CN109943542A - A kind of alcohol dehydrogenase for the production of atazanavir intermediate - Google Patents
A kind of alcohol dehydrogenase for the production of atazanavir intermediate Download PDFInfo
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- CN109943542A CN109943542A CN201811639906.2A CN201811639906A CN109943542A CN 109943542 A CN109943542 A CN 109943542A CN 201811639906 A CN201811639906 A CN 201811639906A CN 109943542 A CN109943542 A CN 109943542A
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- alcohol dehydrogenase
- ala
- val
- gly
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- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
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- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 125000004744 butyloxycarbonyl group Chemical group 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000002389 essential drug Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
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- 235000012208 gluconic acid Nutrition 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
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- 230000031700 light absorption Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
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- 238000004321 preservation Methods 0.000 description 1
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- 230000001177 retroviral effect Effects 0.000 description 1
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- PANBYUAFMMOFOV-UHFFFAOYSA-N sodium;sulfuric acid Chemical compound [Na].OS(O)(=O)=O PANBYUAFMMOFOV-UHFFFAOYSA-N 0.000 description 1
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- FDEMKPDAGFRQTO-LAGVYOHYSA-N tert-butyl (2s,3r)-4-amino-2-benzyl-4-chloro-3-hydroxybutanoate Chemical compound CC(C)(C)OC(=O)[C@H]([C@@H](O)C(N)Cl)CC1=CC=CC=C1 FDEMKPDAGFRQTO-LAGVYOHYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of alcohol dehydrogenase for the production of atazanavir intermediate, belong to technical field of pharmaceutical biotechnology, the alcohol dehydrogenase shows stronger catalytic activity compared with the wild type alcohol dehydrogenase of SEQ ID NO.8, alcohol dehydrogenase can be by obtaining the vitro recombination for encoding the enzyme, polynucleotides mutagenesis, DNA reorganization, fallibility PCR and directed evolution method etc., above-mentioned alcohol dehydrogenase has one or more mutation in following characteristics: T37A, D38E, P41K, D44N, V45K.Reaction condition of the present invention is mild, low for equipment requirements, and production process is not necessarily to high temperature or cooling, and low energy consumption, since enzymatic has efficiently, single-minded selectivity, therefore the key intermediate no coupling product for producing atazanavir in this way generates, purifying is convenient；Furthermore reaction dissolvent is mainly water, and three waste discharge is low, environmentally protective.
Description
Technical field
The present invention relates to a kind of preparation methods of atazanavir intermediate, more particularly to one kind among atazanavir
The alcohol dehydrogenase of body production, belongs to technical field of pharmaceutical biotechnology.
Background technique
Atazanavir is a kind of anti-for treating and preventing AIDS virus/AIDS with trade name Reyataz sale
Retroviral drugs are one of current anti-AIDS drugs main in the world, its essential drugs in the World Health Organization
It is that most important drug, Bristol-Myers Squibb Co. needed for basic health department develop atazanavir earliest on inventory,
Chinese patent CN10282508C has open introduction, and U.S. FDA ratified its listing in 2003, and China also ratified it in 2007
Listing.
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1- is intermediate as the key for preparing atazanavir
Body, main production has chemical synthesis and two kinds of biological synthesis process at present, and wherein bioanalysis can be by corresponding ketoreductase
Or the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of microbe whole-cell bioconversion 3S-1- obtains.
JP4746548B2 proposition earliest is disclosed in Japan Patent JP4746548B2 prepares (2R, 3S)-with bioanalysis
The chloro- 3- tert- butoxy carbonyl amino -4- phenyl -2- butanol of 1-, the patent are asymmetric using a kind of also first ferment of novel carbonyl
Ground restores the chloro- 3- tert- butoxy carbonyl amino -4- Phenyl 2 butanone of (3S) -1- to generate the chloro- 3- tert- fourth oxygen carbonyl of (2R, 3S) -1-
Base amino-4-phenyl -2- butanol.And other similar optical activity alcohol is disclosed in Chinese patent CN1993464B, it should
Patent is protected in China's application simultaneously and in acquirement authorization in 2011, but since the patent has used Coenzyme I INADP, market price
Lattice are expensive, limit its application to a certain extent, subsequent other companies such as U.S. codexis etc. enzyme class, reaction condition, with
And the conditions such as coenzyme circulation optimize promotion, further increase production efficiency, to reduce condition requirement and cost,
It will carry out biocatalysis after apoenzyme and NADP co-immobilization in Chinese patent CN104911224, but the technique
Reaction time too long 48h-60h, production efficiency is lower, further increases production growth；After the preparation and use of immobilised enzymes
Save routine it is cumbersome, and the technique does not provide immobilised enzymes and effectively reuses number.
Use glucose in the technique of Chinese patent CN103468757, the gluconic acid generated at the end of reaction without
Price is recycled, and then generates a large amount of solid wastes, and the technique is reacted using enzyme powder, amplification prospect of production is limited.
Since the enzyme activity of enzyme used is not high in the technique of international monopoly WO2011005527, it is catalyzed 90 grams of substrates and needs to use
To 0.3gNAD, substrate: the ratio of NAD is only 300, causes the NAD cost accounting produced in per kilogram product excessive, so that raw
High expensive is produced, saccharomyces cerevisiae has been used to carry out whole-cell catalytic, but required yeast thallus in Chinese patent CN102732579
Excessively (10g stem cell), catalysis substrate could only convert completely when being not more than 0.1mM, and the production efficiency of the technique is relatively low.
Naturally occurring wild type alcohol dehydrogenase in microorganism Sphingomonas stygia, energy will be by the chloro- 3- of 3S-1-
T-butoxycarbonyl amino -4- phenyl -2- butanol is converted into the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- fourth of (2R, 3S) -1-
Alcohol.The inventor of present disclosure has found alcohol dehydrogenase and Sphingomonas stygia including being mutated in certain position
The wild type alcohol dehydrogenase (SEQ ID NO:8) of generation is compared and shows increased catalytic activity." wild type alcohol dehydrogenase ",
" wild type ADH enzyme " and " wild type ADH alcohol dehydrogenase " refers to by having SEQ ID NO from Sphingomonas stygia:
The alcohol dehydrogenase of eight amino acid sequence.The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1- can be converted by the enzyme
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1-." wild type " refers to as found in nature
The form of material or substance.Such as the protein or nucleic acid sequence of wild type be can from the separation in nature and without
Artificial modification, the original series form present in organism." increased catalytic activity " refers in test in vitro or in vivo
It is measured compared with wild type alcohol dehydrogenase, performance is sent as an envoy to substrate (such as the chloro- 3- t-butoxycarbonyl amino -4- benzene of 3S-1-
Base -2- butanol) increase to product (such as the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1-) conversion rate
Alcohol dehydrogenase.
Summary of the invention
The main object of the present invention is to provide for a kind of alcohol dehydrogenase for the production of atazanavir intermediate, by 3S-
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 1- is converted into the chloro- 3- t-butoxycarbonyl amino -4- benzene of (2R, 3S) -1-
Base -2- butanol, in the wild type alcohol dehydrogenase that the alcohol dehydrogenase and Sphingomonas stygia of certain position mutation generate
(SEQ ID NO:8) is compared and is shown increased catalytic activity.
The purpose of the present invention can reach by using following technical solution:
A kind of alcohol dehydrogenase for the production of atazanavir intermediate, derived from the wild of Sphingomonas stygia
Type alcohol dehydrogenase, performance send as an envoy to substrate to the increased alcohol dehydrogenase of product conversion rate, and the alcohol dehydrogenase is with SEQ ID NO.8's
Wild type alcohol dehydrogenase compared to showing stronger catalytic activity, alcohol dehydrogenase can by make to encode the enzyme vitro recombination,
Polynucleotides mutagenesis, DNA reorganization, fallibility PCR and directed evolution method etc. obtain, and above-mentioned alcohol dehydrogenase has and is selected from following spy
One or more mutation in sign: T37A, D38E, P41K, D44N, V45K.
Above-mentioned alcohol dehydrogenase is selected from sequence SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6.
Several amino acid of above-mentioned alcohol dehydrogenase, C-terminal or N-terminal can be left out；
Any specific truncated analog or segment can use corresponding test to assess catalytic activity；
Additional amino acid residue can be added to one or two end without influencing catalytic activity；
Additional sequences can be functional or non-functional.
Above-mentioned alcohol dehydrogenase is the form of fusion protein, wherein alcohol dehydrogenase such as by hydrotropy label, purification tag and
The example of bacterium positioning signal rather than the mode of limitation is fused to other oroteins.
Above-mentioned alcohol dehydrogenase, alcohol dehydrogenase activity at least enhance 2-10 times than the activity of wild type alcohol dehydrogenase.
The coded sequence of above-mentioned alcohol dehydrogenase preferably is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5.
Above-mentioned alcohol dehydrogenase, polynucleotides include the password for being optimized for expressing in certain types of host cell
Son.
Above-mentioned alcohol dehydrogenase constitutes recombinant plasmid, and control sequence includes promoter, leader sequence, polyadenylation sequence
Column, propeptide sequence, signal peptide sequence and transcription terminator.
For bacterial host cell, instructing the suitable promoter of the transcription of coded sequence includes from Phage T5, phagocytosis
Body T7, bacteriophage lambda, Escherichia coli lacUV5 operon, Escherichia coli trp operon and Escherichia coli tac operon.
Advantageous effects of the invention:
It 1, can biocatalysis general using the enzyme provided by the present invention for the alcohol dehydrogenase of atazanavir intermediate production
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1- is converted into the chloro- 3- t-butoxycarbonyl amino-of (2R, 3S) -1-
4- phenyl -2- butanol；Reaction condition is mild, low for equipment requirements, and production process is not necessarily to high temperature or cooling, and low energy consumption, due to
Enzymatic has efficiently, single-minded selectivity, therefore produces chloro- uncle 3- of key intermediate (2R, 3S) -1- of atazanavir in this way
Butoxy carbonyl amino -4- phenyl -2- butanol no coupling product generates, and purifying is convenient；Furthermore reaction dissolvent is mainly water, three waste discharge
It is low, it is environmentally protective.
Detailed description of the invention
Fig. 1 is the expression plasmid map of Sst-1；
Fig. 2 is Sst-1, Sst-2, Sst-3, and Cod-CK bioconversion reacts 3 hours TLC maps, from left to right successively
For Sst-1, Sst-2, Sst-3, Cod-CK, top band is substrate, and lower section band is product；
Fig. 3 is the TLC map that Sst-1 reacts 20h.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, below with reference to examples and drawings
The present invention is described in further detail, and embodiments of the present invention are not limited thereto.
As shown in Figure 1, in the alcohol dehydrogenase provided in this embodiment for the production of atazanavir intermediate, microorganism
Naturally occurring wild type alcohol dehydrogenase in Sphingomonas stygia, energy will be by the chloro- 3- t-butoxycarbonyl amino-of 3S-1-
4- phenyl -2- butanol is converted into the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1-.Present disclosure
Inventor has found that the wild type alcohol of alcohol dehydrogenase and Sphingomonas stygia generation including being mutated in certain position is de-
Hydrogen enzyme (SEQ ID NO:8) is compared and shows increased catalytic activity." wild type alcohol dehydrogenase ", " wild type ADH enzyme " and " open country
Raw type ADH alcohol dehydrogenase " refers to be taken off by the alcohol with SEQ ID NO:8 amino acid sequence from Sphingomonas stygia
Hydrogen enzyme.The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1- can be converted into chloro- uncle 3- of (2R, 3S) -1- by the enzyme
Butoxy carbonyl amino -4- phenyl -2- butanol." wild type " refers to the form of material or substance as found in nature.
Such as the protein or nucleic acid sequence of wild type be can from nature separation and without artificial modification, in biology
Original series form present in body." increased catalytic activity " refers to measured with wild type alcohol in test in vitro or in vivo
Dehydrogenase is compared, and performance sends as an envoy to substrate (such as the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1-) to product (example
Such as the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1-) the increased alcohol dehydrogenase of conversion rate.
The present invention provides a kind of alcohol dehydrogenase, is derived from the wild type alcohol dehydrogenase of Sphingomonas stygia, performance
Send as an envoy to substrate (such as the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1-) to product (such as (2R, 3S) -1- is chloro-
3- t-butoxycarbonyl amino -4- phenyl -2- butanol) the increased alcohol dehydrogenase of conversion rate.The alcohol dehydrogenase, with SEQ ID
The wild type alcohol dehydrogenase of NO.8 is compared and shows stronger catalytic activity.Alcohol dehydrogenase and the multicore for encoding this alcohol dehydrogenase
The preparation of those skilled in the art's commonly used approach can be used in thuja acid.Alcohol dehydrogenase can be by making to encode the external of the enzyme
Recombination, polynucleotides mutagenesis, DNA reorganization, fallibility PCR and directed evolution method etc. obtain.
Above-mentioned alcohol dehydrogenase, has one or more mutation in following characteristics: T37A, D38E, P41K, D44N,
V45K。
Above-mentioned alcohol dehydrogenase preferably is selected from sequence SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6.Overall length mutation
Alcohol dehydrogenase is for keeping the catalytic activity of enzyme to be not required in that.Correspondingly, it is considered as the truncated analog of alcohol dehydrogenase
With the segment for having catalytic activity.For example, in some embodiments, several amino acid of C-terminal or N-terminal can be left out.It is any
Specific truncated analog or segment can use corresponding test to assess catalytic activity.Likewise, additional amino acid
Residue can be added to one or two end without influencing catalytic activity.Additional sequences can be functional or non-functional
Property.For example, Additional amino acid sequences can be used to assist in purifying, as label, or some other functions are executed.Cause
This, the alcohol dehydrogenase of present disclosure can be the form of fusion protein, and wherein alcohol dehydrogenase (or its segment) is such as by helping
Molten label (such as SUMO albumen), purification tag (the His label as combined metal) and bacterium positioning signal (such as secretion signal)
Example rather than limitation mode be fused to other oroteins.
The present invention provides a kind of alcohol dehydrogenase, and alcohol dehydrogenase activity at least enhances 2- than the activity of wild type alcohol dehydrogenase
10 times.
Above-mentioned alcohol dehydrogenase coded sequence preferably is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5,
It is suitable for by sequence optimisation in expression in escherichia coli.In some embodiments, polynucleotides include being optimized for
The codon expressed in certain types of host cell.The use and preference of codon for various types of microorganism
Property be known because it is the codon for the optimization of the specific amino acid of expression in these microorganisms.The present invention mentions
For a kind of recombinant plasmid, in some embodiments, control sequence includes promoter, leader sequence, Polyadenylation sequences, preceding
Peptide sequence, signal peptide sequence and transcription terminator etc..For bacterial host cell, the suitable of the transcription of coded sequence is instructed to open
Mover includes but is not limited to from Phage T5, phage t7, bacteriophage lambda, Escherichia coli lacUV5 operon, large intestine bar
Bacterium trp operon, Escherichia coli tac operon etc..
Embodiment 1:
By full genome synthetic method, the secondary structure and codon preference of gene are adjusted, to realize
High expression in Escherichia coli.
Using PrimerPremier (http://primer3.ut.ee/) and OPTIMIZER (http: //
Genomes.urv.es/OPTIMIZER/ it) is designed, and guarantees the control of Tm difference within 3 DEG C, primer length control exists
Within 60base, following primer is obtained:
It synthesizes above-mentioned primer, and after adding distilled water to dissolve the primer of acquisition, is added in following reaction system, so that respectively
The final concentration of 30nM of primer, final concentration of 0.6 μM of head and the tail primer.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | So that reaction system total volume is to 50 μ l |
Prepared PCR reaction system is placed in rich day XPcycler gene-amplificative instrament, is expanded by following procedure:
98 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 120s, 35x.It carries out the DNA fragmentation that PCR is obtained to cut glue purification, utilizes the side of homologous recombination
Method clones the site NdeI/XhoI into pET30a.Picking monoclonal is sequenced.It is SEQ ID that successful DNA sequence dna, which is sequenced,
NO.1, is named as Sst-1, and corresponding amino acid sequence is SEQ ID NO.2.
Embodiment 2:
By full genome synthetic method, the secondary structure and codon preference of gene are adjusted, to realize
High expression in Escherichia coli.
Using PrimerPremier (http://primer3.ut.ee/) and OPTIMIZER (http: //
Genomes.urv.es/OPTIMIZER/ it) is designed, and guarantees the control of Tm difference within 3 DEG C, primer length control exists
Within 60base, following primer is obtained:
It synthesizes above-mentioned primer, and after adding distilled water to dissolve the primer of acquisition, is added in following reaction system, so that respectively
The final concentration of 30nM of primer, final concentration of 0.6 μM of head and the tail primer.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | So that reaction system total volume is to 50 μ l |
Prepared PCR reaction system is placed in rich day XP cycler gene-amplificative instrament, is expanded by following procedure
Increase: 98 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 120s, 35x.It carries out the DNA fragmentation that PCR is obtained to cut glue purification, utilizes homologous recombination
Method clones the site NdeI/XhoI into pET30a.Picking monoclonal is sequenced.It is SEQ ID that successful DNA sequence dna, which is sequenced,
NO.3, is named as Sst-2, and corresponding amino acid sequence is SEQ ID NO.4.
Embodiment 3:
By full genome synthetic method, the secondary structure and codon preference of gene are adjusted, to realize
High expression in Escherichia coli.
Using PrimerPremier (http://primer3.ut.ee/) and OPTIMIZER (http: //
Genomes.urv.es/OPTIMIZER/ it) is designed, and guarantees the control of Tm difference within 3 DEG C, primer length control exists
Within 60base, following primer is obtained:
It synthesizes above-mentioned primer, and after adding distilled water to dissolve the primer of acquisition, is added in following reaction system, so that respectively
The final concentration of 30nM of primer, final concentration of 0.6 μM of head and the tail primer.
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | So that reaction system total volume is to 50 μ l |
Prepared PCR reaction system is placed in rich day XPcycler gene-amplificative instrament, is expanded by following procedure:
98 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 120s, 35x.It carries out the DNA fragmentation that PCR is obtained to cut glue purification, utilizes the side of homologous recombination
Method clones the site NdeI/XhoI into pET30a.Picking monoclonal is sequenced.It is SEQ ID that successful DNA sequence dna, which is sequenced,
NO.5, is named as Sst-3, and corresponding amino acid sequence is SEQ ID NO.6.
The synthesis of reference protein Cod-CK gene order；
The sequence according to AJM46704.1, commission Shanghai JaRa biology carry out full genome conjunction to the coded sequence of the albumen
At, and be cloned into pET30a, obtain reference protein expression plasmid Cod-CK (SEQ ID NO.7).
Shaking flask expression test
The Escherichia coli single colonie that picking contains expression vector is inoculated in the culture medium after 10ml high pressure sterilization: tryptose
Peptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate 3.55g/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride 2.68g/L, sulfuric acid
Sodium 0.71g/L, epsom salt 0.493g/L, Iron trichloride hexahydrate 0.027g/L, glycerol 5g/L, glucose 0.8g/L, addition card
That mycin is to 50mg/L.30 DEG C, 250rpm is incubated overnight.Next day takes 1L triangular flask, is linked by the inoculative proportion of 1:100
In culture medium after 100ml high pressure sterilization: tryptone 10g/L, yeast extract 5g/L, disodium hydrogen phosphate 3.55g/L, phosphoric acid
Potassium dihydrogen 3.4g/L, ammonium chloride 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, Iron trichloride hexahydrate 0.027g/
L, glycerol 5g/L, glucose 0.3g/L, addition kanamycins to 50mg/L.Culture is to thallus OD5-6 in 30 DEG C, at once by three
Angle bottle is placed in 25 DEG C of shaking tables, and 250rpm is cultivated 1 hour.Add IPTG to final concentration 0.1mM, and in 25 DEG C, 250rpm continues to train
It supports 16 hours.After culture, 20 minutes collection wet thallus are centrifuged by culture solution in 4 DEG C, under 12000g.Then by bacterial sediment
Wash with distilled water twice, thallus, -70 DEG C of preservations are collected.A small amount of thallus is taken to carry out SDS-PAGE detection simultaneously.
Fed batch fermentation
It is carried out in the bioreactor (Shanghai Guoqiang) that fed batch fermentation is controlled in computer, reactor capacity 15L,
Working volume is 8L, and used culture medium is yeast extract 24g/L, peptone 12g/L, glucose 0.4%, 2.31g/L
Biphosphate enzyme and 12.54g/L dipotassium hydrogen phosphate, pH7.0.Primary inoculation strain prepares 200ml culture, and when OD2.0 is accessed.
In entire fermentation process, temperature is maintained at 37 DEG C, and dissolved oxygen concentration is supplied by stirring rate (rpm) and ventilation in fermentation process
Cascade Mach-Zehnder interferometer is automatically controlled 30%, and the pH value of culture medium is maintained by 50% (v/v) orthophosphoric acid and 30% (v/v) ammonium hydroxide
7.0.In fermentation process, when there is significantly dissolved oxygen rise, start feed supplement.Feed solutions contain 9%w/v peptone, 9%w/
V yeast extract, 14%w/v glycerol.When OD600 is about 35.0 (weight in wet base is about 60g/L), induced with 0.2mM IPTG.
Bioconversion reaction
It is put into magneton stirrer in tri- mouthfuls of beakers of 500ml, and sequentially adds 2.7ml toluene, 32ml isopropanol, 32g3S-
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 1-, mixing melt substrate in advance, add 1mM MgCl2,0.1M PB
PH7.5 makes total system about 190ml, and pH to 7.5 is adjusted after mixing.It is eventually adding 21mg NAD, 6.4ml crude enzyme liquid Sst-1,30C
Shaking table reaction.200ml reaction system.Respectively at 3 hours, sampling in 20 hours is saved.From the figure 3, it may be seen that reaction is complete at 20 hours
Full conversion of substrate.
Bioconversion reaction
It is put into magneton stirrer in tri- mouthfuls of beakers of 500ml, and sequentially adds 2.7ml toluene, 32ml isopropanol, 32g3S-
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 1-, mixing melt substrate in advance, add 1mM MgCl2,0.1M PB
PH7.5 makes total system about 190ml, and pH to 7.5 is adjusted after mixing.It is eventually adding 21mg NAD, 6.4ml crude enzyme liquid Sst-2,30C
Shaking table reaction.200ml reaction system.
Bioconversion reaction
It is put into magneton stirrer in tri- mouthfuls of beakers of 500ml, and sequentially adds 2.7ml toluene, 32ml isopropanol, 32g3S-
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 1-, mixing melt substrate in advance, add 1mM MgCl2,0.1M PB
PH7.5 makes total system about 190ml, and pH to 7.5 is adjusted after mixing.It is eventually adding 21mg NAD, 6.4ml crude enzyme liquid Sst-3,30C
Shaking table reaction.200ml reaction system.
Bioconversion reaction
It is put into magneton stirrer in tri- mouthfuls of beakers of 500ml, and sequentially adds 2.7ml toluene, 32ml isopropanol, 32g3S-
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 1-, mixing melt substrate in advance, add 1mM MgCl2,0.1M PB
PH7.5 makes total system about 190ml, and pH to 7.5 is adjusted after mixing.It is eventually adding 21mg NAD, 6.4ml crude enzyme liquid Cod-CK, 30C
Shaking table reaction.200ml reaction system.
TLC detects product
3 hours reaction converted products in above-described embodiment are subjected to TLC detection, as a result as shown in Figure 2.It can be seen that Sst-1,
Sst-2, Sst-3 reaction speed are all larger than control group Cod-CK.
Enzyme activity assay
6 5ml centrifuge tubes are taken, label 1-6, is separately added into NADH solution 0ul, 40ul, 80ul, the 100ul of 3mM respectively,
120ul, 160ul, then every pipe adds 0.1M, and the phosphate buffer of pH7.0 supplies 3ml, detects simultaneously at 340nm after mixing
Record the value of absorbance；According to above-mentioned measured value, the standard curve Y=k*X of NADH is obtained, wherein Y is the value of absorbance, and X is
The concentration (mM) of NADH, the R of curve2> 99.5%；(extension rate: 600- is referred to by certain multiple dilution enzyme solution with pure water
1000 times), diluted multiple makes between light absorption value variation 0.02-0.04 per minute to be suitable；5ml centrifuge tube is taken, by comparing below
Centrifuge tube is added in example sampling, mixes rapidly, pours into cuvette immediately.
| Detection reagent | Dosage |
| Isopropanol | 500ul |
| 2%NAD | 100uL |
| 100mM PBS(pH7.0) | 2.35mL |
| Enzyme solution after dilution | 50uL |
The variation that absorbance is detected at 340nm records a value every 1min, and change rate per minute is essentially identical,
Wherein the absorbance of 0min is S0, and the absorbance of 3min is S3；
Enzyme activity calculation formula:
Enzyme activity (U/ml)=[(S0-S3) * 3ml*N]/[k × time (t/min) × enzyme concentration (ml)]
Wherein N is the extension rate of enzyme solution.
Testing result is as follows:
| To test sample | Enzyme activity U/ml |
| Sst-1 | 1266 |
| Sst-2 | 277 |
| Sst-3 | 272 |
| Sst | 95 |
As it can be seen that alcohol dehydrogenase provided by the invention, alcohol dehydrogenase activity is than wild type alcohol dehydrogenase Sst (SEQ ID
NO.8 activity) at least enhances 2-10 times.Compared with wild type alcohol dehydrogenase, make substrate (such as the chloro- 3- tertiary butyloxycarbonyl of 3S-1-
Base amino-4-phenyl -2- butanol) to product (such as the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of (2R, 3S) -1-)
Conversion rate is significantly increased.
In conclusion in the present embodiment, the alcohol dehydrogenase provided in this embodiment for the production of atazanavir intermediate,
Using the enzyme can biocatalysis the chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol of 3S-1- is converted into (2R, 3S) -1-
Chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol, reaction condition is mild, low for equipment requirements, and production process is not necessarily to high temperature
Or it is cooling, low energy consumption, since enzymatic has efficiently, single-minded selectivity, therefore produced in the key of atazanavir in this way
The chloro- 3- t-butoxycarbonyl amino -4- phenyl -2- butanol no coupling product of mesosome (2R, 3S) -1- generates, and purifying is convenient；Furthermore it reacts
Solvent is mainly water, and three waste discharge is low, environmentally protective, and whole system is up to 160g/L, substrate using single enzymatic, concentration of substrate
Dosage/NAD dosage is up to 1540:1, and coenzyme cycle-index is high, and reaction condition is mild.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Sequence table
<110>Nanjing Nuoyun Biological Technology Co., Ltd.
<120>a kind of 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 (9)
1. a kind of alcohol dehydrogenase for the production of atazanavir intermediate, which is characterized in that it is derived from
The wild type alcohol dehydrogenase of Sphingomonasstygia, performance send as an envoy to substrate to the increased alcohol dehydrogenase of product conversion rate,
The alcohol dehydrogenase shows stronger catalytic activity compared with the wild type alcohol dehydrogenase of SEQ ID NO.8, and alcohol dehydrogenase can be with
By obtaining the vitro recombination for encoding the enzyme, polynucleotides mutagenesis, DNA reorganization, fallibility PCR and directed evolution method etc., on
Alcohol dehydrogenase is stated, there are one or more mutation in following characteristics: T37A, D38E, P41K, D44N, V45K.
2. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as described in claim 1, which is characterized in that above-mentioned alcohol
Dehydrogenase is selected from sequence SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6.
3. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 2, which is characterized in that above-mentioned alcohol
Several amino acid of dehydrogenase, C-terminal or N-terminal can be left out；
Any specific truncated analog or segment can use corresponding test to assess catalytic activity；
Additional amino acid residue can be added to one or two end without influencing catalytic activity；
Additional sequences can be functional or non-functional.
4. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 2, which is characterized in that above-mentioned alcohol
Dehydrogenase is the form of fusion protein, and wherein alcohol dehydrogenase such as passes through hydrotropy label, purification tag and bacterium positioning signal
Example rather than limitation mode be fused to other oroteins.
5. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 2, which is characterized in that above-mentioned alcohol
Dehydrogenase, alcohol dehydrogenase activity at least enhance 2-10 times than the activity of wild type alcohol dehydrogenase.
6. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as described in claim 1, which is characterized in that above-mentioned alcohol
The coded sequence of dehydrogenase preferably is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5.
7. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 6, which is characterized in that above-mentioned alcohol
Dehydrogenase, polynucleotides include the codon for being optimized for expressing in certain types of host cell.
8. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 6, which is characterized in that above-mentioned alcohol
Dehydrogenase constitutes recombinant plasmid, and control sequence includes promoter, leader sequence, Polyadenylation sequences, propeptide sequence, signal
Peptide sequence and transcription terminator.
9. a kind of alcohol dehydrogenase for the production of atazanavir intermediate as claimed in claim 6, which is characterized in that for thin
Bacterium host cell, instructing the suitable promoter of the transcription of coded sequence includes from Phage T5, phage t7, bacteriophage
Lambda, Escherichia coli lacUV5 operon, Escherichia coli trp operon and Escherichia coli tac operon.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690696A (en) * | 2020-07-07 | 2020-09-22 | 南京朗恩生物科技有限公司 | Method for preparing darunavir intermediate through biocatalysis |
| CN113816836A (en) * | 2021-09-29 | 2021-12-21 | 山东寰酶生物制药有限公司 | Enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342406A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Thermostability enhanced formate dehydrogenase mutant and preparation method thereof |
| CN104342411A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Activity enhanced ketoreductase mutant, coding sequence and preparation method thereof |
| 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 |
| CN104342410A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Ketone reductase mutant and preparation method thereof |
-
2018
- 2018-12-29 CN CN201811639906.2A patent/CN109943542B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342406A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Thermostability enhanced formate dehydrogenase mutant and preparation method thereof |
| CN104342411A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Activity enhanced ketoreductase mutant, coding sequence and preparation method thereof |
| CN104342410A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Ketone reductase mutant and preparation method thereof |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 无: "MULTISPECIES: SDR family oxidoreductase [Novosphingobium]", 《GENBANK:WP_011906790.1》 * |
| 郑昆: "阿扎那韦手性中间体（3S）-3-（叔丁氧羰基）氨基-1-氯-4-苯基-（2R）-丁醇的酶法制备研究", 《中国优秀硕士论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690696A (en) * | 2020-07-07 | 2020-09-22 | 南京朗恩生物科技有限公司 | Method for preparing darunavir intermediate through biocatalysis |
| CN111690696B (en) * | 2020-07-07 | 2021-12-31 | 南京朗恩生物科技有限公司 | Method for preparing darunavir intermediate through biocatalysis |
| CN113816836A (en) * | 2021-09-29 | 2021-12-21 | 山东寰酶生物制药有限公司 | Enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol |
| CN113816836B (en) * | 2021-09-29 | 2024-05-03 | 山东睿鹰制药集团有限公司 | Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol |
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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 |
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