CN109943577B - Biotransformation method of anti-AIDS drug atazanavir intermediate - Google Patents

Abstract

The invention discloses a biotransformation method of an anti-AIDS drug atazanavir intermediate, which belongs to the technical field of medical biology, wherein the secondary structure and codon preference of a gene are adjusted, the length of a primer is controlled within 60base to obtain the primer, the obtained primer is dissolved by double distilled water and then added into a reaction system, a prepared PCR reaction system is amplified, the obtained DNA fragment is subjected to gel cutting and purification, the NdeI/XhoI site of pET30a is cloned by utilizing a homologous recombination method, the successfully sequenced DNA sequence is SEQ ID NO.3 and is named as Sst-2, and the corresponding amino acid sequence is SEQ ID NO. 4. The method has the advantages of mild reaction conditions, low requirements on equipment, no need of high temperature or cooling in the production process, low energy consumption, convenient purification, single enzyme catalysis in the whole system, high coenzyme cycle number and mild reaction conditions.

Description

Biotransformation method of atazanavir intermediate of anti-AIDS drug

Technical Field

The invention relates to a preparation method of an atazanavir intermediate, in particular to a biotransformation method of an atazanavir intermediate of an anti-AIDS drug, 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 virus/aids, is one of the major aids drugs in the world at present, is the most important drug required by the basic health system on the basic drug list of the world health organization, and is developed by the centuries meisha guibao company for the earliest time, and is publicly introduced in the chinese patent CN10282508C, approved to the market by the U.S. FDA in 2003, and approved to the market 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, which first proposed the biological preparation of (2R, 3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol using a novel carbonyl reductase, asymmetrically reduces (3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanone to produce (2R, 3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol in Japanese patent JP4746548B 2. 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 hours, 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 process does not give the effective reuse times of the immobilized enzyme.

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.

Disclosure of Invention

The invention mainly aims to provide a biotransformation method of an anti-AIDS drug atazanavir intermediate, the whole system uses single enzyme catalysis, the concentration of a substrate is up to 160g/L, the dosage of the substrate/NAD dosage is up to 1540:1, the coenzyme cycle number is high, and the reaction condition is mild.

The purpose of the invention can be achieved by adopting the following technical scheme:

1. a biotransformation method of an anti-AIDS drug atazanavir intermediate is characterized in that a whole gene synthesis method is adopted to adjust the secondary structure and codon preference of a gene so as to realize high expression in Escherichia coli, PrimerPremier and optiMIZER are utilized to design, Tm difference is guaranteed to be controlled within 3 ℃, the length of a primer is controlled within 60base, and the following primers are obtained:

1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

2 CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCTT

3 GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAG

4 GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACCGA

5 ACGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTGACAAAGAAGGTGCTGA

6 CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTCAGC

7 CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA

8 CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG

9 CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT

10 AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA

11 GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC

12 AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTG

13 TCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGTGGTCTGCGTGGT

14 GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA

15 TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA

16 GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG

17 GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC

18 GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC

19 CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG

20 GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT

21 CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT

22 CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC

23 GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG

24CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC

synthesizing the primers, dissolving the obtained primers by adding double distilled water, adding the obtained primers into a reaction system to ensure that the final concentration of each primer is 30nM and the final concentration of head and tail primers is 0.6 mu M, placing the prepared PCR reaction system into a Bosun XP cycler gene amplification instrument for amplification, carrying out gel cutting and purification on a DNA fragment obtained by PCR, cloning NdeI/XhoI sites of pET30a by using a homologous recombination method, selecting a single clone for sequencing, wherein 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.

2. The biotransformation method of atazanavir intermediate of anti-AIDS drug as claimed in claim 3, characterized in that the reaction system is:

2mM dNTP mix(2mM each dNTP)，5μl；

10×Pfu buffer，5μl；

Pfu DNA polymerase(10U/μl)，0.5μl；

ddH ₂ o makes the total volume of the reaction system to 50. mu.l ddH ₂ O；

The following procedure was followed for amplification in the reaction system: 30s at 98 ℃; 45s at 55 ℃; 72 ℃ for 120 s; 35 x.

The biotransformation method of the atazanavir intermediate of the anti-AIDS drug comprises the following steps:

step 1: synthesis of reference protein Cod-CK Gene sequence

Carrying out whole-gene synthesis on a coding sequence of the protein according to a sequence shown by AJM46704.1, and cloning into pET30a to obtain a control protein expression plasmid Cod-CK;

step 2: shake flask expression test

Selecting a single escherichia coli colony containing an expression vector, inoculating the escherichia coli colony into 10ml of culture medium after autoclaving, adding kanamycin, culturing overnight, taking a 1L triangular flask the next day, inoculating into the culture medium, and adding kanamycin; adding IPTG for culture, after the culture is finished, centrifuging the culture solution to collect wet thalli, washing thalli sediment twice by using distilled water, collecting thalli, and simultaneously taking a small amount of thalli to carry out SDS-PAGE detection;

and step 3: fed-batch fermentation

The fed-batch fermentation is carried out in a bioreactor controlled by a computer, a strain is inoculated for preparing a culture at the primary stage, the culture is inoculated when OD2.0 is reached, the dissolved oxygen concentration is automatically controlled at 30 percent by the cascade control of stirring rate and ventilation supply in the fermentation process, and the feeding is started when the large dissolved oxygen return is generated in the fermentation process;

and 4, step 4: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, uniformly mixing the mixture to obtain a pre-melting substrate, and adding MgCl ₂ Finally, adding NAD and crude enzyme solution Sst-1, and reacting by a shaking table;

and 5: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, and sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanolMixing the pre-melted substrate, adding MgCl ₂ Finally, NAD and crude enzyme solution Sst-2, 30C are added for shaking table reaction;

and 6: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, uniformly mixing the mixture to obtain a pre-melting substrate, and adding MgCl ₂ Finally, NAD and crude enzyme solution Sst-3, 30C are added for shaking table reaction;

and 7: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, uniformly mixing the mixture to obtain a pre-melting substrate, and adding MgCl ₂ Finally, NAD, crude enzyme liquid Cod-CK and 30C are added for shaking table reaction;

and 8: TLC detection of the product

Performing TLC detection on the reaction conversion product for 3 hours in the reaction;

and step 9: enzyme activity detection

Taking 6 centrifuge tubes, respectively marking the centrifuge tubes with numbers of 1-6, respectively adding NADH solution, then adding phosphate buffer solution into each tube, detecting and recording the value of absorbance to obtain a standard curve Y of NADH (nicotinamide adenine dinucleotide), diluting enzyme solution by pure water according to a certain multiple, taking the centrifuge tubes, sampling according to the following proportion, adding the centrifuge tubes, quickly mixing, and immediately pouring into a cuvette.

In step 2, selecting a single escherichia coli colony containing an expression vector to inoculate in 10ml of culture medium after autoclaving;

the culture medium comprises: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of potassium dihydrogen phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose;

kanamycin was added to 50mg/L, incubated overnight at 30 ℃ and 250 rpm;

taking a 1L triangular flask the next day, and carrying out the following steps: 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;

culturing at 30 deg.C until thallus OD5-6, immediately placing the triangular flask in a shaker at 25 deg.C, and culturing at 250rpm for 1 hr;

IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;

after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli;

the pellet was washed twice with distilled water, and the pellet was collected and stored at-70 ℃. Meanwhile, a small amount of thallus is taken for SDS-PAGE detection.

In step 3, fed-batch fermentation is carried out in a bioreactor controlled by a computer, the volume of the bioreactor is 15L, the working volume is 8L, the used culture medium is 24g/L of yeast extract, 12g/L of peptone, 0.4 percent of glucose, 2.31g/L of catalase phosphate and 12.54g/L of dipotassium phosphate, and the pH value is 7.0;

200ml of culture was prepared for the primary inoculum and inoculated at OD 2.0. The temperature was maintained at 37 ℃ throughout the fermentation, 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;

in the fermentation process, when the large dissolved oxygen rises, feeding materials;

the feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol, and was induced with 0.2mM IPTG when OD600 was 35.0 and wet weight was about 60 g/L.

In step 4, a magneton stirrer is placed into a 500ml three-mouth beaker, 2.7ml of toluene, 32ml of isopropanol and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-melted substrate is uniformly mixed, 1 ml of MgCl2 and 0.1 ml of MPBpH7.5 are added to make the total system about 190ml, and the pH value is adjusted to 7.5 after uniform mixing; finally, 21mg NAD and 6.4ml crude enzyme solution Sst-1, 30C are added for shaking table reaction. 200ml of reaction system; sampling and storing at 3 hours and 20 hours respectively; the reaction had completely converted the substrate within 20 hours.

In step 5, a magneton stirrer is placed into a 500ml three-mouth beaker, 2.7ml of toluene, 32ml of isopropanol and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-melting substrate is uniformly mixed, 1 ml of MgCl2 and 0.1 ml of MPBpH7.5 are added to make the total system about 190ml, the pH is adjusted to 7.5 after uniform mixing, and finally 21mg of NAD, 6.4ml of crude enzyme liquid Sst-2, 30C are added for shaking table reaction and 200ml of reaction system are obtained.

In step 6, a magneton stirrer is placed into a 500ml three-mouth beaker, 2.7ml of toluene, 32ml of isopropanol and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-melting substrate is uniformly mixed, 1 ml of MgCl2 and 0.1 ml of MPBpH7.5 are added to make the total system about 190ml, the pH value is adjusted to 7.5 after uniform mixing, and finally 21mg of NAD, 6.4ml of crude enzyme liquid Sst-3, 30C are added for shaking table reaction and 200ml of reaction system are obtained.

In step 7, a magneton stirrer is placed in a 500ml three-mouth beaker, 2.7ml of toluene, 32ml of isopropanol and 32g of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-melting substrate is uniformly mixed, 1 ml of MgCl2 and 0.1 ml of MPBpH7.5 are added to make the total system about 190ml, the pH value is adjusted to 7.5 after uniform mixing, and finally 21mg of NAD, 6.4ml of crude enzyme liquid Cod-CK, 30C are added for shaking table reaction and 200ml of reaction system are obtained.

In step 9, taking 65 ml centrifuge tubes, respectively marking 1-6, respectively adding 3mM NADH solution 0ul, 40ul, 80ul, 100ul, 120ul and 160ul, then supplementing 0.1M phosphate buffer solution with pH of 7.0 to 3ml each tube, uniformly mixing, detecting at 340nm and recording the absorbance value; from the above measurements, a standard curve Y of NADH is obtained, where Y is the value of absorbance, X is the concentration mM of NADH, and R of the curve is ² >99.5 percent; diluting the enzyme solution with pure water by a certain multiple, wherein the absorbance value per minute is properly changed by 0.02-0.04 by the dilution multiple; taking 5ml of a centrifuge tube, sampling according to the following proportion, adding the samples into the centrifuge tube, quickly mixing, and immediately pouring the mixture into a cuvette;

detecting reagent components and dosage:

isopropanol, 500 ul;

2％NAD，100uL；

100mMPBS(pH7.0)，2.35mL；

diluted enzyme solution, 50 uL;

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 dosage (ml) ]

Wherein N is the dilution multiple of the enzyme solution.

The invention has the beneficial technical effects that:

1. the biotransformation method of the anti-AIDS drug atazanavir intermediate provided by the invention can be used for converting 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol into (2R, 3S) -1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol by utilizing the enzyme under the biocatalysis; the method has the advantages of mild reaction conditions, low requirements on equipment, no need of high temperature or cooling in the production process, low energy consumption, high efficiency and specific selectivity of enzyme catalysis, no by-product generation of the key intermediate (2R, 3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol of the atazanavir and convenient purification; 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.

2. The invention provides an alcohol dehydrogenase mutant, which has the alcohol dehydrogenase activity at least 2-10 times enhanced compared with the activity of wild-type alcohol dehydrogenase, and greatly increases the conversion rate of a substrate (such as 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) to a product (such as (2R, 3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol) compared with the wild-type alcohol dehydrogenase.

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.

Example 1:

the biotransformation method of atazanavir intermediate of anti-aids drug provided in this example 1 includes the following steps:

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 Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled to within 3 ℃ and the Primer length is controlled to within 60 base:

1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

2 CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCTT

3 GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAG

4 GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACCGA

5 ACGCTATCGTTATCGCTGCTGAAATGGCTCCGTCTGCTGACAAAGAAGGTGCTGA

6 CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTCAGC

7 CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA

8 CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG

9 CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT

10 AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA

11 GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC

12 AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTGTCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGT

13 GGTCTGCGTGGT

14 GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA

15 TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA

16 GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG

17 GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC

18 GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC

19 CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG

20 GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT

21 CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT

22 CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC

23 GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG

24 CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC

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-to-tail primer 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 the homologous recombination method. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.1 and named as Sst-1, and the corresponding amino acid sequence is SEQ ID NO. 2.

Example 2:

the biotransformation method of atazanavir intermediate of anti-aids drug provided in this example 2 includes the following steps:

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 Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled to within 3 ℃ and the Primer length is controlled to within 60 base:

1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

2 CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCTT

3 GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAG

4 GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACCGA

5 ACGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTGACAAAGAAGGTGCTGA

6 CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTCAGC

7 CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA

8 CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG

9 CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT

10 AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA

11 GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC

12 AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTGTCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGT

13 GGTCTGCGTGGT

14 GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA

15 TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA

16 GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG

17 GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC

18 GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATAC

19 CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATG

20 GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT

21 CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT

22 CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC

23 GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG

24 CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC

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.3 and is named as Sst-2, and the corresponding amino acid sequence is SEQ ID NO. 4.

Example 3:

the biotransformation method of atazanavir intermediate of anti-aids drug provided in this example 3 includes the following steps:

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 Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) and ensuring that the Tm difference is controlled to within 3 ℃ and the Primer length is controlled to within 60 base:

1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCGC

2 ACCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATATGTATATCTCCT

3 TTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAGC

4 CAGCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCAGTTCACGACC

5 CGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTAACAAAGAAGGTGCTGAC

6 CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCACCTTCTTTGTTAGC

7 CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAA

8 CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCCTGAGCCAGAGCAG

9 CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCT

10 AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGCGGGGTGTCTTCGAA

11 GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGC

12 AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCGGCAGCAGAACCTGTCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGT

13 GGTCTGCGTGGT

14 GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACCACGCAGACCACCAA

15 TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAA

16 GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGCACCCAGGCATTTAG

17 GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGC

18 GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATCGGGGTGTCGATACCAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGC

19 TCAGGCTGCTATG

20 GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGCAGCCTGAGCAACTT

21 CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCT

22 CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCACAGGTAAACAACACC

23 GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAATAACTCG

24 CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAACCTGAGAGAAACC

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-to-tail primer 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.

Example 4:

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).

Example 5:

shake flask expression test

Escherichia coli single colony containing expression vector was picked and inoculated to 10ml

In the 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 overnight at 250rpm and 30 ℃. Taking a 1L triangular flask the next day, and carrying out the following steps: 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 the OD5-6 of the cells became zero, and the cells were immediately placed in a flask in 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.

Example 6 fed-batch fermentation

The fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seleng) with a reactor capacity of 15L and a working volume of 8L, using a medium of 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium 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, 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 was with 0.2mM IPTG when OD600 was approximately 35.0 (wet weight approximately 60 g/L).

Example 7 bioconversion 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 NAD and 6.4ml crude enzyme solution Sst-1, 30C are added for shaking table reaction. 200ml of reaction system. Samples were taken and stored at 3 hours and 20 hours, respectively. As can be seen from FIG. 3, the reaction had completely converted the substrate within 20 hours.

Example 8 bioconversion reactions

A magnetic stirrer was placed in a 500ml three-necked beaker, and 2.7ml of toluene, 32ml of isopropyl alcohol, and 32g of 3S-1-chloro-3-t-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the pre-melt substrate was mixed well, and then 1mM MgCl2 and 0.1M PB pH7.5 were added thereto to give a total of about 190ml, and the pH was adjusted to 7.5 after mixing well. Finally, 21mg NAD and 6.4ml crude enzyme solution Sst-2 and 30C are added for shaking table reaction. 200ml of reaction system.

Example 9 bioconversion reactions

A magnetic stirrer was placed in a 500ml three-necked beaker, and 2.7ml of toluene, 32ml of isopropyl alcohol, and 32g of 3S-1-chloro-3-t-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the pre-melt substrate was mixed well, and then 1mM MgCl2 and 0.1M PB pH7.5 were added thereto to give a total of about 190ml, and the pH was adjusted to 7.5 after mixing well. Finally, 21mg NAD and 6.4ml crude enzyme solution Sst-3 and 30C are added for shaking table reaction. 200ml of reaction system.

Example 10 bioconversion reactions

A magnetic stirrer was placed in a 500ml three-necked beaker, and 2.7ml of toluene, 32ml of isopropyl alcohol, and 32g of 3S-1-chloro-3-t-butoxycarbonylamino-4-phenyl-2-butanol were sequentially added thereto, and the pre-melt substrate was mixed well, and then 1mM MgCl2 and 0.1M PB pH7.5 were added thereto to give a total of about 190ml, and the pH was adjusted to 7.5 after mixing well. Finally, 21mg of NAD, 6.4ml of crude enzyme solution Cod-CK and 30C were added for shaking table reaction. 200ml of reaction system.

EXAMPLE 11 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 higher than the control group Cod-CK.

Example 12 enzyme Activity assay

Taking 65 ml centrifuge tubes, respectively marking 1-6, respectively adding 3mM NADH solution 0ul, 40ul, 80ul, 100ul, 120ul and 160ul, then supplementing 0.1M phosphate buffer solution with pH of 7.0 to 3ml each tube, mixing uniformly, detecting at 340nm and recording the absorbance value; 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 curve ² >99.5 percent; diluting the enzyme solution with pure water by a certain factor (reference dilution factor: 600-1000 times), wherein the dilution factor changes the light absorption value per minute by between 0.02 and 0.04, which is suitable; 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	Amount of the composition
		Isopropanol (I-propanol)	500ul
2％NAD	100uL
		100mM PBS(pH 7.0)	2.35mL
Diluted enzyme solution	50uL

Detecting the change of the absorbance at 340nm, recording a value every 1min, wherein the change rate per minute is basically the same, 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 dosage (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

Therefore, the alcohol dehydrogenase mutant 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 the present embodiment, the enzyme 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 by the biotransformation method of atazanavir intermediate, which is an anti-aids drug, and has the advantages of mild reaction conditions, low equipment requirements, no need of high temperature or cooling in the production process, low energy consumption, and no by-product generation due to high efficiency and specific selectivity of enzyme catalysis, so that the key intermediate (2R, 3S) -1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol in the production of atazanavir by the 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 Nuo cloud Biotechnology Ltd

<120> biotransformation method of atazanavir intermediate of anti-AIDS drug

<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 (8)

1. A biotransformation method of an anti-AIDS drug atazanavir intermediate is characterized in that a whole gene synthesis method is adopted to adjust the secondary structure and codon preference of a gene so as to realize high expression in escherichia coli, Primer Premier and OPTIMIZER are utilized for design, Tm difference is guaranteed to be controlled within 3 ℃, the Primer length is controlled within 60base, and the following primers are obtained:

1TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

2CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCAT

ATGTATATCTCCTT

3GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGT

GAACTGGTTAAAG

4GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACC

AGTTCACGACCGA

5ACGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTGACA

AAGAAGGTGCTGA

6CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCA

CCTTCTTTGTCAGC

7CGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCT

GGCTCAGGAAAAA

8CCAGCGTTGTGAACCAGAGCGTCAACACGACCGTATTTTTCC

TGAGCCAGAGCAG

9CTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCG

AAGACACCCCGCT

10AGTCAACGTTAACGGTGTTAACACGGTGGAAGTCAGACAGC

GGGGTGTCTTCGAA

11GTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAG

GTTCTGCTGCCGC

12AAGCACCACCAGCACGAGCTTTACCACCTTCTTTCAGCAGCG

GCAGCAGAACCTG

13TCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGGT

GGTCTGCGTGGT

14GCAGCTTTAGAGGTGCAGTAAGCAGCGTTGAAAGCAGCACC

ACGCAGACCACCAA

15TTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATG

CCTGGGTGCTGAA

16GAGTTAACACGGATGTTGTAACCCAGAGCAGCGAATTCAGC

ACCCAGGCATTTAG

17GGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATC

GACACCCCGATGC

18GCACCCAGTTCAACGTATTTGTCCATGATAGAACCCAGCATC

GGGGTGTCGATAC

19CAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGC

TCAGGCTGCTATG

20GGACGACCCATACGACCGATCGGGTGACGCATTTCCATAGC

AGCCTGAGCAACTT

21CGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGT

TTACCTGTGCTCT

22CGAATTCGGTGCAGGTAACGAAAGAAGCAGCGTCAGAGCAC

AGGTAAACAACACC

23GTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAG

GTTTAATAACTCG

24CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTAAAC

CTGAGAGAAACC

synthesizing the primers, dissolving the obtained primers by adding double distilled water, adding the obtained primers into a reaction system to ensure that the final concentration of each primer is 30nM and the final concentration of head and tail primers is 0.6 mu M, placing the prepared PCR reaction system into a Bosun XP cycler gene amplification instrument for amplification, carrying out gel cutting purification on a DNA fragment obtained by PCR, cloning into NdeI/XhoI sites of pET30a by using a homologous recombination method, selecting a single clone for sequencing, wherein 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.

2. The biotransformation method of atazanavir intermediate of anti-AIDS drug as claimed in claim 1, characterized in that the reaction system is:

2mM dNTP mix，2mM each dNTP，5μl；

10×Pfu buffer，5μl；

Pfu DNA polymerase，10U/μl，0.5μl；

ddH ₂ o to bring the total volume of the reaction system to 50. mu.l ddH ₂ O；

The following procedure was followed for amplification in the reaction system: 30s at 98 ℃; 45s at 55 ℃; 72 ℃ for 120 s; 35 x.

3. The biotransformation process of atazanavir intermediate as an anti-AIDS drug in claim 2, comprising the following steps:

step 1: synthesis of reference protein Cod-CK Gene sequence

Carrying out whole-gene synthesis on a coding sequence of the protein according to a sequence shown by AJM46704.1, and cloning into pET30a to obtain a control protein expression plasmid Cod-CK;

step 2: shake flask expression test

Selecting a single escherichia coli colony containing the expression plasmid, inoculating the single escherichia coli colony into 10ml of culture medium subjected to autoclaving, adding kanamycin, performing overnight culture, taking a 1L triangular flask the next day, inoculating into the culture medium, and adding kanamycin; adding IPTG for culture, after the culture is finished, centrifuging the culture solution to collect wet thalli, washing thalli sediment twice by using distilled water, collecting thalli, and simultaneously taking a small amount of thalli to carry out SDS-PAGE detection;

and step 3: fed-batch fermentation

The fed-batch fermentation is carried out in a bioreactor controlled by a computer, a strain is inoculated for preparing a culture at the primary stage, the culture is inoculated when OD2.0 is reached, the dissolved oxygen concentration is automatically controlled at 30 percent by the cascade control of stirring rate and ventilation supply in the fermentation process, and the feeding is started when the large dissolved oxygen return is generated in the fermentation process;

and 4, step 4: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, uniformly mixing the mixture to obtain a pre-melting substrate, and adding MgCl ₂ Finally, NAD and crude enzyme solution Sst-2 are added, and shaking table reaction is carried out at 30 ℃;

and 5: biotransformation reactions

Adding a magneton stirrer into a three-opening beaker, sequentially adding toluene, isopropanol and 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol, uniformly mixing the mixture to obtain a pre-melting substrate, and adding MgCl ₂ Finally, NAD and crude enzyme liquid Cod-CK are added, and shaking table reaction is carried out at 30 ℃;

and 6: TLC detection of the product

Performing TLC detection on the reaction conversion product for 3 hours in the reaction;

and 7: enzyme activity detection

Taking 6 centrifuge tubes, respectively marking the centrifuge tubes with numbers of 1-6, respectively adding NADH solution, then adding phosphate buffer solution into each tube, detecting and recording the value of absorbance to obtain a standard curve Y of NADH (nicotinamide adenine dinucleotide), diluting enzyme solution by pure water according to a certain multiple, taking the centrifuge tubes, sampling, adding the centrifuge tubes, rapidly mixing, and immediately pouring into a cuvette.

4. The bioconversion method of atazanavir intermediate of anti-AIDS drug as claimed in claim 3, wherein in step 2, single colony of E.coli containing said expression plasmid is picked and inoculated into 10ml of autoclaved medium;

the culture medium comprises: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of potassium dihydrogen phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose;

kanamycin was added to 50mg/L, incubated overnight at 30 ℃ and 250 rpm;

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 and 0.3g/L glucose, and kanamycin is added to 50 mg/L;

culturing at 30 deg.C until thallus OD5-6, immediately placing the triangular flask in a shaker at 25 deg.C, and culturing at 250rpm for 1 hr;

IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;

after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli;

the bacterial pellet is washed twice with distilled water, and the bacterial pellet is collected and preserved at-70 deg.c while taking small amount of bacterial pellet for SDS-PAGE detection.

5. The bioconversion method of an anti-AIDS drug, atazanavir intermediate, as claimed in claim 3, wherein in step 3, fed-batch fermentation is performed in a computer controlled bioreactor with 15L reactor capacity and 8L working volume, 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;

preparing 200ml culture by primary inoculation strain, inoculating to the whole fermentation process when OD2.0, maintaining the temperature at 37 deg.C, automatically controlling the dissolved oxygen concentration at 30% by stirring speed rpm and aeration supply cascade control during the fermentation process, and maintaining the pH value of the culture medium at 7.0 by 50% v/v orthophosphoric acid and 30% v/v ammonia water;

in the fermentation process, when the large dissolved oxygen rises, feeding materials;

the feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol, induced with 0.2mM IPTG at an OD600 of 35.0 and a wet weight of 60 g/L.

6. The method for bioconversion of the anti-AIDS drug atazanavir intermediate of claim 3, wherein in step 5, a magneton stirrer is placed in a 500ml three-mouth beaker, and 2.7ml toluene, 32ml isopropanol, 32g 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-dissolved substrate is mixed well, and 1mM MgCl is added ₂ 190ml of the total system was mixed with 0.1M PB (pH7.5), the pH was adjusted to 7.5 after mixing, and finally 21mg of NAD and 6.4ml of crude enzyme solution Sst-2 were added, followed by shaking reaction at 30 ℃ to obtain 200ml of the reaction system.

7. The bioconversion method of atazanavir intermediate as an anti-AIDS drug in claim 3, wherein in step 7, a magneton stirrer is placed in a 500ml three-mouth beaker, and 2.7ml toluene, 32ml isopropanol, 32g 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol are sequentially added, the pre-dissolved substrate is mixed well, and 1mM MgCl is added ₂ 0.1M PB pH7.5 gives the totality190ml of NAD, the pH value is adjusted to 7.5 after uniform mixing, finally 21mg of NAD and 6.4ml of crude enzyme liquid Cod-CK are added, shaking table reaction is carried out at 30 ℃, and 200ml of reaction system is obtained.

8. The bioconversion method of atazanavir intermediate as an anti-AIDS drug in claim 3, wherein in step 9, 65 ml centrifuge tubes are taken, respectively labeled 1-6, added with 3mM NADH solution 0ul, 40ul, 80ul, 100ul, 120ul and 160ul, respectively, then supplemented with 0.1M phosphate buffer solution with pH7.0 for 3ml for each tube, and after mixing, the absorbance value is detected and recorded at 340 nm; from the above measurements, a standard curve Y of NADH is obtained, where Y is the value of absorbance, X is the concentration mM of NADH, and R of the curve is ² >99.5 percent; diluting the enzyme solution with pure water by a certain multiple, wherein the absorbance value per minute is properly changed by 0.02-0.04 by the dilution multiple; taking 5ml of a centrifuge tube, sampling according to the following proportion, adding the samples into the centrifuge tube, quickly mixing, and immediately pouring into a cuvette;

detecting reagent components and dosage:

isopropanol, 500 ul;

2％NAD，100uL；

100mM PBS pH7.0，2.35mL；

diluted enzyme solution, 50 uL;

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 dosage (ml) ]

Wherein N is the dilution multiple of the enzyme solution.

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