CN109295019A - A kind of Alcohol dehydrogenase mutant and its application - Google Patents
A kind of Alcohol dehydrogenase mutant and its application Download PDFInfo
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Abstract
The invention discloses a kind of Alcohol dehydrogenase mutant and its application, which is following (1) and/or (2)；(1) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine, methionine or cysteine；(2) the 202nd alanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine or valine.The present invention is transformed the alcohol dehydrogenase LkADH from Kefir grains lactobacillus using half design and rational, obtains multiple Alcohol dehydrogenase mutants.The Alcohol dehydrogenase mutant has the characteristics that high enzyme activity, can chloro- 3, the 5- dicarbapentaborane hecanoic acid t-butyl ester of efficient catalytic 6- prepare the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of (S) -6-, be of great significance to the production of statins drug midbody.
Description
Technical field
The present invention relates to technical field of enzyme engineering more particularly to a kind of Alcohol dehydrogenase mutant and its applications.
Background technique
(S) the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of -6- ((S)-CHOH) is very important chiral intermediate, mainly
For synthesizing HMG-CoA reductase inhibitor, the product of further asymmetric reduction can be through cyano, hydroxyl protection on dehalogenation
And etc. synthesis Atorvastatin calcium (Lipitor), can also by hydroxyl protection, acylation and hydrolysis and etc. synthesize auspicious easypro cut down
Statin calcium (can be determined), and the two is whole world blood lipid-lowering medicine salable.
Currently, mainly synthesizing (S)-CHOH by two kinds of synthetic routes, one is using 4- chloro- 3- carbonyl ethyl butyrate the bottom of as
Object, it is anti-by chemical method or bioanalysis asymmetric syntheses (S) -4- chloro-3-hydroxyl ethyl butyrate, then by a step Claisen condensation
It answers, the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of generation (S) -6- is reacted with tert-butyl acetate；Another method is then chloro- from 6-
3,5- dicarbapentaborane hecanoic acid t-butyl esters (CDOH) set out, and carry out asymmetric reduction to the carbonyl on its position 5- using carbonyl reductase,
Generate (S)-CHOH.
Wherein, route 2 not only reduces step chemical reaction, and enzymatic process reaction condition temperature in reaction step
With product is easily isolated, and either economically or environmental, route 2 all has more potentiality than route 1.However, at present
Industrial major part prepares (S)-CHOH using route 1, and on the low side to the research of route 2.Wherein lack high-performance bio catalysis
Agent is to hinder the bottleneck problem of the route trend industrialization.
In order to enable enzyme to be preferably applied to industrial production, researchers take different molecular modification methods to improve
The catalytic activity of enzyme.According to the difference of library construction principle, can be divided into directed evolution, design and rational and half design and rational these three
Strategy.The directed evolution technologies of enzyme are not required to the relationship it is to be understood that between the structure and reaction mechanism of enzyme, applied widely.But in reality
Since the mutated library of its building is larger in the operation of border, high-throughput screening method is depended on.The design and rational and directed evolution of enzyme
Technology is different, the advantage that this method has purpose strong, high-efficient.Design and rational need in the space structure that fully understands enzyme and
On the basis of catalytic mechanism, the structure and function of enzyme is comprehensively analyzed, then navigates to and carries out essence in crucial structure
True regulation, to obtain desired mutant.And half design and rational is then combined with the advantage of above two enzyme renovation technique
A kind of method.Half design and rational is by the information in Protein Data Bank and identified by means such as computer simulations can
For the key amino acid site of molecular modification, mutation number is reduced, screening operation amount is reduced, improves the success of enzyme molecule transformation
Rate.
Currently, only finding that 3 kinds of alcohol dehydrogenase can be catalyzed CDOH asymmetric reduction and obtain (S)-CHOH.The first comes from
The NADPH dependent form alcohol dehydrogenase LbADH of Lactobacillus brevis DSM 20054；It is derived from for second
The alcohol dehydrogenase LkADH of Lactobacillus kefir DSM 20587, the two have on amino acid sequence 88.6% it is similar
Degree；And the third is the two o'clock of LkADH of the applicant in the patent (application number CN 201410810512.4) of authorization in 2017
Mutant A202L/L199H.For improve the screening efficiency present invention using structure-based " half design and rational " method to its into
Row transformation, has obtained the Alcohol dehydrogenase mutant that enzyme activity significantly improves.
Summary of the invention
The present invention provides a kind of Alcohol dehydrogenase mutant and its application, which has the spy of high enzyme activity
Point, can chloro- 3, the 5- dicarbapentaborane hecanoic acid t-butyl ester of efficient catalytic 6- prepare the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of (S) -6-, it is right
The production of statins drug midbody is of great significance.
Specific technical solution is as follows:
The present invention provides a kind of Alcohol dehydrogenase mutant, the Alcohol dehydrogenase mutant is following (1) and/or (2)；
(1) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, different bright ammonia
Acid, methionine or cysteine；
(2) the 202nd alanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine
Or valine.
Preferably, the Alcohol dehydrogenase mutant is by the 147th benzene of amino acid sequence shown in SEQ ID NO.1
Alanine replaces isoleucine, and the 202nd alanine replaces with leucine；
Also, by one or more mutation in the site as described in (A)~(C) of amino acid sequence shown in SEQ ID NO.1
It is combined；
(A) the 145th glutamic acid is replaced with into leucine；
(B) the 190th tyrosine is replaced with into phenylalanine；
(C) the 96th serine is replaced with into alanine.
It is further preferred that the Alcohol dehydrogenase mutant is by the 147th phenylpropyl alcohol of amino acid sequence shown in SEQ ID NO.1
Propylhomoserin replaces with isoleucine, and the 202nd alanine replaces with leucine, and the 190th tyrosine replaces with phenylalanine,
96th serine replaces with alanine, and the 145th glutamic acid replaces with leucine.
Specifically, it is one of the following that the present invention, which is mutated the Alcohol dehydrogenase mutant obtained:
(I) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, different bright ammonia
Acid, methionine or cysteine；Gained single-point mutants are successively named as F147L, F147I, F147M, F147C；
(II) the 202nd alanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine
Or valine；Gained single-point mutants are successively named as A202L, A202I, A202V:
(III) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, the 202nd
The alanine of position replaces with leucine, is named as F147L/A202L；
Or, the 147th phenylalanine replaces with isoleucine, the 202nd alanine replaces with leucine, is named as
F147I/A202L；
Or, the 147th phenylalanine replaces with isoleucine, the 202nd alanine replaces with isoleucine, name
For F147I/A202I；
Or, the 147th phenylalanine replaces with methionine, the 202nd alanine replaces with isoleucine, name
For F147M/A202I；
Or, the 147th phenylalanine replaces with methionine, the 202nd alanine replaces with valine, is named as
F147M/A202V；
Or, the 147th phenylalanine replaces with isoleucine, the 202nd alanine replaces with valine, is named as
F147I/A202V；
Or, the 147th phenylalanine replaces with cysteine, the 202nd alanine replaces with valine, is named as
F147C/A202V。
(IV) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced into isoleucine, the 202nd
The alanine of position replaces with leucine, and the 145th glutamic acid replaces with leucine；Three point mutation body of gained is named as F147I/
A202L/E145L；
(V) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the 202nd
The alanine of position replaces with leucine, and the 190th tyrosine replaces with phenylalanine；Three point mutation body of gained is named as
F147I/A202L/Y190F；
(VI) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the
202 alanine replace with leucine, and the 96th serine replaces with alanine；Three point mutation body of gained is named as
F147I/A202L/S96A；
(VII) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the
202 alanine replace with leucine, and the 190th tyrosine replaces with phenylalanine, and the 96th serine replaces with
Alanine；Four point mutation body of gained is named as F147I/A202L/Y190F/S96A；
(VIII) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the
202 alanine replace with leucine, and the 190th tyrosine replaces with phenylalanine, and the 145th glutamic acid replaces with
Leucine；Four point mutation body of gained is named as F147I/A202L/Y190F/E145L；
(IX) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the
202 alanine replace with leucine, and the 96th serine replaces with alanine, and the 145th glutamic acid replaces with bright
Propylhomoserin；Four point mutation body of gained is named as F147I/A202L/S96A/E145L；
(X) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into isoleucine, the 202nd
The alanine of position replaces with leucine, and the 190th tyrosine replaces with phenylalanine, and the 96th serine replaces with the third ammonia
Acid, the 145th glutamic acid replace with leucine；Five point mutation body of gained is named as F147I/A202L/Y190F/S96A/
E145L。
The present invention is based on half Rational design methods of structure, to from Kefir grains lactobacillus (Lactobacillus
Kefir DSM 20587), amino acid sequence wild type alcohol dehydrogenase as shown in SEQ ID NO.1 (NCBI accession number:
AY267012.1；Gene size: 759bp) it is transformed, obtain the Alcohol dehydrogenase mutant that enzyme activity significantly improves.
The present invention provides a kind of encoding genes of Alcohol dehydrogenase mutant.
The present invention also provides a kind of expression vectors comprising the encoding gene.
The present invention also provides a kind of genetic engineering bacteriums comprising the encoding gene.
The present invention also provides the Alcohol dehydrogenase mutants or the genetic engineering bacterium in the catalysis chloro- 3,5- dicarbapentaborane of 6-
Hecanoic acid t-butyl ester prepares the application in the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of (S) -6-.
Specifically, the application, comprising: in the presence of co-factor and hydrogen donor, with Alcohol dehydrogenase mutant or gene
Engineering bacteria is catalyst, chloro- 3, the 5- dicarbapentaborane hecanoic acid t-butyl ester of catalysis substrate 6-, the reaction generation chloro- 5- hydroxyl -3- carbonyl of (S) -6-
Base hecanoic acid t-butyl ester.
Preferably, the co-factor is NADPH；Hydrogen donor is isopropanol；The temperature of the reaction is 18~30 DEG C, pH
It is 5.0~7.0.
Compared with prior art, the invention has the following advantages:
The present invention is transformed the alcohol dehydrogenase LkADH from Kefir grains lactobacillus using half design and rational, obtains
The Alcohol dehydrogenase mutant that multiple enzyme activity improve.Wherein, mutant F147L/A202L specific enzyme activity is 18U/mg, F147I/
A202L specific enzyme activity is 23U/mg, is 31.6 times and 40.3 times of protoenzyme LkADH (0.57U/mg) respectively.Combination mutant
F147I/A202L/Y190F/S96A, specific enzyme activity 44U/mg are 77 times of protoenzyme LkADH (0.57U/mg).
Detailed description of the invention
Fig. 1 is alcohol dehydrogenase M in embodiment 1F147I-A202LHomology model configuration schematic diagram.
Fig. 2 is alcohol dehydrogenase MF147I-A202LKey amino acid near catalytic activity pocket.
Specific embodiment
The present invention is described further With reference to embodiment.Wherein, engineering involved in the following example
Bacterium building, enzyme inducing expression and isolate and purify and the measurement of enzyme activity is all made of following method, it is specific as follows:
1, the building of wild type alcohol dehydrogenase recombinant bacterium
According to the gene order design primer of alcohol dehydrogenase, PCR amplification is carried out with primers F-LkADH/R-LkADH to obtain
Target gene.
The sequence of primers F-LkADH are as follows: 5 '-CGCGGATCCATGACTGATCGTTAAAAG-3 '；
The sequence of primer R-LkADH are as follows: 5 '-CCGCTCGAGCTTATTGAGCAGTGTATC CAC-3 '.
PCR reaction system are as follows:
PCR program are as follows: 98 DEG C of 2min；98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 10s (general 5s/kb), 30 circulations；72℃
5min；4 DEG C of heat preservations.
It is purified back after the verifying that the size of PCR product passes through 1% agarose gel electrophoresis with PCR purification kit
It receives.By after purification PCR product and plasmid pET30a (+) carry out double digestion with restriction enzyme BamH I and Xho I respectively.
Sample gel extraction after nucleic acid agarose gel electrophoresis isolates and purifies after digestion carries out enzyme company after measuring respective concentration
Reaction.
Connection product is transformed into e. coli bl21 (DH5 α) competent cell using heat shock method and sends to platinum and is still given birth to
Object technology (Shanghai) Co., Ltd.-Hangzhou sequencing portion sequencing.Recombinant plasmid is extracted from the correct positive transformant of sequence verification
It is transformed into e. coli bl21 (DE3), finally obtains recombinant bacterium.
2, the building of saltant type alcohol dehydrogenase recombinant bacterium
Using recombinant plasmid Pet-30a (+)-LkADH for being connected with alcohol dehydrogenase gene as template, design a pair of comprising mutation
Forward and reverse primer (being specifically shown in embodiment 1) in site carries out PCR, obtains recombination.
Wherein, the reaction system of PCR are as follows:
PCR program: 1) 95 DEG C of preheating 2min；2) 98 DEG C of denaturation 10s；3) 58 DEG C of annealing 15s；4) 72 DEG C of extension 1min；
5) it recycles 30 times；6) 72 DEG C of 3min are re-extended；7) 4 DEG C of heat preservation 2h.
Purification and recovery is carried out after PCR product is correct after the verifying of agarose gel electrophoresis.With restriction enzyme Dpn
I digests PCR product after purification, removes the template plasmid for having methyl.
Digestion system are as follows: 10 μ L of DNA, 2 Buffer μ L, 1 μ L of Dpn I, 7 μ L of deionized water.37 DEG C of 2h, after digestion
Mixture carries out purification and recovery with PCR purification kit.
Finally, purified product is transferred in e. coli bl21 (DE3) competent cell using heat shock method, sequencing is correct
Single colonie as constructs successful muton.
3, the inducing expression of alcohol dehydrogenase with isolate and purify
Picking recombinant bacterium (wild type alcohol dehydrogenase gene engineering bacteria and saltant type alcohol dehydrogenase gene engineering bacteria) single colonie
(contain 50 μ g/mL kanamycins) in 5mL LB liquid medium, after growing 8-10h, connects seed fermentation liquid by 2% inoculum concentration
Into the 50mL LB culture medium containing 50 μ g/mL kanamycins (1% peptone, 0.5% yeast extract, 1% sodium chloride,
PH7.0), revolving speed 200rpm, 37 DEG C of culture about 2-3h (OD600 reaches 0.6-0.8), is added the isopropyl of final concentration of 0.5mM
Thio-β-D- galactoside, induces 14h under 30 DEG C, 200rpm.
Bacterium solution is centrifuged and abandons supernatant, with supernatant is abandoned in centrifugation again after pure water washing wet thallus, is eventually adding a certain amount of phosphorus
Wet thallus is resuspended sour potassium buffer (pH 7.5), and the bacterium solution for being concentrated 10 times is placed in ice-water bath and carries out the broken born of the same parents of ultrasound.It will surpass
The broken broken cytosol of sound is centrifuged, and supernatant is taken to carry out affinity chromatography with Ni column, and with miscellaneous buffer is washed, (20mM potassium phosphate is slow
Fliud flushing, 0.5M NaCl, 50mM imidazoles, pH 7.5) impurity is washed away, with elution buffer (20mM kaliumphosphate buffer, 0.5M
NaCl, 250mM imidazoles, pH 7.5) elution destination protein, the target protein separated is placed in ultra-filtration centrifuge tube and is taken off
Salt concentration, obtains electrophoretically pure target protein.
4, the enzyme activity determination of alcohol dehydrogenase recombinant bacterium
Reaction system is 1mL, and including 1.0mM NADPH, 10mM substrate CDOH (is pre-dissolved in 50%v isopropanol),
Citrate-phosphate disodium hydrogen-NaOH the buffer of pH7 and suitable enzyme, 30 DEG C, oscillating reactions in 600rpm metal bath
20min, then 12,000rpm is centrifuged 2min and (when being reacted with broken cytosol, is added except dereaction thallus terminates full cell effect
0.5mL acetonitrile terminates reaction, and liquid phase detects after filtering), HPLC detection product absorbs the size of peak area after taking supernatant to filter.
Quantitative analysis is carried out to product (S)-CHOH using liquid chromatography.
Column model is PntulipsTM QS-C18 (5 μ m 4.6mm × 250mm).Detection wavelength 225nm, mobile phase
Flow velocity is 1.0mL/min, and acetonitrile: 10mM sodium acetate solution (pH=7)=55:50 (v/v), sample volume are 20 μ L, column temperature 30
℃.Enzyme activity reaction system is depending on experiment demand, after terminating reaction, by the concentration control for the product for needing to detect in detection model
In enclosing.
Enzyme-activity unit (U) is defined as enzyme amount needed for generating 1 μm of ol (S)-CHOH per minute.
Embodiment 1
1, half design and rational of alcohol dehydrogenase LkADH
1.1 147 and 202 rite-directed mutagenesis
(1) F147L, F147I, F147M, F147C, A202L, A202I, A202V mutant
Using recombinant plasmid Pet-30a (+)-LkADH for being connected with alcohol dehydrogenase LkADH gene as template, a pair of of packet is designed
Forward and reverse primers F-Primer/R-Primer (table 1,2) containing mutational site, 147 and 202 are pinpointed by PCR
Mutation.
1 147 site rite-directed mutagenesis primer sequence of table
2 202 site rite-directed mutagenesis primer sequence of table
(2) acquisition of F147L/A202L, F147I/A202L mutant:
To be connected with recombinant plasmid Pet-30a (+)-LkADH (A202L) of alcohol dehydrogenase LkADH (A202L) gene as mould
It is prominent to carry out fixed point by PCR for plate, a pair of forward and reverse primers F-Primer/R-Primer (table 3) comprising mutational site of design
Become.
Table 3 147,202 Sites Combination mutant primer sequences
As shown in table 4, to 147 and 202 progress rite-directed mutagenesis, a series of enzyme activity of mutant is respectively alcohol dehydrogenase
6.40-49.9 times of LkADH.
Table 4 147,202 site mutants and its opposite enzyme activity
1.2 designs and transformation based on structure knowledge
Three-dimensional structure from the alcohol dehydrogenase LkADH of Kefir grains lactobacillus has been parsed, and F147I/A202L is
The two o'clock mutant of LkADH, other mutant also only have the difference of a small amount of amino acid compared with LkADH, and sequence homology is higher than
99%, therefore alcohol dehydrogenase LkADH (PDB ID:4rf2) is selected to be modeled.
Molecular docking is carried out with DS software.By the protein structure file of the resulting F147I/A202L mutant of Blast search
It is directed respectively into DS with the substrate molecule file handled well, water, hydrotreating reservation is carried out to albumen, and retain single subunit and use
In docking.
Using the CDOCKER method in DS software by CDOH to the activated centre for tapping into enzyme, with whether with Ser143With
Tyr156Hydrogen bond is formed to screen the pose of generation.If there are multiple pose to meet, the highest pose of score is selected to do subsequent optimization.
According to molecular docking as a result, discovery substrate be in appended hydrophobic pocket shown in Fig. 2, enzyme and Binding Capacity mouth
The amino acid variation of bag portion position can cause large effect to catalytic pocket, and the amino acid chosen around catalytic activity pocket carries out
Saturation mutation.
Due to lacking effective high-throughput screening method, therefore choose carbonyl (chloromethane cardinal extremity) on 5 carbon of substrate
Site I144, E145, L153 and Y190, G189 carry out fixed point saturation mutation.After determining mutational site, building is corresponding one by one
Muton.
To be connected with recombinant plasmid pET-30a (+)-F147I/A202L of alcohol dehydrogenase gene F147I/A202L as mould
Plate carries out full plasmid PCR.Mutation is introduced purpose by primer by a pair of forward and reverse primer (table 5) comprising mutational site of design
In segment.
Table 5 I144, E145, L153 and Y190, G189 rite-directed mutagenesis primer sequence
Codon at underscore need to be only substituted for for each site takes pair of primers, when being mutated into other amino acid pair
Answer the codon of amino acid.
Purification and recovery is carried out after PCR product is correct after the verifying of agarose gel electrophoresis.With restriction enzyme Dpn
I digests PCR product after purification, removes the template plasmid for having methyl.
Digestion system are as follows: 10 μ L of DNA, 2 Buffer μ L, 1 μ L of Dpn I, 7 μ L of deionized water.37 DEG C of 2h, after digestion
Mixture carries out purification and recovery with PCR purification kit.Purified product is finally transferred to by e. coli bl21 using heat shock method
(DE3) in competent cell, it is to construct successful muton that correct single colonie, which is sequenced,.
E145, L153, Y190 are mutated into 57 mutant of other 19 kinds of amino acid respectively, and I144 is mutated into other 12 kinds
12 mutant, the G189 of amino acid be mutated into other 11 kinds of amino acid 11 mutant (NDT codon coding include F,
L, 12 kinds of amino acid including I, V, Y, H, N, D, C, R, S, G.) after repeated screening is verified, find F147I/A202L/
The enzyme activity of E145L, F147I/A202L/Y190F are improved to some extent.Enzyme activity is respectively 71.2 Hes of alcohol dehydrogenase LkADH
132 times.
6 145,190,96 site mutant of table and its opposite enzyme activity
Since amino acid is more near substrate pocket and at substrate channels, therefore utilize Discovery Studio's
Calculation Mutation Energy (binding) module calculates each site mutation into the mutation energy of alanine.Choosing
6 are gone out and rite-directed mutagenesis are carried out to site M141A, I191A, S96A, M206A, N90A, P188A that affinity is improved.
After being sequenced correctly, to M141A, I191A, S96A, M206A, N90A, P188A, this 6 mutant carry out enzyme activity
Measurement finds that the enzyme activity of S96A is significantly improved after repeated screening is verified；Enzyme activity is 89.8 times of alcohol dehydrogenase LkADH.
3, the combination of mutant
For the enzyme activity for further increasing mutant, obtained forward mutation assay body is combined by we.Primer sequence is as follows:
F-S96A:5 '-GGGATTGCCGTTGCCAAAAGCGTTGAAGAC-3 '；
R-S96A:5 '-GTCTTCAACGCTTTTGGCAACGGCAATCCC-3 '；
F-E145L:5 '-ATGAGCAGTATTCTGGGGATTGTAGGCGAT-3 '；
R-E145L:5 '-ATCGCCTACAATCCCCAGAATACTGCTCAT-3 '；
The mutation of S96A is introduced into mutant F147I/A202L/Y190F and mutant F147I/ by full plasmid PCR
In A202L/E145L, four point mutation body F147I/A202L/Y190F/S96A and F147I/A202L/E145L/S96A are obtained；It will
The mutation of E145L is introduced into mutant F147I/A202L/Y190F, obtains four point mutation body F147I/A202L/Y190F/
E145L；The mutation of S96A is introduced into mutant F147I/A202L/Y190F/E145L and obtains five point mutation body F147I/
A202L/Y190F/E145L/S96A。
7 combination mutant of table and its enzyme activity
Finally, obtaining the highest combination mutant F147I/A202L/Y190F/S96A of enzyme activity.F147I/A202L/
Specific enzyme activity (U/mg) of the Y190F/S96A at 30 DEG C is 44, is 2.44 times of alcohol dehydrogenase F147L-A202L specific enzyme activity, and alcohol is de-
77 times of hydrogen enzyme LkADH specific enzyme activity.
Sequence table
<110>Zhejiang University
<120>a kind of Alcohol dehydrogenase mutant and its application
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 252
<212> PRT
<213>Kefir grains lactobacillus (Lactobacillus kefir)
<400> 1
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 2
<211> 759
<212> DNA
<213>Kefir grains lactobacillus (Lactobacillus kefir)
<400> 2
atgactgatc gtttaaaagg caaagtagca attgtaactg gcggtacctt gggaattggc 60
ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120
gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180
caacacgatg cttctgatga agccggctgg actaagttgt ttgatacgac tgaagaagca 240
tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300
gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgtcttcttc 360
ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420
atgtcatcta tcgaaggttt tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480
ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540
gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600
ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660
aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720
gcagaattcg ttgtcgatgg tggatacact gctcaataa 759
<210> 3
<211> 27
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
cgcggatcca tgactgatcg ttaaaag 27
<210> 4
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
ccgctcgagc ttattgagca gtgtatccac 30
<210> 5
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
agcagtattg aggggctggt aggcgatccg 30
<210> 6
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
cggatcgcct accagcccct caatactgct 30
<210> 7
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 7
agcagtattg aggggatagt aggcgatccg 30
<210> 8
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 8
cggatcgcct actatcccct caatactgct 30
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 9
agcagtattg aggggatggt aggcgatccg 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 10
cggatcgcct accatcccct caatactgct 30
<210> 11
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 11
agcagtattg aggggtgcgt aggcgatccg 30
<210> 12
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 12
cggatcgcct acgcacccct caatactgct 30
<210> 13
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 13
gatgatcttg aaggtctcga ggaaatgatg tca 33
<210> 14
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 14
tgacatcatt tcctcgagac cttcaagatc atc 33
<210> 15
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 15
gatgatcttg aaggtatcga ggaaatgatg tca 33
<210> 16
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 16
tgacatcatt tcctcgatac cttcaagatc atc 33
<210> 17
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 17
gatgatcttg aaggtgtcga ggaaatgatg tca 33
<210> 18
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 18
tgacatcatt tcctcgacac cttcaagatc atc 33
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 19
agcagtattg aggggctggt aggcgatccg 30
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 20
cggatcgcct accagcccct caatactgct 30
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 21
agcagtattg aggggatagt aggcgatccg 30
<210> 22
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 22
cggatcgcct actatcccct caatactgct 30
<210> 23
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 23
atgagcagta ttaacgggat tgtaggcgat 30
<210> 24
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 24
atcgcctaca atcccgttaa tactgctcat 30
<210> 25
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 25
ggcgatccga cgttcggggc atacaacgct 30
<210> 26
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 26
agcgttgtat gccccgaacg tcggatcgcc 30
<210> 27
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 27
gtacatccgg gcttcatcaa gaccccgctg 30
<210> 28
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 28
cagcggggtc ttgatgaagc ccggatgtac 30
<210> 29
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 29
aatatgagca gtgctgaggg gattgtaggc 30
<210> 30
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 30
gcctacaatc ccctcagcac tgctcatatt 30
<210> 31
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 31
acagtacatc cgctgtatat caagaccccg 30
<210> 32
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 32
cggggtcttg atatacagcg gatgtactgt 30
<210> 33
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 33
gggattgccg ttgccaaaag cgttgaagac 30
<210> 34
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 34
gtcttcaacg cttttggcaa cggcaatccc 30
<210> 35
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 35
atgagcagta ttctggggat tgtaggcgat 30
<210> 36
<211> 30
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 36
atcgcctaca atccccagaa tactgctcat 30
Claims (9)
1. a kind of Alcohol dehydrogenase mutant, which is characterized in that the Alcohol dehydrogenase mutant is following (1) and/or (2)；
(1) the 147th phenylalanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine, first
Methyllanthionine or cysteine；
(2) the 202nd alanine of amino acid sequence shown in SEQ ID NO.1 is replaced with into leucine, isoleucine or figured silk fabrics
Propylhomoserin.
2. Alcohol dehydrogenase mutant as described in claim 1, which is characterized in that the Alcohol dehydrogenase mutant is by SEQ ID
147th phenylalanine of amino acid sequence shown in NO.1 replaces isoleucine, and the 202nd alanine replaces with bright ammonia
Acid；
Also, one or more mutation in the site as described in (A)~(C) of amino acid sequence shown in SEQ ID NO.1 are carried out
Combination；
(A) the 145th glutamic acid is replaced with into leucine；
(B) the 190th tyrosine is replaced with into phenylalanine；
(C) the 96th serine is replaced with into alanine.
3. Alcohol dehydrogenase mutant as described in claim 1, which is characterized in that the Alcohol dehydrogenase mutant is by SEQ ID
147th phenylalanine of amino acid sequence shown in NO.1 replaces with isoleucine, and the 202nd alanine replaces with bright ammonia
Acid, the 190th tyrosine replace with phenylalanine, and the 96th serine replaces with alanine, and the 145th glutamic acid replaces
It is changed to leucine.
4. a kind of encoding gene of the Alcohol dehydrogenase mutant as described in any one of claims 1 to 3.
5. a kind of expression vector comprising encoding gene described in claim 4.
6. a kind of genetic engineering bacterium comprising encoding gene described in claim 4.
7. Alcohol dehydrogenase mutant as claimed in any one of claims 1 to 3 or genetic engineering bacterium as claimed in claim 6 are being urged
Change the chloro- 3,5- dicarbapentaborane hecanoic acid t-butyl ester of 6- and prepares the application in the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester of (S) -6-.
8. the use as claimed in claim 7 characterized by comprising in the presence of co-factor and hydrogen donor, with alcohol dehydrogenase
Enzyme mutant or genetic engineering bacterium are catalyst, chloro- 3, the 5- dicarbapentaborane hecanoic acid t-butyl ester of catalysis substrate 6-, reaction generation (S) -6-
Chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester.
9. application as claimed in claim 8, which is characterized in that the co-factor is NADPH；Hydrogen donor is isopropanol；It is described
The temperature of reaction is 18~30 DEG C, and pH is 5.0~7.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941115A (en) * | 2021-03-30 | 2021-06-11 | 宿迁盛基医药科技有限公司 | Preparation method of ticagrelor chiral intermediate |
| CN113528606A (en) * | 2021-07-22 | 2021-10-22 | 湖州颐盛生物科技有限公司 | Method for preparing 17 beta-hydroxysteroid through enzyme catalysis |
| WO2022036662A1 (en) * | 2020-08-21 | 2022-02-24 | 浙江华睿生物技术有限公司 | Method for enzymatic synthesis of 3-hydroxybutyrate |
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| WO2009046153A4 (en) * | 2007-10-01 | 2009-06-25 | Codexis Inc | Ketoreductase polypeptides for the production of azetidinone |
| CN103642765A (en) * | 2013-12-25 | 2014-03-19 | 南京工业大学 | Alcohol dehydrogenase mutant and application thereof |
| CN104531627A (en) * | 2014-12-23 | 2015-04-22 | 浙江大学 | Carbonyl reductase, engineering strain and application of carbonyl reductase |
| CN105861457A (en) * | 2016-05-26 | 2016-08-17 | 无锡佰翱得生物科学有限公司 | Enzyme-activity-improved ethanol dehydrogenase mutant and preparing method and application thereof |
| CN106929490A (en) * | 2017-01-25 | 2017-07-07 | 华东理工大学 | A kind of carbonyl reductase, mutant and its application in statin synthetic intermediate is prepared |
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2018
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| WO2009046153A4 (en) * | 2007-10-01 | 2009-06-25 | Codexis Inc | Ketoreductase polypeptides for the production of azetidinone |
| CN103642765A (en) * | 2013-12-25 | 2014-03-19 | 南京工业大学 | Alcohol dehydrogenase mutant and application thereof |
| CN104531627A (en) * | 2014-12-23 | 2015-04-22 | 浙江大学 | Carbonyl reductase, engineering strain and application of carbonyl reductase |
| CN105861457A (en) * | 2016-05-26 | 2016-08-17 | 无锡佰翱得生物科学有限公司 | Enzyme-activity-improved ethanol dehydrogenase mutant and preparing method and application thereof |
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| WO2022036662A1 (en) * | 2020-08-21 | 2022-02-24 | 浙江华睿生物技术有限公司 | Method for enzymatic synthesis of 3-hydroxybutyrate |
| CN112941115A (en) * | 2021-03-30 | 2021-06-11 | 宿迁盛基医药科技有限公司 | Preparation method of ticagrelor chiral intermediate |
| CN112941115B (en) * | 2021-03-30 | 2024-08-16 | 江苏阿尔法集团盛基药业(宿迁)有限公司 | Ticagrelor chiral preparation method of intermediate |
| CN113528606A (en) * | 2021-07-22 | 2021-10-22 | 湖州颐盛生物科技有限公司 | Method for preparing 17 beta-hydroxysteroid through enzyme catalysis |
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| CN109295019B (en) | 2021-01-12 |
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