.

  (S) -Hydroxynitrile lyase (SHNL) is an enzyme that catalyzes the reaction of hydrocyanic acid with aldehydes or ketones to produce industrially valuable optically active cyanohydrins. Known SHNLs include SHNL derived from cassava (Manihot esculenta), SHNL derived from Hevea brasiliensis, or SHNL derived from sorghum (Sorghum bicolor). Such synthesis of optically active cyanohydrins using SHNL is not necessarily suitable for an industrial production process as it is, and therefore, many attempts have been made to increase the yield and reduce the cost.

  For example, since an organic solvent system is usually used in the reaction of SHNL, SHNL is often used as an immobilized enzyme in order to disperse an organic solvent-insoluble enzyme in the reaction system and perform the reaction efficiently. . Cellulose and silica-based carriers are widely used as carriers for immobilization, but for (R) -hydroxynitrile lyase (RHNL), a method using polyvinyl alcohol as an additive (evaporation with polyvinyl alcohol gel and immobilization) (See Non-Patent Document 1). However, RHNL and SHNL are both identical in that they are enzymes that synthesize cyanohydrins, but their amino acid sequences and three-dimensional structures are completely different, so the immobilization of RHNL and SHNL cannot be discussed equally.

  In addition, in order to stabilize the enzyme, a protein such as albumin or a polymer such as polyethylene glycol is also added (see Patent Document 1).

  On the other hand, because the cost of separating natural SHNL from biological resources is high, industrially, recombinant SHNL is used in addition to natural enzymes. And, by modifying the recombinant SHNL, a highly active modified recombinant SHNL is developed to increase the yield and reduce the cost (see Patent Document 2).

  On the other hand, a technique for immobilizing RHNL or SHNL whose molecular weight has been increased by cross-linking to polyvinyl alcohol gel has also been reported (see Patent Document 3), but immobilization of SHNL having a molecular weight equivalent to that of a natural enzyme is described. It has not been.

Japanese Patent Laid-Open No. 2004-113053 JP 2005-312431 A Special table 2003-514567 Wehtje E., et al, Biotechnology and Bioengineering, 1993, 41 (2), p.171-178

  In the synthesis of optically active cyanohydrin using (S) -hydroxynitrile lyase (SHNL), the lifetime of the immobilized enzyme is extremely short, so there is a limit to the number of repeated reactions, which has been the cause of high production costs. . An object of the present invention is to provide a method for synthesizing optically active cyanohydrin with higher efficiency and lower cost by extending the lifetime of the immobilized enzyme in the synthesis reaction of optically active cyanohydrin.

  As a result of diligent studies to solve the above problems, the inventors of the present invention can dramatically extend the enzyme life by adding a specific polymer such as polyvinyl alcohol when (S) -hydroxynitrile lyase is immobilized. I found.

  That is, the present invention provides an immobilization wherein (S) -hydroxynitrile lyase is adsorbed on the porous carrier by adding polyvinyl alcohol to the solution containing (S) -hydroxynitrile lyase and mixing with the porous carrier. Provided is a method for producing an immobilized enzyme, which comprises obtaining an enzyme.

  The solution containing (S) -hydroxynitrile lyase used in the present invention preferably has a specific activity of 8 to 80 U / mg consisting of enzyme activity with respect to protein mass. This is because within this range, the effect of extending the life of the enzyme by adding polyvinyl alcohol is great. The enzyme solution concentration can be prepared by diluting with pure water or a buffer solution according to the amount of the immobilization carrier used, or concentrating using an ultrafiltration membrane.

  The ratio of the mass of the enzyme solution and the mass of the carrier can be adjusted depending on the water retention ability of the single substance used, and is preferably in the range of 0.5 / 1 to 2/1. This is because in this range, the formation of a racemate can be suppressed and cyanohydrin can be synthesized efficiently.

  In general, polyvinyl alcohol is obtained by saponifying polyvinyl acetate with an alkali or acid. There are fully saponified polyvinyl alcohols and partially saponified polyvinyl alcohols with some vinyl acetate groups left. Further, there is a modified polyvinyl alcohol obtained by saponifying a polyvinyl acetate copolymer obtained by copolymerizing a vinyl monomer having an amino group or an acid group. In this invention, if it has a vinyl alcohol monomer unit in a polymer component, it will not specifically limit. The polyvinyl alcohol used in the present invention is preferably partially saponified, and more preferably partially saponified with a saponification degree of 60 to 90 mol%. The degree of polymerization of polyvinyl alcohol is preferably about 400 to 2500.

  In the said method, it is preferable that polyvinyl alcohol is added at 3-50 mass% with respect to (S) -hydroxy nitrile lyase.

  In the present invention, the porous carrier to be immobilized is not particularly limited, but a carrier made of a porous inorganic material is preferable, and a suitable example thereof is a silica-based carrier.

  The present invention also provides a method for producing an optically active cyanohydrin by bringing the immobilized enzyme produced by the above-described method into contact with a carbonyl compound and a cyanide compound in the presence of an organic solvent hardly soluble or insoluble in water. .

  Furthermore, the present invention also provides an immobilized enzyme produced by the above-described method and having (S) -hydroxynitrile lyase adsorbed on a porous carrier. This immobilized enzyme is a repetitive reaction for synthesizing (S) -2-chloromandelonitrile from hydrocyanic acid and 2-chlorobenzaldehyde with an optical purity exceeding 90% ee even after 10 reactions (S) -2- It is a long-life immobilized enzyme that can synthesize chloromandelonitrile.

  According to the present invention, the life of the enzyme to which (S) -hydroxynitrile lyase is immobilized can be significantly extended. As a result, the number of repeated uses of the immobilized enzyme is increased, and optically active cyanohydrins can be synthesized with higher efficiency and lower costs than in the past.

1. (S) -Hydroxynitrile lyase “(S) -Hydroxynitrile lyase (hereinafter abbreviated as SHNL)” used in the present invention is natural SHNL isolated or purified from plants, or an amino acid equivalent to the SHNL. It includes all natural recombinant SHNL having a sequence, and all modified recombinant SHNL with genetic modifications to improve activity and stability.

  In the case of natural SHNL, its origin is not particularly limited. And SHNL derived from a tattered plant such as Ximenia america. The amino acid sequences of these SHNLs and the base sequences of the genes are already known and can be easily obtained through public databases such as GenBank. For example, the Para rubber tree-derived SHNL gene is registered in GenBank as Accession No. U40402, the cassava-derived SHNL gene is Accession No. Z29091, and the sorghum-derived SHNL gene is Accession No. AJ421152.

  Therefore, recombinant SHNL can be obtained by expressing the SHNL gene prepared on the basis of these sequences as it is or with appropriate modification according to a known method and expressing it in a suitable host such as yeast or Escherichia coli. Specific production methods of natural recombinant SHNL are described in detail in, for example, JP 2000-189159 A, JP 2000-189160 A, JP 2000-245486 A, JP 2004-194550 A, etc. Yes. The modified recombinant SHNL with improved activity is described in detail, for example, in JP-A-2005-312431.

2. Polyvinyl alcohol The polyvinyl alcohol used in the present invention preferably has a high molecular weight of about 10,000 to 200,000. In terms of the degree of polymerization, those having a degree of polymerization of about 400 to 2500 are preferred. In this range, since the viscosity of the mixed enzyme solution does not increase, aggregation of immobilized enzymes hardly occurs.

  In particular, polyvinyl alcohol is preferably partially saponified, more preferably partially saponified at a saponification degree of 60 to 90 mol%. In this range, it is easy to prepare an aqueous solution and can be easily mixed with an enzyme.

  In the present invention, the polyvinyl alcohol can be added in an amount of 1 to 100%, particularly 3 to 50%, with respect to the enzyme (SHNL) protein mass. This is because within this range, polyvinyl alcohol can react without inhibiting the reaction between the substrate and the enzyme.

3. Porous carrier The carrier for immobilizing SHNL used in the present invention is not particularly limited as long as it is a porous carrier that can adsorb SHNL. In particular, an immobilized carrier made of a porous inorganic material such as a silica-based carrier, a silica-alumina-based carrier, an alumina-based carrier, or a clay-based sintered carrier has a specific gravity difference from the reaction solution, and can be easily separated from the immobilized enzyme Therefore, it is preferable.

  The silica-based carrier is a high surface area porous carrier formed by aggregation of silicon dioxide fine particles, and examples thereof include silica gel. Specific examples include Micro Bead Silica Gel (manufactured by Fuji Silysia Chemical Co., Ltd.) and Chromatography Silica Gel (manufactured by Fuji Silysia Chemical Co., Ltd.).

  The silica alumina carrier refers to a porous carrier mainly composed of aluminum oxide and silicon dioxide. Specifically, Mizuka Sieves Y-540 Y-type zeolite (manufactured by Mizusawa Chemical Industry Co., Ltd.), Mizuka Sieves 13X-488 zeolite 13X (manufactured by Mizusawa Chemical Industry Co., Ltd.), HSZ-630HOA H-mordenite (manufactured by Tosoh Corporation), Na-mordenite ( Catalytic Chemical Co., Ltd.).

  The alumina carrier is a porous carrier mainly composed of aluminum oxide. Specific examples include NeoBead DL (Mizusawa Chemical Co., Ltd.), γ-alumina KHA-34 (Sumitomo Chemical Co., Ltd.), and the like.

  The clay-based sintered carrier is a silicate raw material (for example, a kaolinite-based mineral such as kaolinite, dickite, nacrite, halloysite, clay-based materials such as pyrophyllite, montmorillonite, sericite, talc, chlorite, etc. Is a porous carrier obtained by granulating and sintering. Specific examples include Toyonite200 (manufactured by Toyo Denka Kogyo Co., Ltd.), Toyonite200A (manufactured by Toyo Denka Kogyo Co., Ltd.), and the like.

As the porous carrier, since the amount of enzyme adsorbed depends on the pore size of the carrier, it is preferable to select a carrier having a pore size effective for sufficiently immobilizing the enzyme. Specifically, those having a pore diameter of 10 to 80 nm, preferably 10 to 60 nm, and most preferably 10 to 40 nm are selected. Furthermore, the material constituting the porous carrier preferably has a larger specific surface area, specifically 20 m ² / g or more, so that more enzyme can be immobilized. The shape of the carrier used for immobilization is not particularly limited as long as it is porous, but it is preferably spherical when producing an immobilized enzyme for a packed reaction tank. In consideration of the separation workability of the immobilized enzyme, or in the case of a packed reactor, the particle size is relatively narrow, and the particle size is 10 μm to 5 mm, preferably 100 μm to Although it is preferable that it is 2 mm, it is not the limitation. For the type, pore size, and particle size of the immobilization support, refer to JP-A-2001-190275.

4). Method for Producing Immobilized Enzyme A suitable porous carrier is mixed with an enzyme composition obtained by adding polyvinyl alcohol to a solution containing (S) -hydroxynitrile lyase to immobilize S-hydroxynitrile lyase on the carrier. Polyvinyl alcohol is added in the desired amount as described above after adjusting the solution containing S-hydroxynitrile lyase to a pH within a range where the enzyme activity is not impaired. Here, polyvinyl alcohol is simply added as an additive to a solution containing SHNL. The addition of polyvinyl alcohol to the solution containing S) -hydroxynitrile lyase and the mixing of the porous carrier may be performed first or simultaneously.

  The mixture is agitated and allowed to stand until the adsorption rate reaches a maximum to immobilize the enzyme on the carrier. Usually, the maximum adsorption rate can be achieved with stirring and standing time of 1 to 24 hours.

  After the immobilization treatment, the obtained immobilized enzyme can be used as it is. However, when the immobilized enzyme contains excessive moisture, carriers are aggregated in the reaction solvent during the synthesis of the optically active cyanohydrin, so that the moisture contained in the immobilized enzyme can be dispersed to a level that can be dispersed. It is preferable to remove. The removal of water from the immobilized enzyme can be performed by drying under reduced pressure, drying by ventilation, or the like.

5. Synthesis of optically active cyanohydrin by immobilized enzyme The synthesis of optically active cyanohydrin using the immobilized enzyme of the present invention is carried out by a known method (for example, JP 2002-355085, JP 2002-176974, JP 2001-346596). No. 2001-190275, 2000-245486, 2001-120289, 2000-217590, etc.).

That is, an optically active cyanohydrin can be synthesized by adding the immobilized enzyme of the present invention and a reaction substrate to a reaction solvent and reacting at a reaction temperature of 0 to 40 ° C. for 20 minutes to 24 hours. The reaction time is appropriately adjusted according to the conversion rate of the substrate. As the reaction substrate, a carbonyl compound and a cyanide compound can be used. The carbonyl compound is an aldehyde or ketone represented by COR1R2, wherein R1 and R2 are hydrogen atoms, substituted or unsubstituted linear or branched saturated alkyl groups having 1 to 18 carbon atoms, or substituted or unsubstituted. An aromatic group having 5 to 22 ring members (however, R1 and R2 do not represent a hydrogen atom at the same time). When hydrogen cyanide is used as the cyan compound, the supply method may be either a liquid supply method or a gas supply method. Moreover, it will not specifically limit if it is a substance which produces cyanide ion (CN < ^- >), For example, cyanohydrins, such as hydrogen cyanide salts, such as sodium cyanide and potassium cyanide, and acetone cyanohydrin, can be used.

  As a reaction solvent, when a large amount of water is present in the reaction system, racemization of the optically active cyanohydrin generated by the enzyme reaction is likely to occur. In addition, when an aldehyde or ketone having a low solubility in water is used as a raw material, it is preferable to use a reaction solvent whose main component is an organic solvent that is hardly soluble or insoluble in water from the viewpoint of decreasing production efficiency. The organic solvent is not particularly limited as long as it does not affect the synthesis reaction of the optically active cyanohydrin by the enzymatic reaction, the physical properties of the raw material aldehyde or ketone used in the synthesis reaction, and the physical properties of the product cyanohydrin. It can be selected as appropriate according to the conditions. Specifically, aliphatic or aromatic linear, branched or cyclic saturated or unsaturated hydrocarbon solvents which may be halogenated, such as pentane, hexane, toluene, xylene, methylene chloride, etc. An aliphatic or aromatic linear or branched or cyclic saturated or unsaturated alcohol solvent which may be halogenated, such as isopropyl alcohol, n-butanol, isobutanol, t-butanol, Hexanol, cyclohexanol, n-amyl alcohol, etc .; aliphatic or aromatic linear or branched or cyclic saturated or unsaturated ether solvents which may be halogenated, such as diethyl ether, dipropyl ether , Diisopropyl ether, dibutyl ether, methyl t-butyl ether, etc .; optionally halogenated fat Or aromatic linear or branched or cyclic saturated or unsaturated ester solvents such as methyl formate, methyl acetate, ethyl acetate, butyl acetate, methyl propionate, etc., and these can be used alone. Alternatively, a plurality of them may be mixed and used. Moreover, the said solvent can also use what contained or was saturated with water or an aqueous buffer solution.

6). Lifetime of immobilized enzyme In this specification, “lifetime of immobilized enzyme” is obtained in a reaction of synthesizing (S) -2-chloromandelonitrile from hydrocyanic acid and 2-chlorobenzaldehyde (S) -2. -Evaluated by the number of repetitions until the optical purity of chloromandelonitrile is 90% ee or less.

  As a result, it was confirmed that the immobilized enzyme according to the present invention showed an optical purity exceeding 90% ee even after 10 repetitions. On the other hand, in the case where polyvinyl alcohol is not added, or the immobilized enzyme produced by adding polyvinylpyrrolidone and albumin, (S) -2-chloromandelonitrile can be synthesized with a purity of 90% ee or more. The reaction was 6 times, 1 time, and 2 times.

  That is, the immobilized enzyme of the present invention is an immobilized enzyme that has a remarkably long life in a repetitive reaction as compared with a conventionally used immobilized enzyme, and can be said to be useful for industrial production of optically active cyanohydrin. .

1. Preparation of S-hydroxynitrile lyase
S-hydroxynitrile lyase was prepared by genetic engineering using the yeast Saccharomyces cerevisiae as a host (see JP 2001-190275 A). That is, first, total mRNA was extracted from cassava leaves according to a conventional method. Next, cDNA synthesis was performed using the obtained mRNA as a template to prepare cDNA. On the other hand, the following primers were synthesized based on the cassava-derived S-hydroxynitrile lyase gene sequence described in the literature [Arch. Biochem. Biophys. 311, 496-502 (1994)].
Sense primer: ggggaattcatggtaactgcacattttgttctgattc (SEQ ID NO: 1)
Antisense primer: ggggtcgacctcacggattagaagccgccg (SEQ ID NO: 2)

  Using the synthesized primer, PCR (90 ° C., 30 seconds; 55 ° C., 30 seconds; 72 ° C., 60 seconds; total 35 cycles) was performed to obtain the S-hydroxynitrile lyase gene. Analysis of the gene sequence was consistent with the sequence shown in the literature.

  Next, the obtained PCR fragment was inserted between the promoter and terminator of the expression vector YEp352-GAP to prepare a yeast episomal expression vector YEp352-GC. This is transformed into a yeast Saccharomyces cerevisiae Inv-Sc1 strain by a conventional method, and a recombinant yeast YEp352-GC-containing the expression vector YEp352-GC- is selected by selecting a strain that grows in a minimal selection medium not containing uracil. S2 strain was obtained.

  Subsequently, the obtained recombinant yeast strain YEp352-GC-S2 was obtained by using YNBDCas liquid medium (6.7 g / L Yeast nitrogen base without amino acid (Difco), 20 g / L glucose, 20 g / L casamino acid, 40 mg / L S-hydroxynitrile lyase was produced in the cells by culturing in mL L-tryptophan) for 24 hours. The bacterial cells were collected from the recombinant bacterial culture by centrifugation, and the bacterial cells were crushed using a bead mill. The crushed cell fluid was centrifuged to prepare a crude enzyme solution, which was purified by ammonium sulfate fractionation and used as an S-hydroxynitrile lyase solution in the following examples.

  Subsequently, the protein concentration of the enzyme solution obtained in (1) was measured. The protein concentration was measured using BCA Protein Assay Reagent Kit (PIERCE). The protein concentration of the enzyme solution obtained in the above (1) was 37.5 mg / mL. The enzyme activity (volume activity) of (S) -hydroxynitrile lyase in the enzyme composition was 495 U / mL. Therefore, the specific activity of the enzyme solution obtained in the above (1) was calculated to be 13.2 U / mg.

2. Preparation of immobilized enzyme (Example 1)
To 1 mL of the enzyme solution prepared in Example 1, 0.2 mL of an aqueous solution of partially saponified polyvinyl alcohol (trade name Kuraray Poval PVA217: polymerization degree 1700, manufactured by Kuraray Co., Ltd.) adjusted to 10 wt% was mixed. Prepared.

  Next, 1 mL of the obtained enzyme composition and 1 g of an immobilization carrier (porous silica gel, trade name MB-5D, manufactured by Fuji Silysia Chemical Ltd.) were mixed. Thereby, the immobilized enzyme in which the enzyme composition was immobilized was prepared.

(Example 2)
An enzyme composition was prepared by mixing 0.2 mL of an aqueous solution (partially saponified polyvinyl alcohol, trade name Kuraray Poval PVA205: polymerization degree 500, manufactured by Kuraray Co., Ltd.) having a different degree of polymerization of the polyvinyl alcohol of Example 1. Next, in the same manner as in Example 1, an immobilized enzyme in which this enzyme composition was immobilized on porous silica gel was prepared.

(Comparative Example 1)
An enzyme composition was prepared by mixing 0.2 mL of 0.2 M sodium citrate buffer instead of the polyvinyl alcohol of Example 1. Next, in the same manner as in Example 1, an immobilized enzyme in which this enzyme composition was immobilized on porous silica gel was prepared.

(Comparative Example 2)
Instead of the polyvinyl alcohol of Example 1, 0.2 mL of a 10 wt% polyvinylpyrrolidone aqueous solution (molecular weight 30000) was mixed to prepare an enzyme composition. Next, in the same manner as in Example 1, an immobilized enzyme in which this enzyme composition was immobilized on porous silica gel was prepared.

(Comparative Example 3)
Instead of the polyvinyl alcohol of Example 1, 20 mg of albumin (Albumin, Bovine, manufactured by SIGMA CHEMICAL) and 0.2 mL of 0.2 M sodium citrate buffer were mixed to prepare an enzyme composition. Next, in the same manner as in Example 1, an immobilized enzyme in which this enzyme composition was immobilized on porous silica gel was prepared.

3. Synthesis of optically active cyanohydrin First, 0.6 g of the immobilized enzyme prepared in Example 1 was added to a mixed solution of 3 g of t-butyl methyl ether saturated with 0.2 M citrate buffer and 0.2 g of hydrocyanic acid. Then 0.7 g of 2-chlorobenzaldehyde was added. This was stirred at 20 ° C. to synthesize (S) -2-chloromandelonitrile. The reaction solution was sampled, the conversion rate of aldehyde was measured by HPLC, and the reaction was carried out until the conversion rate reached 95% or more. The reaction solution was collected, and the optical purity of (S) -2-chloromandelonitrile was measured by HPLC. Furthermore, the immobilized enzyme after completion of the reaction was recovered, and the reaction was repeated under the same conditions as described above. The repeated reaction was performed until the optical purity was less than 90% ee.

  Similarly, (S) -2-chloromandelonitrile was synthesized using each of the immobilized enzymes prepared in Example 2 and Comparative Examples 1 to 3.

4). Results Regarding the synthesis of (S) -2-chloromandelonitrile with each immobilized enzyme, the reaction time for each number of repetitions (number of batches) and the optical purity of (S) -2-chloromandelonitrile were measured. It is shown in Table 1.

  As is clear from the results in Table 1, the immobilized enzyme prepared by adding polyvinyl alcohol synthesizes (S) -2-chloromandelonitrile with a purity of 92% ee or more even after 10 repeated reactions. I was able to. On the other hand, the immobilized enzyme prepared by adding sodium citrate buffer, polyvinylpyrrolidone, and albumin can synthesize (S) -2-chloromandelonitrile with a purity of 90% ee or more. 1 to 2 times. The reason for this effect is not clear, but it is considered that polyvinyl alcohol contributes to the protection of the enzyme in the immobilized enzyme preparation stage.

  From the above, it was confirmed that by adding polyvinyl alcohol to prepare an immobilized enzyme, it is possible to obtain an immobilized enzyme having a significantly longer life than the conventional one.

  According to the present invention, optically active cyanohydrins can be synthesized at a lower cost than before by significantly extending the lifetime of the immobilized S-hydroxynitrile lyase. Therefore, the present invention is useful for industrial synthesis of optically active cyanohydrins.

SEQ ID NO: 1-description of artificial sequence: primer SEQ ID NO: 2-description of artificial sequence: primer

Claims (5)

  By adding polyvinyl alcohol to a solution containing (S) -hydroxynitrile lyase and mixing with a porous carrier, an immobilized enzyme having (S) -hydroxynitrile lyase adsorbed on the porous carrier is obtained. A method for producing an immobilized enzyme.   The method according to claim 1, wherein the polyvinyl alcohol is partially saponified at a saponification degree of 60 to 90 mol%.   The method according to claim 1 or 2, wherein polyvinyl alcohol is added in the solution in an amount of 3 to 50% based on the mass of (S) -hydroxynitrile lyase.   An immobilized enzyme comprising (S) -hydroxynitrile lyase, polyvinyl alcohol, and a porous carrier produced by the method according to any one of claims 1 to 3.   A method for producing an optically active cyanohydrin, comprising contacting the immobilized enzyme according to claim 4 with a carbonyl compound and a cyanide compound.