CN114752574B - Enzyme catalysis system, catalase, preparation method and application - Google Patents

Enzyme catalysis system, catalase, preparation method and application Download PDF

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CN114752574B
CN114752574B CN202111070631.7A CN202111070631A CN114752574B CN 114752574 B CN114752574 B CN 114752574B CN 202111070631 A CN202111070631 A CN 202111070631A CN 114752574 B CN114752574 B CN 114752574B
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周硕
劳淑华
雷军林
赖敦岳
陈振明
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Zhejiang Zanyuan Biotechnology Co ltd
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Abstract

The application discloses an enzyme catalysis system, a catalase, a preparation method and an application. The enzyme catalysis system comprises: catalase, organic solvent, coenzyme and coenzyme regenerant for catalyzing a substrate to generate a product through reduction reaction. The amino acid sequence of the catalase is shown as SEQ ID NO:1 or a sequence similar to SEQ ID NO:1 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar. The (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one which can be used as an ezetimibe intermediate is obtained by an enzyme catalytic reduction reaction. The ezetimibe intermediate prepared by the catalase has the advantages of low enzyme consumption and high product conversion rate.

Description

Enzyme catalysis system, catalase, preparation method and application
Technical Field
The application relates to the technical field of enzyme engineering and biological pharmacy, in particular to an enzyme catalysis system, catalase, a preparation method and application.
Background
Ezetimibe, also known as ezetimibe and ezetimibe, the english name is: ezetimibe, chemical name: 1- (4-fluorophenyl) - (3R) -3- (4-fluorophenyl) - (3S-hydroxypropyl)]- (4S (4-hydroxyphenyl) -2-propiolactam, CAS:163222-33-1, molecular formula C 24 H 21 F 2 NO 3 The compound has the molecular weight of 409.43, is white crystalline powder, is a highly purified and highly selective cholesterol absorption inhibitor drug, and is the first drug approved by the U.S. FDA to be marketed and capable of selectively inhibiting cholesterol absorption. Mainly used for reducing the content of cholesterol in human body, and has certain effect on treating primary hypercholesterolemia, familial homozygous hypercholesterolemia (HoFH) and homozygous sitosterolemia (or phytosterolemia); for atheromatous diseases and coronary heart diseaseThe prevention and treatment of the diseases have important significance.
The ezetimibe molecular structure has a chiral benzylic hydroxyl group and two stereocenters for modifying a rigid 2-azetidinone parent nucleus, and 8 stereoisomers can exist, so that the establishment and control of the absolute configuration become one of the most challenging works in the synthesis field.
In most routes to ezetimibe synthesis, (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepin-2-one serves as a key chiral intermediate for ezetimibe, which can be prepared by chemical and enzymatic kinetic resolution of its precursor ketone. The target product obtained by the chemical asymmetric reduction method has low optical purity, the chemical method has high cost, and the chemical catalyst is easy to cause serious pollution to the environment. Compared with chemical method, the biological method has the advantages of strong stereospecificity, high catalytic efficiency, no pollution and the like. However, the current biological preparation method still has some defects, such as serious enzyme activity loss, reduced conversion rate and continuous increase of enzyme quantity for maintaining the reaction. Therefore, finding a system which can reduce the dosage of enzyme and improve the product conversion rate has great significance for reducing the cost and improving the product recovery.
Disclosure of Invention
The application provides an enzyme catalysis system, a catalase, a preparation method and an application, which can solve the problems of large enzyme dosage, low conversion rate and the like in the existing reaction.
In a first aspect, the present application provides an enzymatic system comprising: catalase, organic solvent, coenzyme and coenzyme regenerant for catalyzing a substrate to generate a product through reduction reaction; the substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1):
Figure BDA0003260108410000021
the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, and the structure of the product is shown as the formula (2):
Figure BDA0003260108410000022
further, the amino acid sequence of the catalase is shown in SEQ ID NO:1, or a sequence identical to SEQ ID NO:1 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar.
Further, the concentration of the substrate may be 50g/L to 250g/L, 100g/L to 200g/L, or 120g/L to 180g/L.
Further, the concentration of the organic solvent may be from 20% (v/v) to 50% (v/v), may be from 25% (v/v) to 45% (v/v), and may be from 30% (v/v) to 40% (v/v), based on the total volume of the reaction system; the reaction system is a system in the process of catalytic reduction reaction, and comprises an enzyme catalysis system, a substrate, water and the like.
Further, the organic solvent includes at least one of dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, propyl acetate, butyl acetate, N-dimethylformamide, dichloromethane, or toluene.
Further, the coenzyme is selected from any one of Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP).
Further, the concentration of the coenzyme may be 0.2 to 2g/L, or 0.5 to 1.7g/L, or 1.0 to 1.5g/L.
Further, the coenzyme regenerating agent includes: isopropanol and carbonyl reductase.
Further, the amino acid sequence of the carbonyl reductase is shown as SEQ ID NO:2, respectively.
Furthermore, the content of the carbonyl reductase in percentage by volume can be 0.5-1%, also can be 0.6-0.9%, also can be 0.7-0.8% based on the total volume of the reaction system.
Further, the coenzyme regenerating agent includes: glucose and glucose dehydrogenase.
Further, the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO:3, respectively.
Further, the content of the glucose dehydrogenase may be 0.2 to 0.6% by volume, or 0.3 to 0.5% by volume, or 0.4% by volume based on the total volume of the reaction system.
Further, the concentration of glucose may be 30 to 50g/L, 35 to 45g/L, or 37 to 43g/L.
In a second aspect, the present application provides a catalase, the amino acid sequence of the catalase is set forth in SEQ ID NO:1 or a sequence similar to SEQ ID NO:1 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar.
Further, the amino acid sequence of the catalase is represented by SEQ ID NO:1 by one or more point mutations.
Further, the point-mutated amino acids include SEQ ID NO:1, amino acid F at position 30, amino acid V at position 50, amino acid D at position 94, amino acid H at position 158, amino acid A at position 169, amino acid S at position 172, amino acid D at position 229, amino acid L at position 241, and amino acid R at position 243.
Further, the point mutation is carried out by F30T, V50P, D94Q, H158I, A169T, S172R, D229V, L I or R243Y.
Further, the amino acid sequence of the catalase is SEQ ID NO: 5. SEQ ID NO: 6. the amino acid sequence of SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13, or a pharmaceutically acceptable salt thereof.
As an alternative embodiment, the point mutations occur in any of the following combinations:
(1) F30T and S172R;
(2) H158I, L I and R243Y;
(3) V50P, D Q and a169T;
(4) A169T, S R, D V and L241I; or
(5) F30T, D Q, A169T, L I and R243Y.
Further, the amino acid sequence of the catalase is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18, or a pharmaceutically acceptable salt thereof.
In a third aspect, the present application provides the use of a catalase enzyme to catalyze the reduction of a substrate to produce a product; the substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxolyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1):
Figure BDA0003260108410000041
the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, and the structure of the product is shown as the formula (2):
Figure BDA0003260108410000042
Figure BDA0003260108410000051
in a fourth aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the catalase of the second aspect.
Further, the nucleotide sequence is any one of:
(a) The nucleotide sequence is shown as SEQ ID NO:19 is shown in the figure;
(b) Nucleotide sequence and SEQ ID NO:19 is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar; or
(c) The nucleotide sequence is obtained by codon optimization of the nucleotide sequence in (a) or (b).
Further, the nucleotide sequence is as shown in SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. the amino acid sequence of SEQ ID NO: 24. SEQ ID NO: 25. the amino acid sequence of SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. the amino acid sequence of SEQ ID NO: 30. the amino acid sequence of SEQ ID NO: 31. SEQ ID NO:32 or SEQ ID NO:33, or a nucleotide sequence shown in the specification.
In a fifth aspect, the present application provides a process for the preparation of (4S) -3- [ (5S) -5- (4-fluorophenyl) 5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one comprising: the substrate (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone is mixed with the enzymatic catalytic system of the first aspect to produce an enzymatically catalyzed reduction reaction to give (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one.
Further, the organic solvent includes at least one of dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, propyl acetate, butyl acetate, N-dimethylformamide, dichloromethane, or toluene.
Furthermore, the mass ratio of the substrate to the catalase in the enzyme catalysis system can be (120-210) to 1, can also be (150-200) to 1, and can also be (160-180) to 1.
Further, the concentration of the substrate may be 50g/L to 250g/L, 100g/L to 200g/L, or 120g/L to 180g/L.
Further, the concentration of the organic solvent in the enzyme-catalyzed system may be from 20% (v/v) to 50% (v/v), from 25% (v/v) to 45% (v/v), or from 30% (v/v) to 40% (v/v), based on the total volume of the reaction system.
Further, the enzyme-catalyzed reaction is carried out in the presence of a coenzyme which is NADP or NAD and a coenzyme-regenerating agent which is isopropanol and carbonyl reductase, or glucose and glucose dehydrogenase.
Further, the amino acid sequence of the carbonyl reductase is shown as SEQ ID NO:2, the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO:3, respectively.
Further, the pH of the enzymatic reduction reaction may be 6.0 to 9.0, or 6.5 to 8.5, or 7.0 to 8.0.
Further, the temperature of the enzyme catalytic reduction reaction is 25-65 ℃, also can be 30-60 ℃, and also can be 35-55 ℃.
Further, the enzyme-catalyzed reduction reaction is carried out under shaking conditions: the rotating speed can be 150 to 500 revolutions per minute, 200 to 450 revolutions per minute, and 250 to 400 revolutions per minute.
In a sixth aspect, the present application provides the use of (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, prepared as in the process of the fifth aspect, as an intermediate for the preparation of ezetimibe; the structure of ezetimibe is shown as formula (3):
Figure BDA0003260108410000061
the method for producing the ezetimibe intermediate by using the catalysis of catalase has the following beneficial effects:
(1) According to the application, hydrogenase is contacted with a substrate (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone to perform an enzyme catalytic reaction, so that (4S) -3- [ (5S) -5- (4-fluorophenyl) 5-hydroxypentanoyl ]4 phenyl-1,3-oxazepan-2-one is prepared, the enzyme amount of the hydrogenase is small, the conversion rate is high, and the method has the advantages of improving the product conversion rate and reducing the production cost.
(2) Experiments show that the technical scheme that ethyl acetate and butyl acetate are used as organic solvents, and a coenzyme regeneration system is added in a premixed system containing catalase so that the catalase and the coenzyme regeneration system are compounded according to a specific proportion is favorable for improving the substrate conversion rate, and the substrate conversion rate is at least 97%. In addition, the technical means that the mixed solution obtained by mixing ethyl acetate and a substrate is added into the premixing system in a coenzyme circulation mode matching with glucose and glucose dehydrogenase can further improve the substrate conversion rate, and the substrate conversion rate can reach 99.5%.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is an HPLC profile of a substrate and an ezetimibe intermediate standard;
FIG. 2 is a graph of the amino acid sequence of example four containing ethyl acetate and SEQ ID NO: the catalase reaction system of 18 is on an HPLC chart of 1 h;
FIG. 3 is a graph of the amino acid sequence of example four containing ethyl acetate and SEQ ID NO: the catalase reaction system of 18 was on a 24h HPLC profile;
FIG. 4 is a graph of the sequence of example eleven containing ethyl acetate and SEQ ID NO:18 on an HPLC profile of 1 h;
FIG. 5 is a graph of the sequence of example eleven containing ethyl acetate and SEQ ID NO: the catalase reaction system of 18 showed an HPLC profile at 24 h.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The application provides an enzyme catalysis system, a catalase, a preparation method and an application. The following are detailed descriptions. It should be noted that the following description of the embodiments is not intended to limit the preferred order of the embodiments.
Experimental procedures without specific conditions noted in the examples below, generally following conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred embodiments and materials described herein are exemplary only, and are not intended to limit the scope of the present application.
Unless otherwise indicated, the reagents and materials referred to herein are either commercially available or can be prepared by one of ordinary skill in the art on the basis of common general knowledge.
Definition and description:
as used herein, "recombinant expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences that enable expression of the nucleic acid molecule in a suitable expression system. In the examples of the present application, the recombinant expression vector refers to a DNA construct formed by inserting a foreign gene into a vector using molecular biology techniques, and exemplary pET24a-1 is a recombinant expression vector.
As used herein, "enzyme preparation" refers to a functionalized product obtained by extracting a biocatalytically active substance from a living organism (e.g., a microorganism) with the aid of other components. In the examples of the present application, the active ingredient of the enzyme preparation is an enzyme, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2, the enzyme preparation can be applied to the production process of ezetimibe. In some embodiments, the enzyme preparation may include necessary excipients, stabilizers, protectants, etc. food or pharmaceutically acceptable adjuvants and/or adjuvants.
As used herein, "nucleotide sequence" refers to a sequence of nucleotides from the 5 'to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded.
As used herein, "codon" refers to a trinucleotide sequence specifying a particular amino acid. "codon bias" refers to the frequency of codon usage in an organism's coding sequence that encodes an amino acid.
As used herein, amino acids are represented by the single or three letter codes and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).
The embodiment of the application provides a catalase, and the amino acid sequence of the catalase is shown as SEQ ID NO:1, or a sequence identical to SEQ ID NO:1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity.
In some embodiments of the application, the amino acid sequence of the catalase is represented by SEQ ID NO:1 by one or more point mutations.
In some embodiments of the present application, the point mutated amino acid comprises SEQ ID NO:1, amino acid F at position 30, amino acid V at position 50, amino acid D at position 94, amino acid H at position 158, amino acid A at position 169, amino acid S at position 172, amino acid D at position 229, amino acid L at position 241, and amino acid R at position 243.
In some embodiments of the present application, the point mutations occur in a manner of F30T, V50P, D Q, H I, A169T, S R, D229V, L I or R243Y.
In some embodiments of the application, the amino acid sequence of the catalase is SEQ ID NO: 5. the amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13, or a pharmaceutically acceptable salt thereof.
In some embodiments of the present application, the point mutations occur in any of the following combinations:
(1) F30T and S172R;
(2) H158I, L I and R243Y;
(3) V50P, D Q and a169T;
(4) A169T, S R, D V and L241I; or
(5) F30T, D Q, A169T, L I and R243Y.
In some embodiments of the application, the amino acid sequence of the catalase is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO:17 or SEQ ID NO:18, or a pharmaceutically acceptable salt thereof.
The present examples also provide a nucleic acid molecule comprising a nucleotide sequence encoding the above-described catalase.
In some embodiments of the application, the nucleotide sequence is any one of:
(a) The nucleotide sequence is shown as SEQ ID NO:19 is shown at °
(b) Nucleotide sequence and SEQ ID NO:19 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity or
(c) The nucleotide sequence is obtained by codon optimization of the nucleotide sequence in (a) or (b).
In some embodiments of the present application, the nucleotide sequence is as set forth in SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. the amino acid sequence of SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. the amino acid sequence of SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO:32 or SEQ ID NO:33, or a nucleotide sequence shown in seq id no.
The embodiment of the application also provides an application of catalase in catalyzing a substrate to generate a product through a reduction reaction, wherein the catalase can catalyze carbonyl in the substrate to form chiral benzylic hydroxyl. The substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxolyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1):
Figure BDA0003260108410000101
the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepin-2-one, and the structure of the product is shown as the formula (2):
Figure BDA0003260108410000102
the embodiments of the present application further provide an enzymatic system, comprising: the above-mentioned catalase, organic solvent, coenzyme and coenzyme-regenerating agent; the substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxolyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1); the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, and the structure of the product is shown as the formula (2).
In some embodiments of the present disclosure, the substrate concentration can be from 50g/L to 250g/L, from 100g/L to 200g/L, or from 120g/L to 180g/L.
In some embodiments of the present application, the concentration of the organic solvent may be from 20% (v/v) to 50% (v/v), from 25% (v/v) to 45% (v/v), or from 30% (v/v) to 40% (v/v), based on the total volume of the reaction system.
In some embodiments herein, the organic solvent comprises at least one of dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, propyl acetate, butyl acetate, N-dimethylformamide, dichloromethane, or toluene; preferably, the organic solvent is at least one of ethyl acetate or butyl acetate.
In some embodiments of the present application, the coenzyme is selected from any of Nicotinamide Adenine Dinucleotide (NAD) or Nicotinamide Adenine Dinucleotide Phosphate (NADP). When the coenzyme is added to the enzyme catalytic system, the coenzyme receives H provided by the coenzyme regenerant, thereby forming reduced coenzyme. The reduced coenzyme can provide H for the reduction reaction of a substrate. Specifically, nicotinamide Adenine Dinucleotide (NAD) can accept H provided by the coenzyme regenerating agent to form reduced Nicotinamide Adenine Dinucleotide (NADH). Nicotinamide Adenine Dinucleotide Phosphate (NADP) is capable of accepting H provided by the coenzyme regenerant to form reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH). After the reduction reaction of the substrate is finished, the reduced coenzyme loses H to form the coenzyme again, and then the reduced coenzyme is formed under the action of a coenzyme regenerant, so that the cyclic regeneration of the coenzyme is realized.
In some embodiments of the present application, the concentration of the coenzyme may be 0.2 to 2g/L, or 0.5 to 1.7g/L, or 1.0 to 1.5g/L.
In some embodiments of the present application, the coenzyme regenerant comprises: isopropanol and carbonyl reductase. Carbonyl reductases are capable of donating H from isopropanol to the coenzyme. When the H in the isopropanol is completely consumed, new isopropanol is added to supplement new H, so that the cyclic regeneration process of the coenzyme is continuously promoted.
In some embodiments of the present application, the amino acid sequence of the carbonyl reductase is as set forth in SEQ ID NO:2, respectively.
In some embodiments of the present disclosure, the carbonyl reductase may be present in an amount of 0.5% to 1%, or 0.6% to 0.9%, or 0.7% to 0.8% by volume based on the total volume of the reaction system.
In some embodiments of the present application, the coenzyme regenerant comprises: glucose and glucose dehydrogenase. Glucose dehydrogenase is capable of supplying H of glucose to a coenzyme. When H in the glucose is completely consumed, new H can be supplemented by adding new glucose, so that the cyclic regeneration process of the coenzyme is continuously promoted.
In some embodiments of the present application, the amino acid sequence of the glucose dehydrogenase is as set forth in SEQ ID NO:3, respectively.
In some embodiments of the present disclosure, the glucose dehydrogenase may be present in an amount of 0.2% to 0.6%, 0.3% to 0.5%, and 0.4% by volume based on the total volume of the reaction system.
In some embodiments of the present application, the concentration of glucose may be 30 to 50g/L, or 35 to 45g/L, or 37 to 43g/L.
The embodiment of the application also provides a preparation method of (4S) -3- [ (5S) -5- (4-fluorophenyl) 5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, which comprises the following steps: the substrate (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone was mixed with the above enzyme catalyzed system to produce an enzyme catalyzed reduction reaction to give (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one.
In some embodiments of the present application, the mass ratio of substrate to catalase in the enzymatic system may be (120-210) to 1, may be (150-200) to 1, or may be (160-180) to 1.
In some embodiments of the present application, the enzyme-catalyzed reaction is carried out in the presence of a coenzyme and a coenzyme regenerant, wherein the coenzyme is NADP or NAD, and the coenzyme regenerant is isopropanol and carbonyl reductase, or glucose and glucose dehydrogenase; the coenzyme regenerant is used for providing cyclically regenerated reduced Nicotinamide Adenine Dinucleotide (NADH) or reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), and the NADH and the NADPH are used as hydrogen donors to participate in the reduction reaction.
In some embodiments of the present application, the pH of the enzymatic reduction reaction is between 6.0 and 9.0, or between 6.5 and 8.5, or between 7.0 and 8.0.
In some embodiments of the present application, the temperature of the enzymatic reduction reaction is from 25 ℃ to 65 ℃, and may be from 30 ℃ to 60 ℃, and may be from 35 ℃ to 55 ℃.
In some embodiments of the present application, the enzyme-catalyzed reduction reaction is performed under shaking conditions: the rotating speed can be 150-500 rpm, 200-450 rpm, 250-400 rpm.
In some embodiments of the present application, the manner of addition of the catalase may be: adding directly catalase or a catalase preparation comprising catalase to the premix system; or adding wet thallus obtained by fermenting and culturing a transformant containing a gene encoding the catalase into a premixed system; or adding a cell disruption solution obtained by disrupting wet thalli into a premixed system; or adding the product of the separation and purification treatment of the cell disruption solution into a premixed system.
The embodiment of the application also provides application of the (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepine-2-ketone prepared by the method as an intermediate for preparing ezetimibe; the structure of ezetimibe is shown as formula (3):
Figure BDA0003260108410000131
description of the examples of the present application relating to the culture Medium
(1) LB liquid culture Medium
Comprises 1% NaCl,1% tryptone, and 0.5% yeast extract, mixing well, adjusting pH to 7.0, and sterilizing with high pressure steam autoclave at 121 deg.C for 30min. Kanamycin was added to a final concentration of 50ug/mL prior to use.
(2) Self-induction culture medium
120g of yeast powder, 32.25g of peptone, 0.75g of magnesium sulfate (MgSO 4), and 16.5g of ammonium sulfate ((NH 4) 2 SO 4), 32.5g of monopotassium phosphate (KH) 2 PO 4), 35.5g disodium hydrogen phosphate (Na) 2 HPO 4), 2.5g of glucose and 10g of alpha-lactose, then all the weighed components are added into a pulverizer to be uniformly stirred, 50g of self-induced culture medium powder is weighed and dissolved in 1L of water, after complete mixing, the pH is adjusted to 7.0, and sterilization is carried out for 30min at 121 ℃.
The details of the description relating to plasmids and competent cells in the examples of the present application are given in table 1 below:
TABLE 1
Figure BDA0003260108410000141
Description of gene fragments and reagents referred to in the examples of the present application:
the gene segment related in the embodiment of the application comprises a primer, a primer shown as SEQ ID NO:19 and the like were synthesized by Biotechnology engineering (Shanghai) Ltd.
Molecular reagents such as restriction enzymes (e.g., bamH I, nde I and Dpn I), T4 ligase, KOD Hi-Fi enzyme kit and the like, which are referred to in the examples of the present application, are commercially available from Takara Bio Inc.
The technical solutions and advantages of the present application are further described below with reference to examples.
Example one Source of Catalase and construction of recombinant expression vector containing Catalase-encoding Gene
This example provides a recombinant expression vector, which can be obtained by a method comprising the steps of:
the amino acid sequences of the Galactomyces reussii strains dug out by the NCBI database are respectively shown as SEQ ID No. 1. Carrying out codon optimization according to E.coli codon preference, and synthesizing a nucleotide sequence of the catalase with the sequence of SEQ ID No.1 by a whole-gene synthesis method, wherein the synthesized nucleotide sequence is shown as SEQ ID No. 19. Enzyme cutting sites Nde I and BamH I are added at two ends of the nucleotide sequence, and the gene is cloned to Nde I and BamH I sites corresponding to pET24a to obtain a recombinant expression vector pET24a-1.
Example II construction of genetically engineered bacteria containing Catalase-encoding Gene
The construction method of the gene engineering bacteria containing the catalase coding gene of the embodiment is as follows:
will have the sequence shown in SEQ ID NO:1 to obtain a plurality of catalase mutants, wherein the specific mutation mode is detailed in table 2. And (3) designing a point mutation primer by using Oligo7 software according to the amino acid site to be mutated by adopting a site-directed mutation strategy, and introducing mutation by inserting, replacing or deleting a base at the 5' end of the upstream and downstream mutation primers.
(1) The recombinant expression vector pET24a-1 constructed in the first example was selected as a template, and inverse PCR was performed using a KOD high fidelity enzyme kit to obtain a catalase mutation sequence.
(2) Treating with Dpn I restriction endonuclease to obtain catalase mutation sequence, and converting the enzyme digestion product into Escherichia coli BL21 (DE 3) competence after T4 ligase ligation.
(3) Then coating an LB resistant plate containing kanamycin, placing the plate at 37 ℃ for inverted culture for 18h, selecting a single colony to be transferred into an LB liquid culture medium containing kanamycin, selecting a culture solution, sending the sample for sequencing, storing a clone with correct sequencing for later use, thereby obtaining the genetic engineering bacteria which takes escherichia coli as a host and contains a catalase coding gene, and carrying out strain activity determination on the obtained genetic engineering bacteria containing the catalase coding gene, wherein the results are shown in Table 2:
TABLE 2
Figure BDA0003260108410000151
Figure BDA0003260108410000161
Note: the relative activity is the activity of the mutant strain relative to the activity of the wild strain;
+ means 1-5 times increase in vitality, + + means 5-10 times, + +++ means 10-20 times, + ++++ means 20-30 times, and ++++ means more than 30 times.
In table 2, the catalase shown in seq id No.5 to seq id No.18 is obtained by mutating one or more sites on the basis of the catalase shown in seq id No.1, and the strain activities of the catalase shown in seq id No.5 to seq id No.18 are all significantly improved compared to the catalase shown in seq id No. 1. The strains of the catalase obtained by adopting the combined mutation mode of F30T, D94Q, A169T, L241I and R243Y have the highest activity. Secondly, the catalase is obtained by adopting the combined mutation mode of A169T, S172R, D229V and L241I, and the strain activity of the catalase obtained by adopting the combined mutation mode is generally higher than that of the catalase obtained by adopting the single-site mutation mode.
Example three, induced expression and post-treatment of genetically engineered bacteria containing Catalase encoding Gene
This example provides an enzyme powder of catalase, the specific method for obtaining it includes the following steps:
(1) Inoculating the genetic engineering bacteria to an LB liquid culture medium containing 50ug/mL kanamycin at the final concentration, and culturing at 37 ℃ at 180r/min until OD600 is about 0.6-0.8 to obtain seed bacteria liquid; the seed culture was inoculated at a volume concentration of 1% to fresh autoinduction medium containing kanamycin at a final concentration of 50ug/mL and cultured at 30 ℃ for 18 hours. Centrifuging at 25 deg.C and 8000r/min for 10min, discarding supernatant, washing twice with PB pH7.0, and collecting wet thallus.
(2) And crushing the bacterial liquid by an ultrasonic crushing method. The wet cells were resuspended in ultrapure water to prepare a 20% bacterial solution. The crushing conditions were as follows: working for 1 second, pause for 2s,180W, and crushing for 10min. The conditions of high-pressure homogeneous crushing can also be adopted: 50HZ,800bar, crushing twice.
(3) Removing cell debris and macromolecular impurities in the cell disruption solution through high-speed centrifugation to obtain a crude enzyme solution, wherein the centrifugation conditions are as follows: centrifuging at 12000r/min at 4 deg.C for 10-15min.
(4) 120ml of crude enzyme solution with the concentration of 20 percent is frozen and dried into 3 to 4g of enzyme powder by vacuum freeze drying at the temperature of minus 25 ℃.
EXAMPLE IV tolerance test for different organic solvents
In this example, the detection of the tolerance of the catalase having the sequences of SEQ ID No.1 and SEQ ID No.18 in different solvents includes the following steps:
1) Adding 1.5mL of isopropanol into a 30mL reaction flask, adding 1.5mL of organic solvent, adding 0.3g of substrate, mixing and dissolving to obtain a mixed solution with degree of
2) Adding 0.003g of catalase, adding 3mL of water, and adding 10mg of NADP + The initial pH was adjusted to 6.5.
3) Continuously reacting for 24h under the oscillating condition of 180r/min at 30 ℃, sampling and detecting the product conversion rate by HPLC.
The reaction system comprises 25% (v/v) of isopropanol, 25% (v/v) of organic solvent, 50g/L of substrate, substrate: catalase =100:1.
the organic solvent is: dimethyl sulfoxide, tetrahydrofuran, N-dimethylformamide, ethyl acetate, butyl acetate, propyl acetate, dichloromethane or toluene.
The reaction product was detected and analyzed by High Performance Liquid Chromatography (HPLC).
The model of the HPLC apparatus is Agilent 1260 (Agilent), and the working conditions of the HPLC are as follows:
(1) Preparation of a sample solution: taking 100 mu L of reaction product, adding 1mL of ethyl acetate for extraction, centrifuging for 1min at 12000rmp/min, taking supernatant, diluting by 60 times with mobile phase, mixing uniformly, and injecting sample at 20uL °
(2) A chromatographic column: a large xylonite IC column, 4.6 × 150mm,5 μm;
(3) Preparation of mobile phase: the normal hexane and the ethanol are mixed according to the volume ratio of 92: 8 to prepare the oil-water mixture.
(4) Flow rate: 1mL/min.
(5) Detection wavelength: 268nm.
(6) Column temperature: at 30 ℃.
The substrate conversion (%) was calculated from the amount of the substrate compound reduced, and the results of the measurement of the substrate conversion (%) are shown in Table 2, and the calculation formula of the substrate conversion (%) is shown in the following formula (1):
Figure BDA0003260108410000181
in the formula (1), A 1 Area of peak of substrate Compound after reaction, A 2 The peak area of the substrate compound before the reaction.
The stereoselectivity (e.e value) was calculated according to the amount of the product with different configuration, and the results of the stereoselectivity measurement are shown in Table 2, and the formula for calculating the stereoselectivity (e.e%) is shown in the following formula (2):
Figure BDA0003260108410000182
in formula (2), A3 is the peak area of the S-type product (the configuration required for the present reaction), and A4 is the peak area of the R-type product.
The results of the experiment are shown in table 3:
TABLE 3
Figure BDA0003260108410000183
Figure BDA0003260108410000191
From the results in Table 3, it can be seen that the conversion rate of catalase is highest when the organic solvent is ethyl acetate and butyl acetate, which indicates that when the catalytic reaction is carried out by using the catalase of the present application, the organic solvent in the system is preferably ethyl acetate or butyl acetate, and a higher product conversion rate can be obtained. And as can be seen from the results in table 1, SEQ id no: the stereoselectivity (e.e value) of the catalase of 1 in different organic solvents can reach 98 percent, and the stereoselectivity of the catalase of SEQ ID NO: the stereoselectivity (e.e value) of the catalase of 18 in different organic solvents can reach 99%, and the stereoselectivity of the catalase of SEQ ID NO:18 to the catalase of SEQ ID NO:18, the conversion of the catalase product was higher.
The HPLC profile of the substrate and ezetimibe intermediate standard is shown in fig. 1, and the HPLC profile of the substrate and ezetimibe intermediate standard is shown in fig. 2 as the HPLC profile of the sample containing ethyl acetate and SEQ ID NO:18 on an HPLC profile at 1h, as shown in fig. 3, containing ethyl acetate and SEQ ID NO: the HPLC profile of the catalase reaction system of 18 at 24h, as can be seen from comparison of fig. 2 and fig. 3, the peak area of the substrate is reduced and the peak area of the ezetimibe intermediate is significantly increased after 24h reaction, which indicates that in ethyl acetate solvent, the peak area of SEQ ID NO: the catalase of 18 can better catalyze the substrate conversion to generate the ezetimibe intermediate.
EXAMPLE V different concentrations of Ethyl acetate
In this example, the specific experimental method for detecting the conversion rate of the catalase having the sequences of SEQ ID No.1 and SEQ ID No.18 at different concentrations of ethyl acetate when the organic solvent is selected as ethyl acetate comprises the following steps:
1) Adding 10mL of isopropanol into a 100mL reaction bottle, adding ethyl acetate with different concentrations (v/v), adding 5g of substrate, and uniformly mixing to obtain a mixed solution;
2) Adding 0.0375g of catalase, adding 0.5mL of carbonyl reductase, and adding proper amount of water to make up the total amount to be 50mL; then 10mg of NADP was added + Adjusting the initial pH to 7.5;
3) Continuously reacting for 24h under the oscillating condition of 35 ℃ and 180r/min, sampling and detecting the product conversion rate by HPLC.
The reaction system comprises 20% (v/v) isopropanol, 20% -50% (v/v) organic solvent, 100g/L substrate, substrate: catalase =133:1.
The HPLC detection conditions and conversion rate were calculated as in example one, and the experimental results are shown in table 4:
TABLE 4
Figure BDA0003260108410000201
As is clear from the results in Table 4, the highest conversion rate of catalase was obtained when the concentration of ethyl acetate as an organic solvent was 40% (v/v), which indicates that when the catalytic reaction was carried out by using the catalase of the present application, a higher conversion rate of the product could be obtained when the concentration of ethyl acetate as an organic solvent in the system was controlled to 40% (v/v).
EXAMPLE six different concentrations of butyl acetate
This example tests the presence of a mixture of butyl acetate having the amino acid sequences shown in SEQ ID No.1 and SEQ ID NO:18 sequence of catalase, the specific experimental method comprises the following steps:
1) Adding 10mL of isopropanol into a 100mL reaction bottle, adding butyl acetate with different concentrations (v/v), adding 5g of substrate, and uniformly mixing to obtain a mixed solution;
2) Adding 0.0375g of catalase, adding 0.5mL of carbonyl reductase, and adding proper amount of water to make the total amount to be 50mL; then 10mg of NADP was added + Adjusting the initial pH to 7.5;
3) Continuously reacting for 24h under the oscillating condition of 35 ℃ and 180r/min, sampling and detecting the product conversion rate by HPLC.
The reaction system comprises 20% (v/v) isopropanol, 20% -50% (v/v) organic solvent, 100g/L substrate, substrate: catalase =133:1.
The HPLC detection conditions and the conversion rate were calculated in the same manner as in example one, and the experimental results are shown in Table 5:
TABLE 5
Figure BDA0003260108410000211
As is clear from the results in Table 5, the highest conversion of catalase was obtained at a concentration of 30% (v/v) of butyl acetate as the organic solvent, indicating that when the catalytic reaction was carried out using the catalase of the present application, a higher conversion of the product was obtained when the concentration of butyl acetate as the organic solvent in the system was controlled to 30% (v/v).
EXAMPLE seven dissolution of various concentrations of substrate with ethyl acetate
This example examined the appropriate concentration of substrate added when the solvent was chosen to be ethyl acetate, and the specific experimental procedure included the following steps:
1) Adding 10mL of isopropanol into a 100mL reaction bottle, adding 15mL of ethyl acetate, adding substrates with different masses, and uniformly mixing to obtain a mixed solution;
2) Adding 0.0375g of catalase, adding 10mg of NADP + Adding 0.5mL carbonyl reductase and 24.5mL water; adjusting the initial pH to 7.0;
3) Continuously reacting for 24h under the oscillating condition of 35 ℃ and 180r/min, sampling and detecting the product conversion rate by HPLC.
The reaction system comprises 20% (v/v) of isopropanol and 30% (v/v) of organic solvent.
The HPLC detection conditions and the conversion rate were calculated in the same manner as in example one, and the experimental results are shown in Table 6:
TABLE 6
Figure BDA0003260108410000221
As can be seen from the results in table 6, for SEQ ID NO:1, the higher the substrate concentration is, the lower the conversion rate is, which indicates that the reaction has substrate inhibition, and the high substrate concentration can inhibit enzyme activity, so that the conversion rate is reduced; and for SEQ ID NO:18, when the substrate concentration reaches 150g/L, the conversion rate can still reach 98.8%, which indicates that the substrate inhibition is relieved, namely the catalase prepared by adopting the nucleotide sequence shown in SEQ ID NO:18, the same enzyme amount can be used for catalyzing a substrate with higher concentration, when the organic solvent in the system is ethyl acetate, when the substrate concentration is 150g/L, the mass ratio of the substrate to the catalase is 200:1, and higher product conversion rate can be obtained.
EXAMPLE eight dissolution of substrates of different concentrations with butyl acetate
This example examines the appropriate concentration of substrate added when the solvent is selected to be butyl acetate, and the specific experimental procedure comprises the following steps:
1) Adding 10mL of isopropanol into a 100mL reaction bottle, adding 15mL of butyl acetate, adding substrates with different masses, and uniformly mixing to obtain a mixed solution;
2) Adding 0.0375g of catalase, adding 10mg of NADP + Adding 0.5mL carbonyl reductase and 24.5mL water; adjusting the initial pH to 7.0;
3) Continuously reacting for 24h under the oscillating condition of 35 ℃ and 180r/min, sampling and detecting the product conversion rate by HPLC.
The reaction system comprises 20% (v/v) of isopropanol and 30% (v/v) of organic solvent.
The HPLC detection conditions and conversion rate were calculated as in example one, and the experimental results are shown in table 7:
TABLE 7
Figure BDA0003260108410000231
As can be seen from the results in table 7, for SEQ ID NO:1, the higher the substrate concentration is, the lower the conversion rate is, which indicates that the reaction has substrate inhibition, and the high substrate concentration can inhibit enzyme activity, so that the conversion rate is reduced; and for SEQ ID NO:18, when the substrate concentration reaches 100g/L, the conversion rate can still reach 96.8%, which indicates that the substrate inhibition is relieved, namely the method adopts the sequence shown in SEQ ID NO:18, when butyl acetate is used as an organic solvent in a substrate system with higher concentration and the substrate concentration is 100g/L, the mass ratio of the substrate to the catalase is 133:1, and higher product conversion rate can be obtained.
EXAMPLE nine Ethyl acetate and Isopropanol System
This example tests that when the coenzyme regeneration system added is isopropanol and carbonyl reductase, the organic solvent is ethyl acetate and has the amino acid sequences of SEQ ID No.1 and SEQ ID NO:18 sequence of catalase, the specific experimental method comprises the following steps:
1) 50mL of isopropyl alcohol, 100mL of ethyl acetate and 37.5g of a substrate were added to a 500mL reaction flask to obtain a mixture of [ deg. ] C
2) Adding 0.188g of catalase, and adding 50mg of NADP + 2.5mL of carbonyl reductase was added, 97.5mL of water was added, and the pH was adjusted to 7.8-8.0 with 10% NaOH during the reaction.
3) The reaction is continued for 24h under the oscillating condition of 37 ℃ and 400r/min, and the product conversion rate is detected by HPLC after sampling.
The reaction system contained 40% (v/v) ethyl acetate, 150g/L substrate, substrate: catalase =200: 1.
The HPLC detection conditions and the conversion rate were calculated in the same manner as in example one.
The experimental results are as follows: SEQ ID NO: the catalase 24h product conversion of 1 is 25.5%;
SEQ ID NO: the catalase 24h product conversion of 18 was 98.9%.
As can be seen from the experimental results, when the coenzyme regeneration system is isopropanol and carbonyl reductase, and the organic solvent is ethyl acetate, and the system employs the organic solvent and substrate concentrations preferred in the foregoing experimental results, the concentrations of SEQ ID NO: the conversion rate of the product of the catalase of 18 can reach 98.9 percent.
The experimental procedure of comparative example one is the same as that of example nine, except that the enzyme added is an enzyme having an amino acid sequence shown in SEQ ID NO:4, the 24h product conversion was 97.6%. Description of the invention SEQ ID NO: the catalase of 18 achieved higher product conversion at the same amount relative to the enzyme of the comparative example, suggesting that the catalase of the present application can achieve higher product conversion at lower amounts.
EXAMPLE ten butyl acetate and Isopropanol System
This example demonstrates that when the coenzyme regeneration system added is isopropanol and carbonyl reductase, and the organic solvent is butyl acetate, the enzyme has the amino acid sequences shown in SEQ ID No.1 and SEQ ID NO:18 sequence of catalase, the specific experimental method comprises the following steps:
1) 50mL of isopropyl alcohol, 75mL of butyl acetate and 25g of a substrate were added to a 500mL reaction flask to obtain a mixture of ° C
2) Adding 0.188g of catalase, adding 50mg of NADP + 2.5mL of carbonyl reductase was added, 122.5mL of water was added, and the pH was adjusted to 7.8-8.0 with 10% NaOH during the reaction.
3) The reaction is continued for 24h under the oscillating condition of 37 ℃ and 400r/min, and the product conversion rate is detected by HPLC after sampling.
The reaction system contained 30% (v/v) butyl acetate, 100g/L substrate, substrate: catalase =133:1.
the HPLC detection conditions and the conversion rate were calculated in the same manner as in example one.
The experimental results are as follows: SEQ ID NO: the catalase 24h product conversion of 1 was 33%;
the amino acid sequence of SEQ ID NO: the catalase 24h product conversion of 18 was 97%.
As can be seen from the experimental results, when the coenzyme regeneration system is isopropanol and carbonyl reductase, and the organic solvent is butyl acetate, and the system employs the organic solvent and substrate concentrations preferred in the foregoing experimental results, SEQ ID NO: the product conversion of the catalase of 18 can reach 97%.
The experimental procedure of comparative example two is the same as that of example ten, except that the enzyme added is an enzyme whose amino acid sequence is as shown in SEQ ID NO:4, the product conversion rate in 24h is 96.1%. Description of the SEQ ID NO:18 at the same amount, higher product conversion relative to the enzyme of the comparative example was achieved, suggesting that the catalase of the present application could achieve higher product conversion at a lower amount.
EXAMPLE eleven, ethyl acetate and glucose System
This example examined the results of the assays of the compounds having the amino acid sequences of SEQ ID No.1 and SEQ ID NO:18 sequence of catalase, the specific experimental method comprises the following steps:
1) Adding 100mL of ethyl acetate into a 500mL reaction bottle, adding 37.5g of substrate, and stirring and dissolving at 37 ℃ to obtain a mixed solution;
2) Weighing 10g of glucose and 105mL of water, completely dissolving, adding into a reaction bottle, and adjusting the pH value to 7.5;
3) Adding 0.188g of catalase; immediately, 1mL of glucose dehydrogenase was added, and 50mg of NADP was added + ；
4) The reaction is continued for 24h under the oscillating condition of 37 ℃ and 400r/min, and the product conversion rate is detected by HPLC after sampling.
The reaction system contains 40% (v/v) ethyl acetate, 150g/L substrate, substrate: catalase =200:1, controlling the pH value in the reaction process to be 7.5.
The HPLC detection conditions and the conversion rate were calculated in the same manner as in example one.
The experimental results are as follows: SEQ ID NO: the 24h product conversion of the catalase of 1 is 38 percent;
SEQ ID NO: the 24h product conversion of catalase 18 was 99.5%.
As can be seen from the experimental results, when the coenzyme regeneration system is glucose and glucose dehydrogenase, and the organic solvent is ethyl acetate, and the organic solvent and substrate concentrations preferred in the foregoing experimental results are adopted in the system, the concentrations of SEQ ID NO: the conversion rate of the product of the catalase of 18 can reach 99.5 percent, and the conversion rate is the highest, so the system of the embodiment is the optimal system.
As shown in fig. 4, when the coenzyme regeneration system is glucose and glucose dehydrogenase, the enzyme containing ethyl acetate and SEQ ID NO:18 on an HPLC profile at 1h, shown in fig. 5 as containing ethyl acetate and SEQ ID NO: the HPLC profile of the catalase reaction system of 18 at 24h, as can be seen from comparison between FIG. 4 and FIG. 5, after 24h of reaction, the peak area of the substrate is reduced, even the substrate peak is substantially disappeared, and the peak area of the ezetimibe intermediate is significantly increased, which indicates that when the coenzyme regeneration system is glucose and glucose dehydrogenase, the ratio of SEQ ID NO:18 was very high in the conversion capacity of the catalase product.
The experimental procedure of comparative example three is the same as that of example eleven except that the enzyme added is an enzyme having an amino acid sequence shown in SEQ ID NO:4, the 24h product conversion was 97.8%. Description of the SEQ ID NO: the catalase of 18 achieved higher product conversion at the same amount relative to the enzyme of the comparative example, suggesting that the catalase of the present application can achieve higher product conversion at lower amounts.
Example twelve, butyl acetate and glucose System
This example examined the results of the assays of the enzyme molecules having the amino acid sequences shown in SEQ ID No.1 and SEQ ID NO:18 sequence of catalase, the specific experimental method comprises the following steps:
1) Adding 75mL of ethyl acetate into a 500mL reaction bottle, adding 25g of substrate, and stirring at 37 ℃ to dissolve to obtain a mixed solution;
2) Weighing 10g of glucose and 140mL of water, completely dissolving, adding into a reaction bottle, and adjusting the pH value to 7.5;
3) Adding 0.188g of catalase; immediately, 1mL of glucose dehydrogenase was added, and 50mg of NADP was added + ；
4) The reaction is continued for 24h under the oscillating condition of 37 ℃ and 400r/min, and the product conversion rate is detected by HPLC after sampling.
The reaction system contained 30% (v/v) butyl acetate, 100g/L substrate, substrate: catalase =133:1, controlling the pH value in the reaction process to be 7.5.
The HPLC detection conditions and the conversion rate were calculated in the same manner as in example one.
The experimental results are as follows: SEQ ID NO: the 24h product conversion of the catalase of 1 is 36 percent;
SEQ ID NO: the catalase 24h product conversion of 18 was 98%.
As can be seen from the experimental results, when the coenzyme regeneration system is glucose and glucose dehydrogenase, and the organic solvent is butyl acetate, and the system employs the organic solvent and substrate concentrations preferred in the foregoing experimental results, SEQ ID NO: the product conversion of the catalase of 18 can reach 98 percent.
The experimental procedure of comparative example four is the same as that of example twelve, except that the enzyme added has an amino acid sequence shown in SEQ ID NO:4, the product conversion rate in 24h is 96.8%. Description of the invention SEQ ID NO:18 at the same amount, higher product conversion relative to the enzyme of the comparative example was achieved, suggesting that the catalase of the present application could achieve higher product conversion at a lower amount.
The catalase and a corresponding enzyme catalysis system are adopted, and a substrate (4S) -3- [5- (4-fluorophenyl) -1,5-dioxolyl ] -4-phenyl-2-oxazolidinone can be efficiently catalyzed to be reduced to obtain (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazacyclopentane-2-one serving as an ezetimibe intermediate.
While the above detailed description of an enzyme catalysis system, a catalase, a preparation method and applications provided in the examples of the present application have been described, the principles and embodiments of the present application are described herein by using specific examples, and the description of the above examples is only for the purpose of helping understanding the method and the core concept of the present application, and the skilled in the art may change the specific embodiments and the application scope according to the concept of the present application, and the content of the present description should not be construed as limiting the present application.
Sequence listing
<110> Taizhou enzyme Biotechnology Ltd
<120> catalase, enzyme catalysis system, preparation method and application
<141> 2021-08-17
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 279
<212> PRT
<213> Galactomyces reessii
<400> 1
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 2
<211> 283
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ala Lys Asn Phe Ser Asn Val Glu Tyr Pro Ala Pro Pro Pro Ala
1 5 10 15
His Thr Lys Asn Glu Ser Leu Gln Val Leu Asp Leu Phe Lys Leu Asn
20 25 30
Gly Lys Val Ala Ser Ile Thr Gly Ser Ser Ser Gly Ile Gly Tyr Ala
35 40 45
Leu Ala Glu Ala Phe Ala Gln Val Gly Ala Asp Val Ala Ile Trp Tyr
50 55 60
Asn Ser His Asp Ala Thr Gly Lys Ala Glu Ala Leu Ala Lys Lys Tyr
65 70 75 80
Gly Val Lys Val Lys Ala Tyr Lys Ala Asn Val Ser Ser Ser Asp Ala
85 90 95
Val Lys Gln Thr Ile Glu Gln Gln Ile Lys Asp Phe Gly His Leu Asp
100 105 110
Ile Val Val Ala Asn Ala Gly Ile Pro Trp Thr Lys Gly Ala Tyr Ile
115 120 125
Asp Gln Asp Asp Asp Lys His Phe Asp Gln Val Val Asp Val Asp Leu
130 135 140
Lys Gly Val Gly Tyr Val Ala Lys His Ala Gly Arg His Phe Arg Glu
145 150 155 160
Arg Phe Glu Lys Glu Gly Lys Lys Gly Ala Leu Val Phe Thr Ala Ser
165 170 175
Met Ser Gly His Ile Val Asn Val Pro Gln Phe Gln Ala Thr Tyr Asn
180 185 190
Ala Ala Lys Ala Gly Val Arg His Phe Ala Lys Ser Leu Ala Val Glu
195 200 205
Phe Ala Pro Phe Ala Arg Val Asn Ser Val Ser Pro Gly Tyr Ile Asn
210 215 220
Thr Glu Ile Ser Asp Phe Val Pro Gln Glu Thr Gln Asn Lys Trp Trp
225 230 235 240
Ser Leu Val Pro Leu Gly Arg Gly Gly Glu Thr Ala Glu Leu Val Gly
245 250 255
Ala Tyr Leu Phe Leu Ala Ser Asp Ala Gly Ser Tyr Ala Thr Gly Thr
260 265 270
Asp Ile Ile Val Asp Gly Gly Tyr Thr Leu Pro
275 280
<210> 3
<211> 261
<212> PRT
<213> Bacillus subtilis
<400> 3
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Val Ile Thr Gly Ser Ser
1 5 10 15
Thr Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Ala Thr Ala Lys Ala
20 25 30
Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val
35 40 45
Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly
50 55 60
Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile
65 70 75 80
Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Ile Lys Val
100 105 110
Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Ser Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Thr
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 4
<211> 352
<212> PRT
<213> Thermoanaerobacter brockii
<400> 4
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe Gln Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Ser Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> 5
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 5
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 6
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 6
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Pro Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 7
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 7
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 8
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 8
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg Ile Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 9
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 9
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 10
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 10
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 11
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 11
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Val Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 12
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 12
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 13
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 13
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 14
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 14
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 15
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 15
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg Ile Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 16
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 16
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Pro Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 17
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 17
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Val Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 18
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 18
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 19
<211> 837
<212> DNA
<213> Galactomyces reessii
<400> 19
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 20
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 20
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 21
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 21
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctcct gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 22
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 22
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 23
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 23
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tattttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 24
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 24
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 25
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 25
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 26
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 26
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 27
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 27
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 28
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 28
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 29
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 29
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 30
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 30
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tattttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
attggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 31
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 31
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctcct gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 32
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 32
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
attggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 33
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 33
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
attggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837

Claims (9)

1. An enzymatic catalysis system, comprising: catalase, organic solvent, coenzyme and coenzyme regenerant for catalyzing a substrate to generate a product through reduction reaction;
the substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1):
Figure FDA0004103679330000011
the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, and the structure of the product is shown as the formula (2):
Figure FDA0004103679330000012
the amino acid sequence of the catalase is shown as SEQ ID NO:18, respectively.
2. The enzymatic system of claim 1, wherein the substrate is present at a concentration of 50g/L to 250g/L;
based on the total volume of the reaction system, the volume percentage content of the organic solvent is 20-50%;
the organic solvent is at least one selected from dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, propyl acetate, butyl acetate, N-dimethyl formyl, dichloromethane or toluene;
the coenzyme is selected from any one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate;
the concentration of the coenzyme is 0.2-2 g/L;
the coenzyme regenerating agent comprises: isopropanol and carbonyl reductase, or, glucose and glucose dehydrogenase.
3. The enzyme catalysis system according to claim 2, wherein the carbonyl reductase has an amino acid sequence as set forth in SEQ ID NO:2 is shown in the specification;
the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO:3 is shown in the figure;
the concentration of the glucose is 30-50 g/L.
4. The catalase is characterized in that the amino acid sequence of the catalase is shown in SEQ ID NO:18, respectively.
5. Use of the catalase of claim 4 to catalyze a reduction of a substrate to produce a product;
the substrate is (4S) -3- [5- (4-fluorophenyl) -1,5-dioxolyl ] -4-phenyl-2-oxazolidinone, and the structure of the substrate is shown as the formula (1):
Figure FDA0004103679330000021
the product is (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one, and the structure of the product is shown as the formula (2):
Figure FDA0004103679330000022
6. a nucleic acid molecule comprising a nucleotide sequence encoding the catalase of claim 4.
7. The nucleic acid molecule of claim 6, wherein the nucleotide sequence is as set forth in SEQ ID NO:33, or a nucleotide sequence shown in seq id no.
8. A process for preparing (4S) -3- [ (5S) -5- (4-fluorophenyl) 5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one comprising:
mixing the substrate (4S) -3- [5- (4-fluorophenyl) -1,5-dioxopentyl ] -4-phenyl-2-oxazolidinone with the enzymatic system of any of claims 1 to 3 to produce an enzymatically catalyzed reduction reaction to give (4S) -3- [ (5S) -5- (4-fluorophenyl) -5-hydroxypentanoyl ] -4-phenyl-1,3-oxazepan-2-one.
9. The method according to claim 8, wherein the mass ratio of the substrate to the catalase in the enzyme-catalyzed system is (120-210): 1;
the concentration of the substrate is 50 g/L-250 g/L;
based on the total volume of the reaction system, the volume percentage content of the organic solvent in the enzyme catalysis system is 20-50%;
the pH value of the enzyme catalytic reduction reaction is 6.0-9.0;
the temperature of the enzyme catalytic reduction reaction is 25-65 ℃.
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