CN109943542B - Alcohol dehydrogenase for producing atazanavir intermediate - Google Patents
Alcohol dehydrogenase for producing atazanavir intermediate Download PDFInfo
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- CN109943542B CN109943542B CN201811639906.2A CN201811639906A CN109943542B CN 109943542 B CN109943542 B CN 109943542B CN 201811639906 A CN201811639906 A CN 201811639906A CN 109943542 B CN109943542 B CN 109943542B
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- alcohol dehydrogenase
- ala
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an alcohol dehydrogenase for producing atazanavir intermediate, belonging to the field of medical biotechnology, the alcohol dehydrogenase has stronger catalytic activity compared with wild alcohol dehydrogenase of SEQ ID NO.8, the alcohol dehydrogenase can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA reorganization, error-prone PCR and directed evolution method, etc. of the encoded enzyme, the alcohol dehydrogenase has one or more mutations selected from the following characteristics: T37A, D38E, P41K, D44N and V45K. The method has mild reaction conditions, low requirements on equipment, no need of high temperature or cooling in the production process and low energy consumption, and the enzyme catalysis has high efficiency and specific selectivity, so that the key intermediate for producing the atazanavir by the method has no byproduct and is convenient to purify; in addition, the reaction solvent is mainly water, the discharge of three wastes is low, and the method is green and environment-friendly.
Description
Technical Field
The invention relates to a preparation method of an atazanavir intermediate, in particular to alcohol dehydrogenase for producing the atazanavir intermediate, belonging to the technical field of medical biology.
Background
Atazanavir, sold under the trade name Reyataz, is an antiretroviral drug for treating and preventing aids/aids, is one of the major aids drugs in the world at present, is the most important drug required by basic health systems on the basic drug list of the world health organization, and has been developed by atazanavir at the earliest by the company of centurie meissnobao, publicly described in chinese patent CN10282508C, approved by the FDA in us in 2003, and approved by china in 2007.
(2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol is used as a key intermediate for preparing atazanavir, and the main production processes at present comprise a chemical synthesis method and a biological synthesis method, wherein the biological method can be obtained by performing whole-cell biotransformation on 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol by using corresponding ketoreductase or microorganisms.
JP4746548B2, disclosed in Japanese patent JP4746548B2, originally proposed the biological preparation of (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol by asymmetrically reducing (3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanone with a novel carbonyl-acyl enzyme to produce (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. And other similar optically active alcohols are disclosed in chinese patent CN1993464B, which is simultaneously applied for protection in china and granted in 2011, but because the patent uses coenzyme IINADP, the market price is expensive, the application of the patent is limited to a certain extent, and other subsequent companies such as U.S. codexis and the like optimize and promote the conditions such as enzyme types, reaction conditions, coenzyme circulation and the like, further improve the production efficiency, and hopefully reduce the condition requirements and the cost,
in Chinese patent CN104911224, the main enzyme and NADP are co-immobilized and then biocatalysis is carried out, but the reaction time of the process is too long for 48-60 h, the production efficiency is low, and the production growth is further increased; the preparation of the immobilized enzyme and the storage procedure after the immobilized enzyme is used are complicated, and the effective reuse times of the immobilized enzyme are not given by the process.
Glucose is used in the process of Chinese patent CN103468757, the gluconic acid generated after the reaction is finished has no recycling price, so a large amount of solid wastes are generated, and the process uses enzyme powder for the reaction, so that the amplification production prospect is limited.
In the process of international patent WO2011005527, since the enzyme activity of the enzyme used is not high, 0.3g nad is required to catalyze 90 g of substrate: the proportion of NAD is only 300, which causes the cost of NAD in each kilogram of products to be too high, so that the production cost is higher, in Chinese patent CN102732579, Saccharomyces cerevisiae is used for whole-cell catalysis, but the required yeast thallus is too much (10g of dry cells), the catalytic substrate can be completely converted only when the catalytic substrate is not more than 0.1mM, and the production efficiency of the process is lower.
The wild-type alcohol dehydrogenase naturally present in the microorganism Sphingomonas stygia is capable of converting 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol into (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. The inventors of the present disclosure have found that an alcohol dehydrogenase including a mutation at a certain position exhibits increased catalytic activity compared to a wild-type alcohol dehydrogenase (SEQ ID NO: 8) produced by Sphingomonas styragia. "wild-type alcohol dehydrogenase", "wild-type ADH enzyme", and "wild-type ADH alcohol dehydrogenase" refer to a polypeptide consisting of a polypeptide having the sequence of SEQ ID NO: 8 amino acid sequence of alcohol dehydrogenase. The enzyme can convert 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol to produce (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. "wild-type" refers to the same form of material or substance as found in nature. For example, a wild-type protein or nucleic acid sequence is the original sequence form that can be isolated from nature and exists in an organism without artificial modification. "increased catalytic activity" refers to an alcohol dehydrogenase that exhibits an increased rate of conversion of a substrate (e.g., 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) to a product (e.g., (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) as compared to the wild-type alcohol dehydrogenase as measured in an in vitro or in vivo assay.
Disclosure of Invention
The main object of the present invention is to provide an alcohol dehydrogenase for use in the production of atazanavir intermediate, converting 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol to (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, the alcohol dehydrogenase mutated at a certain position showing increased catalytic activity compared to the wild-type alcohol dehydrogenase produced by Sphingomonas stygia (SEQ ID NO: 8).
The purpose of the invention can be achieved by adopting the following technical scheme:
an alcohol dehydrogenase for use in the production of an atazanavir intermediate, which is derived from a wild-type alcohol dehydrogenase of Sphingomonas stygia, exhibits an alcohol dehydrogenase that increases the rate of conversion of a substrate to a product, the alcohol dehydrogenase exhibiting a stronger catalytic activity than the wild-type alcohol dehydrogenase of SEQ ID NO.8, the alcohol dehydrogenase being obtainable by subjecting an enzyme encoding the same to in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, directed evolution methods, and the like, the alcohol dehydrogenase having one or more mutations selected from the following characteristics: T37A, D38E, P41K, D44N and V45K.
The alcohol dehydrogenase is selected from the sequences SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6.
The alcohol dehydrogenase, C terminal or N terminal several amino acids can be deleted;
any particular truncated analog or fragment can be used to assess catalytic activity using a corresponding assay;
additional amino acid residues may be added to one or both termini without affecting catalytic activity;
the additional sequences may be functional or non-functional.
The above alcohol dehydrogenases, in the form of fusion proteins, are fused to other proteins, such as by way of example and not limitation, a solubilizing tag, a purification tag and a bacterial localization signal.
The alcohol dehydrogenase has an alcohol dehydrogenase activity at least 2-10 times higher than that of a wild-type alcohol dehydrogenase.
The coding sequence of the alcohol dehydrogenase is preferably selected from SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO. 5.
The above alcohol dehydrogenase, polynucleotide, comprises codons optimized for expression in a particular type of host cell.
The alcohol dehydrogenase constitutes recombinant plasmid, and its control sequence includes promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.
For bacterial host cells, suitable promoters to direct transcription of the coding sequence include those from bacteriophage T5, bacteriophage T7, bacteriophage lambda, E.coli lacUV5 operon, E.coli trp operon, and E.coli tac operon.
The invention has the beneficial technical effects that:
1. the alcohol dehydrogenase for producing the atazanavir intermediate can be used for biologically catalyzing and converting 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol into (2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol; the reaction condition is mild, the requirement on equipment is low, the production process does not need high temperature or cooling, the energy consumption is low, and the enzyme catalysis has high efficiency and specific selectivity, so that the key intermediate (2R,3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol for producing the atazanavir by the method has no byproduct and is convenient to purify; in addition, the reaction solvent is mainly water, the discharge of three wastes is low, and the method is green and environment-friendly.
Drawings
FIG. 1 is an expression plasmid map of Sst-1;
FIG. 2 is a TLC pattern of Sst-1, Sst-2, Sst-3, and Cod-CK bioconversion reactions for 3 hours, from left to right for Sst-1, Sst-2, Sst-3, Cod-CK, with the top band as substrate and the bottom band as product;
FIG. 3 is a TLC pattern of the Sst-1 reaction for 20 h.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
As shown in FIG. 1, among the alcohol dehydrogenases used in the production of atazanavir intermediate provided in this example, the wild-type alcohol dehydrogenase naturally occurring in the microorganism Sphingomonas stygia was able to convert 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol into (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. The inventors of the present disclosure have found that an alcohol dehydrogenase including a mutation at a certain position exhibits increased catalytic activity compared to a wild-type alcohol dehydrogenase (SEQ ID NO: 8) produced by Sphingomonas stygia. "wild-type alcohol dehydrogenase", "wild-type ADH enzyme" and "wild-type ADH alcohol dehydrogenase" refer to a polypeptide consisting of a polypeptide derived from Sphingomonas stygia having the sequence of SEQ ID NO: 8 amino acid sequence of alcohol dehydrogenase. The enzyme can convert 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol to produce (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. "wild-type" refers to the same form of material or substance as found in nature. For example, a wild-type protein or nucleic acid sequence is the original sequence form that can be isolated from nature and exists in an organism without artificial modification. "increased catalytic activity" refers to an alcohol dehydrogenase that exhibits an increased rate of conversion of a substrate (e.g., 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) to a product (e.g., (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) as compared to a wild-type alcohol dehydrogenase as measured in an in vitro or in vivo assay.
The present invention provides an alcohol dehydrogenase derived from a wild-type alcohol dehydrogenase of Sphingomonas stygia, which exhibits an alcohol dehydrogenase that increases the conversion rate of a substrate (e.g., 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) to a product (e.g., (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol). The alcohol dehydrogenase shows stronger catalytic activity compared with the wild-type alcohol dehydrogenase of SEQ ID NO. 8. Alcohol dehydrogenases and polynucleotides encoding such alcohol dehydrogenases may be prepared using methods commonly used by those skilled in the art. Alcohol dehydrogenases can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods, etc., which encode the enzyme.
The above alcohol dehydrogenase having one or more mutations selected from the following features: T37A, D38E, P41K, D44N and V45K.
The alcohol dehydrogenase is preferably selected from the sequences SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6. The full-length mutant alcohol dehydrogenase is not essential for maintaining the catalytic activity of the enzyme. Accordingly, truncated analogs and catalytically active fragments of alcohol dehydrogenases are contemplated. For example, in some embodiments, several amino acids may be deleted from the C-terminus or N-terminus. Any particular truncated analog or fragment can be used in a corresponding assay to assess catalytic activity. Likewise, additional amino acid residues may be added to one or both termini without affecting catalytic activity. The additional sequences may be functional or non-functional. For example, the additional amino acid sequence may be used to aid in purification, as a marker, or to perform some other function. Thus, the alcohol dehydrogenases of the present disclosure may be in the form of fusion proteins, wherein the alcohol dehydrogenase (or fragment thereof) is fused to other proteins, such as by way of example and not limitation, a solubilizing tag (e.g., SUMO protein), a purification tag (e.g., metal-binding His tag), and a bacterial localization signal (e.g., secretion signal).
The present invention provides an alcohol dehydrogenase whose alcohol dehydrogenase activity is enhanced by at least 2 to 10 times as compared with that of a wild-type alcohol dehydrogenase.
The above alcohol dehydrogenase encoding sequence, preferably selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, has been sequence optimized for expression in E.coli. In some embodiments, the polynucleotide comprises codons optimized for expression in a particular type of host cell. The codon usage and preferences for each different type of microorganism are known as are optimized codons for the expression of a particular amino acid in these microorganisms. The present invention provides a recombinant plasmid, and in some embodiments, the control sequence includes a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, a transcription terminator, and the like. For bacterial host cells, suitable promoters for directing transcription of the coding sequence include, but are not limited to, the genes selected from bacteriophage T5, bacteriophage T7, bacteriophage lambda, E.coli lacUV5 operon, E.coli trp operon, E.coli tac operon, and the like.
Example 1:
the secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli.
The following primers were obtained by designing with PrimePremier (http:// primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled within 3 ℃ and the primer length is controlled within 60 base:
1TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG
2CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCTT
3GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAG
4GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACCGA
5ACGCTATCGTTATCGCTGCTGAAATGGCTCCGTCTGCTGACAAAGAAGGTGCTGA
6CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTCAGC
7CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA
8CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG
9CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT
10AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA
11GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC
12AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTG
13TCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGTGGTCTGCGTGGT
14GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA
15TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA
16GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG
17GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC
18GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC
19CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG
20GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT
21CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT
22CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC
23GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG
24CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC
the above primers were synthesized, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | The total volume of the reaction system was adjusted to 50. mu.l |
The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.1 and is named as Sst-1, and the corresponding amino acid sequence is SEQ ID NO. 2.
Example 2:
the secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli.
The following primers were obtained by designing with PrimePremier (http:// primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled within 3 ℃ and the primer length is controlled within 60 base:
1TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG
2CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCTT
3GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAG
4GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACCGA
5ACGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTGACAAAGAAGGTGCTGA
6CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTCAGC
7CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA
8CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG
9CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT
10AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA
11GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC
12AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTG
13TCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGTGGTCTGCGTGGT
14GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA
15TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA
16GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG
17GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC
18GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC
19CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG
20GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT
21CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT
22CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC
23GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG
24CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC
the above primers were synthesized, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | The total volume of the reaction system was adjusted to 50. mu.l |
The prepared PCR reaction system is placed in a Bori XPcycler gene amplification instrument, and amplification is carried out according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by the homologous recombination method. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.3 and is named as Sst-2, and the corresponding amino acid sequence is SEQ ID NO. 4.
Example 3:
the secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli.
The following primers were obtained by designing with PrimePremier (http:// primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled within 3 ℃ and the primer length is controlled within 60 base:
1TGTTTAACTTTAAGAAGGAGATATACATATGACCATCGC
2ACCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCT
3TTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAGC
4CAGCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACC
5CGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTAACAAAGAAGGTGCTGAC
6CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTTAGC
7CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA
8CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG
9CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT
10AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA
11GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC
12AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTG
13TCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGTGGTCTGCGTGGT
14GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA
15TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA
16GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG
17GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC
18GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC
19CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG
20GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT
21CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT
22CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC
23GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG
24CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC
the above primers were synthesized, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | The total volume of the reaction system was adjusted to 50. mu.l |
The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.5 and is named as Sst-3, and the corresponding amino acid sequence is SEQ ID NO. 6.
Synthesizing a reference protein Cod-CK gene sequence;
based on the sequence shown by AJM46704.1, the Shanghai Czeri organism was assigned to perform whole gene synthesis of the coding sequence of the protein, and cloned into pET30a to obtain a control protein expression plasmid Cod-CK (SEQ ID NO. 7).
Shake flask expression test
Coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L. The culture was carried out at 30 ℃ and 250rpm overnight. The next day, take 1L triangular flask, press 1: 100 into 100ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L. The cells were cultured at 30 ℃ until OD5-6 was obtained, and the flasks were immediately placed on a shaker at 25 ℃ and cultured at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 hours at 250 rpm. After completion of the culture, the culture was centrifuged at 12000g at 4 ℃ for 20 minutes to collect wet cells. Then the bacterial pellet is washed twice with distilled water, and the bacterial is collected and preserved at-70 ℃. Meanwhile, a small amount of thallus is taken for SDS-PAGE detection.
Fed-batch fermentation
The fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate, pH 7.0. 200ml of culture was prepared for the primary inoculum and inoculated at OD 2.0. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation process, when a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. Induction with 0.2mM IPTG occurred at an OD600 of about 35.0 (wet weight of about 60 g/L).
Biotransformation reactions
A500 ml three-necked beaker was charged with a magneton stirrer, and 2.7ml of toluene, 32ml of isopropanol, and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the mixture was mixed with the pre-melting substrate, 1mM MgCl2 and 0.1M PB (pH7.5) were added thereto to give a total of about 190ml, and the mixture was mixed with pH adjusted to 7.5. Finally, 21mg of NAD and 6.4ml of crude enzyme solution Sst-1 and 30C are added for shaking table reaction. 200ml of reaction system. Samples were taken at 3 hours and 20 hours for storage. As can be seen from FIG. 3, the reaction had completely converted the substrate within 20 hours.
Biotransformation reactions
A500 ml three-necked beaker was charged with a magneton stirrer, and 2.7ml of toluene, 32ml of isopropanol, and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the mixture was mixed with the pre-melting substrate, 1mM MgCl2 and 0.1M PB (pH7.5) were added thereto to give a total of about 190ml, and the mixture was mixed with pH adjusted to 7.5. Finally, 21mg of NAD and 6.4ml of crude enzyme solution Sst-2 are added for shaking table reaction at 30C. 200ml of reaction system.
Biotransformation reactions
A500 ml three-necked beaker was charged with a magneton stirrer, and 2.7ml of toluene, 32ml of isopropanol, and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the mixture was mixed with the pre-molten substrate, 1mM MgCl2 and 0.1M PBpH7.5 were added thereto to give a total of about 190ml, and the mixture was mixed with PBpHs adjusted to 7.5. Finally, 21mg of NAD and 6.4ml of crude enzyme solution Sst-3 are added for shaking table reaction at 30C. 200ml of reaction system.
Biotransformation reactions
A500 ml three-necked beaker was charged with a magneton stirrer, and 2.7ml of toluene, 32ml of isopropanol, and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the mixture was mixed with the pre-melting substrate, 1mM MgCl2 and 0.1M PB (pH7.5) were added thereto to give a total of about 190ml, and the mixture was mixed with pH adjusted to 7.5. Finally, 21mg of NAD, 6.4ml of crude enzyme solution Cod-CK and 30C were added for shake reaction. 200ml of reaction system.
TLC detection of the product
The conversion products of the reaction in the above examples were subjected to TLC detection, and the results are shown in FIG. 2. It can be seen that the Sst-1, Sst-2 and Sst-3 reaction rates are all greater than the control Cod-CK.
Enzyme activity detection
Taking 65 ml centrifuge tubes, respectively labeling 1-6, adding 3mM NADH solution 0ul, 40ul, 80ul, 100ul, 120ul, 160ul, supplementing 0.1M phosphate buffer solution with pH of 7.0 to 3ml, mixingDetecting and recording the absorbance value at 340nm after the mixture is homogenized; from the above measured values, a standard curve Y ═ k × X of NADH was obtained, where Y is the value of absorbance, X is the concentration (mM) of NADH, R of the curve2>99.5 percent; diluting the enzyme solution with pure water by a certain dilution ratio (reference dilution ratio: 600-1000 times), wherein the dilution ratio is suitable for changing the light absorption value per minute by 0.02-0.04; 5ml of centrifuge tube is taken, the samples are added into the centrifuge tube according to the following proportion, the mixture is quickly mixed, and the mixture is immediately poured into a cuvette.
| Detection reagent | Dosage of |
| Isopropanol (I-propanol) | 500ul |
| 2％NAD | 100uL |
| 100mM PBS(pH7.0) | 2.35mL |
| Diluted enzyme solution | 50uL |
Detecting the change of absorbance at 340nm, recording a value every 1min, and keeping the change rate basically the same every minute, wherein the absorbance at 0min is S0, and the absorbance at 3min is S3;
the enzyme activity calculation formula is as follows:
enzyme activity (U/ml) [ (S0-S3) × 3ml × N ]/[ kXtime (t/min) × enzyme addition (ml) ]
Wherein N is the dilution multiple of the enzyme solution.
The detection results are as follows:
| sample to be tested | Enzyme activity U/ml |
| Sst-1 | 1266 |
| Sst-2 | 277 |
| Sst-3 | 272 |
| Sst | 95 |
As can be seen, the alcohol dehydrogenase provided by the invention has the alcohol dehydrogenase activity which is at least 2-10 times enhanced compared with the activity of the wild-type alcohol dehydrogenase Sst (SEQ ID NO. 8). The conversion rate of a substrate (e.g., 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) to a product (e.g., (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) is greatly increased as compared to a wild-type alcohol dehydrogenase.
In summary, in this example, the alcohol dehydrogenase for producing atazanavir intermediate provided in this example can be used to biologically catalyze the conversion of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol into (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, the reaction conditions are mild, the equipment requirement is low, the production process does not need high temperature or cooling, the energy consumption is low, and due to the high efficiency and specific selectivity of enzyme catalysis, there is no byproduct generated in the production of (2R,3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol which is the key intermediate of atazanavir by this method, the purification is convenient; in addition, the reaction solvent is mainly water, the discharge of three wastes is low, the method is green and environment-friendly, the whole system is catalyzed by single enzyme, the concentration of the substrate is up to 160g/L, the dosage of the substrate/NAD is up to 1540:1, the cycle number of the coenzyme is high, and the reaction condition is mild.
The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.
Sequence listing
<110> Nanjing Noroton Biotechnology Ltd
<120> an alcohol dehydrogenase for the production of atazanavir intermediate
<130> 2018
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 780
<212> DNA
<213> Artificial Sequence
<400> 1
atgaccatcg ctctgaacaa cgttgttgct gttgttaccg gtgctgctgg tggtatcggt 60
cgtgaactgg ttaaagctat gaaagctgct aacgctatcg ttatcgctgc tgaaatggct 120
ccgtctgctg acaaagaagg tgctgaccac tacctgcagc acgacgttac ctctgaagct 180
ggttggaaag ctgttgctgc tctggctcag gaaaaatacg gtcgtgttga cgctctggtt 240
cacaacgctg gtatctctat cgttaccaaa ttcgaagaca ccccgctgtc tgacttccac 300
cgtgttaaca ccgttaacgt tgactctatc atcatcggta cccaggttct gctgccgctg 360
ctgaaagaag gtggtaaagc tcgtgctggt ggtgcttctg ttgttaactt ctcttctgtt 420
ggtggtctgc gtggtgctgc tttcaacgct gcttactgca cctctaaagc tgctgttaaa 480
atgctgtcta aatgcctggg tgctgaattc gctgctctgg gttacaacat ccgtgttaac 540
tctgttcacc cgggtggtat cgacaccccg atgctgggtt ctatcatgga caaatacgtt 600
gaactgggtg ctgctccgtc tcgtgaagtt gctcaggctg ctatggaaat gcgtcacccg 660
atcggtcgta tgggtcgtcc ggctgaaatg ggtggtggtg ttgtttacct gtgctctgac 720
gctgcttctt tcgttacctg caccgaattc gttatggacg gtggtttctc tcaggtttaa 780
<210> 2
<211> 259
<212> PRT
<213> Artificial Sequence
<400> 2
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Ala Glu Met Ala Pro Ser Ala Asp Lys Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 3
<211> 780
<212> DNA
<213> Artificial Sequence
<400> 3
atgaccatcg ctctgaacaa cgttgttgct gttgttaccg gtgctgctgg tggtatcggt 60
cgtgaactgg ttaaagctat gaaagctgct aacgctatcg ttatcgctgc tgaaatggct 120
aaatctgctg acaaagaagg tgctgaccac tacctgcagc acgacgttac ctctgaagct 180
ggttggaaag ctgttgctgc tctggctcag gaaaaatacg gtcgtgttga cgctctggtt 240
cacaacgctg gtatctctat cgttaccaaa ttcgaagaca ccccgctgtc tgacttccac 300
cgtgttaaca ccgttaacgt tgactctatc atcatcggta cccaggttct gctgccgctg 360
ctgaaagaag gtggtaaagc tcgtgctggt ggtgcttctg ttgttaactt ctcttctgtt 420
ggtggtctgc gtggtgctgc tttcaacgct gcttactgca cctctaaagc tgctgttaaa 480
atgctgtcta aatgcctggg tgctgaattc gctgctctgg gttacaacat ccgtgttaac 540
tctgttcacc cgggtggtat cgacaccccg atgctgggtt ctatcatgga caaatacgtt 600
gaactgggtg ctgctccgtc tcgtgaagtt gctcaggctg ctatggaaat gcgtcacccg 660
atcggtcgta tgggtcgtcc ggctgaaatg ggtggtggtg ttgtttacct gtgctctgac 720
gctgcttctt tcgttacctg caccgaattc gttatggacg gtggtttctc tcaggtttaa 780
<210> 4
<211> 259
<212> PRT
<213> Artificial Sequence
<400> 4
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Ala Glu Met Ala Lys Ser Ala Asp Lys Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 5
<211> 780
<212> DNA
<213> Artificial Sequence
<400> 5
atgaccatcg ctctgaacaa cgttgttgct gttgttaccg gtgctgctgg tggtatcggt 60
cgtgaactgg ttaaagctat gaaagctgct aacgctatcg ttatcgctgc tgaaatggct 120
aaatctgcta acaaagaagg tgctgaccac tacctgcagc acgacgttac ctctgaagct 180
ggttggaaag ctgttgctgc tctggctcag gaaaaatacg gtcgtgttga cgctctggtt 240
cacaacgctg gtatctctat cgttaccaaa ttcgaagaca ccccgctgtc tgacttccac 300
cgtgttaaca ccgttaacgt tgactctatc atcatcggta cccaggttct gctgccgctg 360
ctgaaagaag gtggtaaagc tcgtgctggt ggtgcttctg ttgttaactt ctcttctgtt 420
ggtggtctgc gtggtgctgc tttcaacgct gcttactgca cctctaaagc tgctgttaaa 480
atgctgtcta aatgcctggg tgctgaattc gctgctctgg gttacaacat ccgtgttaac 540
tctgttcacc cgggtggtat cgacaccccg atgctgggtt ctatcatgga caaatacgtt 600
gaactgggtg ctgctccgtc tcgtgaagtt gctcaggctg ctatggaaat gcgtcacccg 660
atcggtcgta tgggtcgtcc ggctgaaatg ggtggtggtg ttgtttacct gtgctctgac 720
gctgcttctt tcgttacctg caccgaattc gttatggacg gtggtttctc tcaggtttaa 780
<210> 6
<211> 259
<212> PRT
<213> Artificial Sequence
<400> 6
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Ala Glu Met Ala Lys Ser Ala Asn Lys Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 7
<211> 263
<212> PRT
<213> Artificial Sequence
<400> 7
Met Pro Leu Glu Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val
1 5 10 15
Thr Gly Ala Ala Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys
20 25 30
Ala Ala Asn Ala Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp
35 40 45
Val Glu Gly Ala Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala
50 55 60
Gly Trp Lys Ala Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val
65 70 75 80
Asp Ala Leu Val His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu
85 90 95
Asp Thr Pro Leu Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp
100 105 110
Ser Ile Ile Ile Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly
115 120 125
Gly Lys Ala Arg Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val
130 135 140
Ala Gly Leu Arg Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys
145 150 155 160
Ala Ala Val Lys Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala
165 170 175
Leu Gly Tyr Asn Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp
180 185 190
Thr Pro Met Leu Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala
195 200 205
Ala Pro Ser Arg Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro
210 215 220
Ile Gly Arg Met Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr
225 230 235 240
Leu Cys Ser Asp Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met
245 250 255
Asp Gly Gly Phe Ser Gln Val
260
<210> 8
<211> 259
<212> PRT
<213> Sphingomonas stygia
<400> 8
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
Claims (7)
1. An alcohol dehydrogenase for use in the production of an atazanavir intermediate, said alcohol dehydrogenase being selected from the group consisting of the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No. 6.
2. An alcohol dehydrogenase useful in the production of atazanavir intermediate as claimed in claim 1, wherein said alcohol dehydrogenase is in the form of a fusion protein, wherein the alcohol dehydrogenase is fused to other proteins such as by way of example and not limitation of a solubilizing tag, a purification tag and a bacterial localization signal.
3. The alcohol dehydrogenase for use in the production of atazanavir intermediate of claim 1 wherein said alcohol dehydrogenase has an alcohol dehydrogenase activity which is at least 2-10 fold greater than the activity of the wild-type alcohol dehydrogenase.
4. The alcohol dehydrogenase for use in the production of atazanavir intermediate as claimed in claim 1, wherein the coding sequence of said alcohol dehydrogenase is SEQ ID No.1, SEQ ID No.3, SEQ ID No. 5.
5. An alcohol dehydrogenase for use in the production of an atazanavir intermediate as claimed in claim 4 wherein said alcohol dehydrogenase, polynucleotide comprises codons optimized for expression in a particular type of host cell.
6. The alcohol dehydrogenase for use in the production of atazanavir intermediate of claim 4 wherein said alcohol dehydrogenase comprises a recombinant plasmid having control sequences comprising a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence and a transcription terminator.
7. The alcohol dehydrogenase useful in the production of an atazanavir intermediate of claim 4, wherein suitable promoters for directing transcription of coding sequences for a bacterial host cell include bacteriophage T5, bacteriophage T7, bacteriophage lambda, E.coli lacUV5 operon, E.coli trp operon, and E.coli tac operon.
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| CN104342412A (en) * | 2013-08-09 | 2015-02-11 | 南京朗恩生物科技有限公司 | Ketoreductase mutant for producing (S)-4-chloro-3-hydroxy ethyl butyrate |
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| CN109943542A (en) | 2019-06-28 |
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Denomination of invention: An alcohol dehydrogenase used for the production of azanavir intermediates Granted publication date: 20220712 Pledgee: Bank of China Limited Nanjing Chengdong Branch Pledgor: NANJING NUOYUN BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980013829 |