CN106636020A - Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application - Google Patents
Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application Download PDFInfo
- Publication number
- CN106636020A CN106636020A CN201610902479.7A CN201610902479A CN106636020A CN 106636020 A CN106636020 A CN 106636020A CN 201610902479 A CN201610902479 A CN 201610902479A CN 106636020 A CN106636020 A CN 106636020A
- Authority
- CN
- China
- Prior art keywords
- mutant
- short
- chain dehydrogenase
- amino acid
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a mutant of short-chain dehydrogenase, a recombinant expression vector, a genetic engineering bacterium, a preparation method of the mutant and application of the mutant or the genetic engineering bacterium generating the mutant in asymmetrically reducing a series of prochiral ketones to prepare optical homochiral alcohol. The chiral alcohol prepared by asymmetric reduction of the mutant of the short-chain dehydrogenase or the genetic engineering bacterium containing the mutant has high catalytic activity, and high-optical-purity chiral alcohol (ee>99%) can be synthesized.
Description
Technical field
The invention belongs to technical field of enzyme engineering, and in particular to a kind of molecular modification of short-chain dehydrogenase, while being related to phase
The recombinant expression carrier and recombinant expression transformants of enzyme gene, and above-mentioned mutant enzyme or the weight containing mutant enzyme should be mutated
Group cell prepares the application in optical homochiral alcohol in a series of prochiral ketones of asymmetric reduction.
Background technology
Chiral alcohol is the optically active compounds that a class is connected with hydroxyl in chiral carbon.Hydroxyl can be converted into easily various
The property of other functions group makes chiral alcohol become one of most important chiral building block.It is extensive with optically active chiral alcohol
For synthesis of chiral medicine, fine chemicals and agricultural chemicals etc..The asymmetric reduction of prochiral ketone is to prepare optical activity
The important method of chiral alcohol, substrate ketone 100% can be converted in theory the chiral alcohol of single enantiomer, with very high industry
Using value.Wherein, living things catalysis prochiral ketone asymmetric reduction synthesis of chiral alcohol because with theoretical yield it is high, it is selective it is good,
Accessory substance is few and becomes the optimization approach of chiral alcohol green syt the advantages of gentle reaction condition.
In the enzyme with catalytic asymmetric reduction prochiral ketone activity, short-chain dehydrogenase because its catalytic substrate spectrum is wide,
Heat endurance is good and the features such as strong organic solvent tolerance and receives much attention.It is prepared by the reduction of short-chain dehydrogenase enzymatic prochiral ketone
The existing many reports of high-optical-purity chiral alcohol.However, most of biocatalysts are catalyzed prochiral ketone asymmetric reduction process
Follow Prelog regular, it then follows the biocatalyst of anti-Prelog rule catalysis prochiral ketone reduction is relatively fewer.This will limit
It is made as the chiral drug synthesis preparation of important intermediate anti-Prelog chiral alcohol and pushing away for living things catalysis green synthesis techniques
Extensively.The early stage screening of this seminar obtains one plant of bacterial strain that can be catalyzed prochiral ketone reduction with anti-Prelog stereoselectivities
Empedobacter brevis ZJUY-1401 (Empedobacter brevis ZJUY-1401), in being preserved in China typical culture collection
The heart, deposit number CCTCC NO:M 2014520 (A of patent publication No. CN 105316250)；And excavate simultaneously from its genome
Clonal expression follows the short-chain dehydrogenase EbSDR8 (patents that anti-Prelog rule highly-solid selectivelies reduce prochiral ketone
The A of publication number CN 105238768).But, the catalysis activity of the short-chain dehydrogenase far from meeting industrialization production requirements, because
This, it is necessary to the catalytic efficiency of the enzyme is improved fully to excavate its using value by correlation technique.
(3) content of the invention
It is an object of the invention to provide mutant and its recombination engineering of the short-chain dehydrogenase EbSDR8 of enzyme activity raising etc.,
Synthesis for chiral alcohol provides strong biocatalyst.
To achieve these goals, the present invention is employed the following technical solutions：
A first aspect of the present invention provides the mutant of the short-chain dehydrogenase EbSDR8 that catalysis activity is significantly improved, and these are dashed forward
Variant is built on the basis of short-chain dehydrogenase EbSDR8 amino acid sequences shown in SEQ ID No.2, the short-chain dehydrogenase
In nucleotide sequence such as sequence table (patent publication No. CN 105238768A) shown in SEQ ID No.1.The mutant be
Containing the simple point mutation or many in following several sites on the basis of short-chain dehydrogenase EbSDR8 amino acid sequences shown in SEQ ID No.2
Point combinatorial mutagenesis：94 glycine (Gly94), 145 hyte propylhomoserins (His145), 153 serines (Ser153), 188 junket
Propylhomoserin (Tyr188) and 193 leucines (Leu193).
Preferably, the mutant is enterprising in two sites of Gly94 and Ser153 of short-chain dehydrogenase EbSDR8 respectively
Row replaces, and it is furthermore preferred that with more excellent catalysis activity above-mentioned two preferred sites are replaced simultaneously when.
Preferably, the mutant of the short-chain dehydrogenase EbSDR8 is respectively：It is alanine by 94 glycine mutations
(Gly94Ala, i.e. G94A), its amino acid sequence shown in SEQ ID No.4 is constituted；It is bright by 153 mutant serines
Propylhomoserin (Ser153Leu, i.e. S153L), its amino acid sequence shown in SEQ ID No.6 is constituted；And above-mentioned two is preferred
The combinatorial mutagenesis (Gly94Ala/Ser153Leu, i.e. G94A/S153L) of single-point mutants has more excellent catalysis activity, and it is
Amino acid sequence shown in SEQ ID No.8 is constituted.
It is any that disappearance is passed through to amino acid in above-mentioned variant amino acid sequence, one or several amino acid are inserted or replace
And with short-chain dehydrogenase enzymatic activity, still fall within protection scope of the present invention.
A second aspect of the present invention provides the nucleotide sequence of above-mentioned short-chain dehydrogenase enzyme mutant.Wherein mutant G94A cores
In nucleotide sequence such as sequence table shown in SEQ ID No.3, its encoding amino acid sequence is as shown in sequence table SEQ ID No.4；It is prominent
In variant S153L nucleotide sequences such as sequence table shown in SEQ ID No.5, its encoding amino acid sequence such as sequence table SEQ ID
Shown in No.6；In mutant G94A/S153L nucleotide sequences such as sequence table shown in SEQ ID No.7, its encoding amino acid sequence
As shown in sequence table SEQ ID No.8.
As it is known by the man skilled in the art, the nucleotide sequence of the short-chain dehydrogenase enzyme mutant of the present invention can also be coding
Other any nucleotide sequences of the protein of the composition of amino acid shown in sequence table.
Any replacement that one or more nucleotides are carried out to shown mutant nucleotide sequence, disappearance or insertion process are obtained
The nucleotide sequence for obtaining, as long as it has more than 90% homology with nucleotides, belongs to protection scope of the present invention.
A third aspect of the present invention provides a kind of nucleotide sequence of the short-chain dehydrogenase enzyme mutant gene comprising the present invention
Recombinant expression carrier.These recombinant vectors can pass through this area conventional method by the short-chain dehydrogenase enzyme mutant nucleosides of the present invention
Acid sequence is connected to built-up on various carriers.The carrier can be the conventional various carriers in this area, such as various plasmids, bite
Thalline or viral vector etc., preferred pET-30a.
A fourth aspect of the present invention provides a kind of genetic engineering bacterium of expression restructuring short-chain dehydrogenase enzyme mutant, and can pass through will
The recombinant expression carrier of the present invention converts into host microorganism acquisition.Described host microorganism can be each of this area routine
Host microorganism is planted, as long as meeting recombinant expression carrier can stablize self-replacation and entrained short-chain dehydrogenase of the invention
Mutant gene can be with effective expression.Preferably Escherichia coli of the invention, more preferably E. coli BL21 (DE3).
A fifth aspect of the present invention provides a kind of preparation method of restructuring short-chain dehydrogenase enzyme mutant, comprises the steps：
The recombinant expression transformants of the culture present invention, induction obtains restructuring short-chain dehydrogenase mutant protein.Wherein, described culture weight
Group expression transformant used by culture medium can be this area can make transformants grew and produce the present invention short-chain dehydrogenase dash forward
The culture medium of misfolded proteins, preferred LB culture mediums：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.2.Culture
Method and condition of culture are not particularly limited, as long as enabling transformant to grow and producing short-chain dehydrogenase mutant protein i.e.
Can.It is preferred that following methods：Recombination bacillus coli according to the present invention is seeded in the LB culture mediums containing kanamycins and is cultivated, when
When optical density OD600 of nutrient solution reaches 0.5～0.7, in final concentration of 0.1～1.0mM isopropyl-beta D-thios galactopyranosyl
Under the induction of glucosides (IPTG), you can the restructuring short-chain dehydrogenase mutant protein of the high efficient expression present invention.
It is latent in asymmetry catalysis that a sixth aspect of the present invention provides the short-chain dehydrogenase enzyme mutant or its genetic engineering bacterium
Chiral ketone prepares the application in optical activity chirality alcohol.
Specifically, described application is：With prochiral ketone (I) as substrate, the short-chain dehydrogenase enzyme mutant or its restructuring
Cell, with NADH or NADPH as coenzyme, at 20～50 DEG C, in conversion reaction system a that the buffer solution of pH 5.5～10.5 is constituted
Middle reaction, after reaction completely, reactant liquor is isolated and purified and obtains corresponding product.
Wherein, R1And R2For hydrogen, alkyl, haloalkyl, halogen, alkoxyl and nitro.
Specific R1And R2Can be-H ,-CH3,-CH2CH3,-CH2CH2CH3,-CH2X ,-CX3,-X ,-OCH3,-
OCH2CH3；X represents F, Cl, Br, OH, NO2。
Described reaction condition can be as used by this area normal condition selected.
Further, initial substrate concentration is 5～1000mmol/L in the transformation system a.
Further, recombinating in the transformation system a, preferably concentration is the pure enzyme of short-chain dehydrogenase enzyme mutant in reactant liquor
0.1～2.0mg/mL.The quality consumption of thalline is calculated as 1～400g/L with thalline weight in wet base in the transformation system a.
Further, the transformation system a also include organic solvent, by substrate, catalyst, organic solvent and pH 5.5～
10.5 buffer solution constitutes transformation system b, and organic solvent accounts for transformation system b cumulative volumes 1～20%, in transformation system b at the beginning of substrate
Beginning concentration is 5～1000mmol/L, and the quality consumption of thalline is calculated as 10～400g/L with thalline weight in wet base.
Further, the reaction is carried out in the buffer solution of pH 6.5～10.5.
Further, reaction system can also add 1～15% alcohol or sugar as auxiliary substrate, can significantly improve the vigor of reaction
And stereoselectivity.The auxiliary substrate includes but is not limited to one of following：1. ethanol, 2. isopropanol, 3. glucose, 4. sucrose
Deng.
Further, the auxiliary substrate in reaction system is isopropanol.
Further, the concentration of isopropanol is 10% in reaction system.
Further, the conversion reaction solution isolation and purification method is：After reaction terminates, conversion reaction solution is centrifuged, is taken
Clear liquid is extracted with isopyknic ethyl acetate, the organic layer as crude product containing corresponding chiral alcohol, and crude product purification is obtained into corresponding
Chiral alcohol.The method of the crude product purification is known in the art technology, usually organic solvent extraction, chromatographic isolation and absorption point
From etc..
Transformation system a of the present invention and transformation system b are transformation system, for ease of distinguishing different step transformation system
Composition it is different and name, letter itself does not have implication.
The beneficial effects are mainly as follows：The invention provides the short-chain dehydrogenase that catalysis activity is significantly improved is dashed forward
Variant and its nucleotide sequence, and the recombinant expression carrier and recombinant expression transformants containing corresponding mutant gene, pass through
These short-chain dehydrogenase enzyme mutants or the recombinant cell asymmetric reduction containing corresponding mutant protein can prepare high-optical-purity
Chiral alcohol；It is prepared by heretofore described short-chain dehydrogenase enzyme mutant or the recombinant cell asymmetric reduction containing mutant protein
Chiral alcohol has high catalytic activity, can synthesize high-optical-purity chiral alcohol (ee>99%).Catalyst is easily prepared, reaction bar
Part is gentle, substrate wide adaptability, environmental friendliness, and its recombinant cell can be in the body containing isopropanol reaction of not additional any coenzyme
The asymmetric reduction of efficient catalytic prochiral ketone in system, with good industrial applications DEVELOPMENT PROSPECT.
(4) illustrate
Fig. 1 is short-chain dehydrogenase EbSDR8 and its mutant isolates and purifies rear SDS-PAGE figures.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The structure of mutant
With the oligonucleotide fragment containing catastrophe point as primer (table 1), using QuickChangeTM methods
(Stratagene, La Jolla, CA) expands the pET-30a recombinant plasmids containing short-chain dehydrogenase EbSDR8 genes.
The mutation construction primer of table 1
aUnderscore is denoted as mutational site
PCR reaction systems：5 × PrimerSTAR buffer (Mg2+plus), 5 μ L；DNTPs (each 2.5mM), 2.0 μ L；
Upstream primer (10 μM), 1.0 μ L；Downstream primer (10 μM), 1.0 μ L；Recombinant plasmid template, 15ng；PrimerSTAR
PolymeraseTM HS (2.5U/ μ L), 0.5 μ L；Plus ddH2O is 25 μ L to cumulative volume.
PCR programs：(1) 98 DEG C, 1min；(2) 98 DEG C, 10s；(3) 55 DEG C, 10s；(4) 72 DEG C, 7min.Step (2)-(4)
4 DEG C are cooled to after circulating 20 times.
PCR primer is once purged, using the restriction enzyme DpnI of specific recognition methylation sites digested with
Degraded template plasmid.Endonuclease reaction system and condition：The PCR primer of the cleaned process of 17 μ L, 2.0 μ 10 × buffer solutions of L, 1.0 μ
L restriction enzyme DpnI, 37 DEG C of insulation 1h.
The PCR primer that above-mentioned Jing digestions are processed is converted into e. coli bl21 (DE3), large intestine of being recombinated accordingly
Bacillus, coats the flat board containing kanamycins, and overnight incubation at 37 DEG C, random picked clones carry out bacterium colony PCR identifications and sequencing
Checking, as a result shows the recombinant expression carrier successful conversion containing short-chain dehydrogenase enzyme mutant gene to expressive host E.coli
In BL21 (DE3).It is final to obtain mutant G94A, S153L and G94A/S153L nucleotide sequence sequencing result respectively such as sequence
In table shown in SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, the such as sequence table of corresponding encoded protein amino acid sequence
Shown in middle SEQ ID No.4, SEQ ID No.6 and SEQ ID No.8.
Embodiment 2：The abduction delivering of short-chain dehydrogenase enzyme mutant
The engineering bacteria that embodiment 1 builds is seeded in the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, and 37 DEG C were cultivated
Night, then be inoculated in the 50mL LB culture mediums containing 50 μ g/mL kanamycins with 1% inoculum concentration (v/v), 37 DEG C, 200rpm cultures
To cell concentration OD600 to 0.6 or so, the IPTG of final concentration of 0.1mM, after 26 DEG C of Fiber differentiation 6h, 4 DEG C, 8000rpm are added
Centrifugation 10min collects thallines, it is standby in -80 DEG C of storages.
Embodiment 3：Short-chain dehydrogenase enzyme mutant is isolated and purified
The somatic cells that embodiment 2 is collected are suspended in 10mL Na2HPO4-NaH2PO4In buffer solution (100mM, pH 8.0),
Vibration to shake up and crush (effective time 8min) under rearmounted ultrasonic wave.It is broken that broken liquid removes cell in 12,000rpm centrifugation 10min
Piece, collects supernatant (crude enzyme liquid) and isolates and purifies for the follow-up of enzyme.Purification column is Ni-NTA, and packed column volume is 5mL, is first used
Loading level pad (20mM sodium phosphates, 500mM NaCl and 20mM imidazoles, pH 7.4) balances Ni-NTA posts, with 5mL/min
Speed loading crude enzyme liquid, with loading level pad elute to remove unadsorbed albumen, finally with elution buffer (20mM
T sodium phosphates, 500mM NaCl and 500mM imidazoles, pH 7.4) wash-out collection target protein.Enzyme liquid is carried out with HiTrap desalting columns
Desalination, desalination buffer solution is Na2HPO4-NaH2PO4(100mM, pH 7.5) buffer solution, the pure enzyme liquid of gained is standby in 4 DEG C of storages.
Enzyme liquid after purification is analyzed with SDS-PAGE, and SDS-PAGE electrophoresis is shown in Fig. 1, and passage 1 is albumen Maker；Passage 2 is
The pure enzymes of EbSDR8；Passage 3 is the pure enzymes of mutant G94A；Passage 4 is the pure enzymes of mutant S153L；Passage 5 is mutant G94A/
The pure enzymes of S153L.As a result show Jing Ni-NTA affinity chromatographys, obtain electrophoretically pure restructuring short-chain dehydrogenase EbSDR8 and its mutation
Body.
Embodiment 4：The ratio of short-chain dehydrogenase EbSDR8 and its mutant is lived
Detect NADH extinctions value changes at 340nm to calculate carbonyl reduction enzyme activity by spectrophotometer.Enzyme activity list
Position (U) definition be：Enzyme amount at 30 DEG C, needed for 1 μm of ol NADH oxidation of catalysis per minute；Had by every milligram of albumen than work
Some enzyme activity (U/mg).Reaction system is (1.0mL)：0.3mM NADH, 100 μ L isopropanols, Na2HPO4-NaH2PO4Buffer solution
(100mM, pH 7.5) and appropriate pure enzyme.Substrate be respectively acetophenone 1a, 4 '-fluoro acetophenone 1b, 4 '-chloro-acetophenone 1c, 4 '-
The trifluoro-benzene second of bromoacetophenone 1d, 4 '-methyl acetophenone 1e, 4 '-methoxyacetophenone 1f, 4 '-acetophenone 1g, 2,2,2-
The chloro- 4 '-fluoro acetophenone 1i of ketone 1h, 2-, 3 '-chloro- 4 '-fluoro acetophenone 1j, 2,3 ', 4 '-trichloroacetophenone 1k, 2,2 ', 4 '-trichlorine
Acetophenone 1l.Wild type EbSDR8 and its mutant catalytic phase answer substrate more as shown in table 2 than living and stereoselectivity.
The EbSDR8 of table 2 and its mutant catalysis activity
Embodiment 5：The kinetic parameter of short-chain dehydrogenase enzyme mutant EbSDR8 and its mutant
At the standard conditions, enzyme activity determination is carried out by the concentration of substrate in change reaction system, according to double works reciprocal
Figure method calculates corresponding kinetic constant.Substrate used and its concentration are as follows in kinetic constant calculating：Acetophenone 1a (0～
10mM), 4 '-fluoro acetophenone 1b (0～10mM), 4 '-chloro-acetophenone 1c (0～1.0mM), 4 '-bromoacetophenone 1d (0～0.5mM),
4 '-methyl acetophenone 1e (0～0.5mM), 4 '-methoxyacetophenone 1f (0～0.2mM), 4 '-acetophenone 1g (0～
0.2mM), the chloro- 4 '-fluoro acetophenone 1i (0～0.5mM) of 2,2,2- trifluoroacetophenone 1h (0～0.3mM), 2-, 3 '-chloro- 4 '-fluorine
Acetophenone 1j (0～1.5mM), 2,3 ', 4 '-trichloroacetophenone 1k (0～1.0mM), 2,2 ', 4 '-trichloroacetophenone 1l (0～
1.0mM).Wild type EbSDR8 and its mutant catalytic phase answer the apparent kinetics parameter of substrate as shown in table 3.
The EbSDR8 of table 3 and its mutant asymmetric reduction prochiral ketone apparent kinetics parameter
Embodiment 5：Short-chain dehydrogenase enzyme mutant EbSDR8 and its mutant G94A/S153L conversion high concentrations 2,2,2- tri-
Fluoro acetophenone
Reaction system (10.0mL)：0.4g wet thallus cells, variable concentrations 2,2,2- trifluoroacetophenones, 1.0mL isopropanols,
9.0mL Na2HPO4-NaH2PO4Buffer solution (100mM, pH 7.5) and appropriate pure enzyme.In 35 DEG C, react under the conditions of 200rpm.
Wild type EbSDR8 recombination bacillus coli whole-cell catalytic activity is significantly lower than mutant G94A/S153L, after reaction 1h, mutation
Body G94A/S153L whole-cell catalytic reaction yields reach 95.2%, and wild type EbSDR8 reaction group yield is only 69.9%；
Under similarity condition, the time required to wild type EbSDR8 and the mutant G94A/S153L whole-cell catalytics reaction yields reach 99%
Respectively 5.5 and 1.5h.When concentration of substrate reaches 800mM, the full cell of the mutant remains to effective catalytic reaction to be carried out, reaction
9h, yield reaches 95.1%, and the concentration of product reaches 760.7mM, and the mutant catalysis high concentration substrate still shows when converting
Good stereoselectivity, product ee values keep more than 99%.The full cells of mutant G94A/S153L can be without external source coenzyme
In the case of addition, realize that coenzyme is circulated as auxiliary substrate using isopropanol, be effectively catalyzed the trifluoroacetophenone of high concentration 2,2,2- not
Asymmetric reduction, shows that the whole-cell biocatalyst has wide industrial applications prospect.
Claims (9)
1. a kind of mutant of short-chain dehydrogenase, it is characterised in that：Mutant is in short-chain dehydrogenase shown in SEQ ID No.2
Simple point mutation containing following site or multi-point combination mutation on the basis of EbSDR8 amino acid sequences：94 glycine, 145
Hyte propylhomoserin, 153 serines, 188 tyrosine and 193 leucines.
2. the mutant of short-chain dehydrogenase according to claim 1, it is characterised in that the mutant is in SEQ ID
94 glycine and the one of site of 153 serines are entered on the basis of the amino acid sequence of short-chain dehydrogenase EbSDR8 shown in No.2
Row replaces or two sites are replaced simultaneously.
3. the mutant of short-chain dehydrogenase according to claim 2, it is characterised in that the mutant is in SEQ ID
Two sites on 94 glycine and 153 serine sites on the basis of the amino acid sequence of short-chain dehydrogenase EbSDR8 shown in No.2
Replaced simultaneously.
4. the mutant of short-chain dehydrogenase according to claim 2, it is characterised in that the amino acid sequence of the mutant
For SEQ ID No.4,94 glycine mutations are alanine.
5. the mutant of short-chain dehydrogenase according to claim 2, it is characterised in that the amino acid sequence of the mutant
For SEQ ID No.6,153 mutant serines are leucine.
6. the mutant of short-chain dehydrogenase according to claim 2, it is characterised in that the amino acid sequence of the mutant
For SEQ ID No.8,94 glycine mutations are alanine, and 153 mutant serines are leucine.
7. recombinant expression carrier, it is characterised in that the recombinant expression carrier is comprising described in coding claim 1-6 any one
Amino acid sequence nucleotide sequence.
8. genetic engineering bacterium, it is characterised in that the genetic engineering bacterium by the recombinant expression carrier belonging to claim 7 convert to
Obtain in host microorganism.
9. the mutant of the short-chain dehydrogenase as described in claim 1-6 any one or the gene work as belonging to claim 8
Journey bacterium prepares the application in optical activity chirality alcohol in asymmetry catalysis prochiral ketone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902479.7A CN106636020A (en) | 2016-10-17 | 2016-10-17 | Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902479.7A CN106636020A (en) | 2016-10-17 | 2016-10-17 | Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106636020A true CN106636020A (en) | 2017-05-10 |
Family
ID=58856479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610902479.7A Pending CN106636020A (en) | 2016-10-17 | 2016-10-17 | Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636020A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546690A (en) * | 2018-04-24 | 2018-09-18 | 沈阳药科大学 | The preparation and application of a kind of short-chain dehydrogenase and its mutant and gene |
| CN108570460A (en) * | 2018-04-24 | 2018-09-25 | 沈阳药科大学 | Short-chain dehydrogenase enzyme mutant and application thereof |
| CN109468346A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | The biological preparation method of one kind (S) -1- (the iodo- 5- fluorophenyl of 2-) ethyl alcohol |
| CN109468290A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | A kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application |
| CN109468291A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | A kind of carbonyl reductase EbSDR8 mutant and its construction method and application |
| CN110129382A (en) * | 2019-01-23 | 2019-08-16 | 西北工业大学 | A kind of carbonyl reductase catalytically synthesizing chiral ortho position is halogenated-method of alpha-phenyl ethyl alcohol |
| CN111041008A (en) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | Short-chain dehydrogenase mutant and application thereof |
| CN111433357A (en) * | 2018-02-12 | 2020-07-17 | 江南大学 | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827850A (en) * | 2012-08-31 | 2012-12-19 | 杭州师范大学 | Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application |
| CN102952814A (en) * | 2012-11-13 | 2013-03-06 | 杭州师范大学 | Recombined heat-resisting short-chain dehydrogenase gene, codase, carrier, engineering bacterium and application |
| CN103421823A (en) * | 2013-05-20 | 2013-12-04 | 浙江工业大学 | Leifsonia xyli HSO904-based short chain dehydrogenase, and encoding gene, carrier, engineering bacteria and application thereof |
| CN104212841A (en) * | 2014-08-22 | 2014-12-17 | 浙江工业大学 | Method for preparing (R)-3,5-bis(trifluoromethyl)phenethyl alcohol in ionic liquid-containing cosolvent medium |
| CN105238768A (en) * | 2015-08-07 | 2016-01-13 | 浙江大学 | Short-chain dehydrogenase, gene of short-chain dehydrogenase, recombinant expression vector, genetically engineered bacterium and application |
-
2016
- 2016-10-17 CN CN201610902479.7A patent/CN106636020A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827850A (en) * | 2012-08-31 | 2012-12-19 | 杭州师范大学 | Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application |
| CN102952814A (en) * | 2012-11-13 | 2013-03-06 | 杭州师范大学 | Recombined heat-resisting short-chain dehydrogenase gene, codase, carrier, engineering bacterium and application |
| CN103421823A (en) * | 2013-05-20 | 2013-12-04 | 浙江工业大学 | Leifsonia xyli HSO904-based short chain dehydrogenase, and encoding gene, carrier, engineering bacteria and application thereof |
| CN104212841A (en) * | 2014-08-22 | 2014-12-17 | 浙江工业大学 | Method for preparing (R)-3,5-bis(trifluoromethyl)phenethyl alcohol in ionic liquid-containing cosolvent medium |
| CN105238768A (en) * | 2015-08-07 | 2016-01-13 | 浙江大学 | Short-chain dehydrogenase, gene of short-chain dehydrogenase, recombinant expression vector, genetically engineered bacterium and application |
Non-Patent Citations (2)
| Title |
|---|
| AIPENG LI等: "Reconstruction of the Catalytic Pocket and Enzyme-Substrate Interactions To Enhance the Catalytic Efficiency of a Short-Chain Dehydrogenase/Reductase", 《CHEMCATCHEM》 * |
| NCBI: "Empedobacter brevis strain ZJUY-1401 short-chain dehydrogenase/reductase (sdr8) gene, complete cds", 《GENBANK DATABASE》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111433357A (en) * | 2018-02-12 | 2020-07-17 | 江南大学 | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol |
| CN111433357B (en) * | 2018-02-12 | 2022-11-11 | 江南大学 | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol |
| CN108570460A (en) * | 2018-04-24 | 2018-09-25 | 沈阳药科大学 | Short-chain dehydrogenase enzyme mutant and application thereof |
| CN108546690A (en) * | 2018-04-24 | 2018-09-18 | 沈阳药科大学 | The preparation and application of a kind of short-chain dehydrogenase and its mutant and gene |
| CN113293152B (en) * | 2018-04-24 | 2022-05-20 | 沈阳药科大学 | Short-chain dehydrogenase mutant and use thereof |
| CN113293152A (en) * | 2018-04-24 | 2021-08-24 | 沈阳药科大学 | Short-chain dehydrogenase mutants and uses thereof |
| CN108546690B (en) * | 2018-04-24 | 2021-08-10 | 沈阳药科大学 | Short-chain dehydrogenase and mutant thereof, and preparation and application of gene |
| CN108570460B (en) * | 2018-04-24 | 2021-07-30 | 沈阳药科大学 | Short-chain dehydrogenase mutants and uses thereof |
| CN109468290A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | A kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application |
| CN109468291B (en) * | 2018-06-01 | 2020-03-20 | 杭州馨海生物科技有限公司 | Carbonyl reductase EbSDR8 mutant and construction method and application thereof |
| CN109468291A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | A kind of carbonyl reductase EbSDR8 mutant and its construction method and application |
| CN109468346A (en) * | 2018-06-01 | 2019-03-15 | 杭州馨海生物科技有限公司 | The biological preparation method of one kind (S) -1- (the iodo- 5- fluorophenyl of 2-) ethyl alcohol |
| CN111041008A (en) * | 2018-10-11 | 2020-04-21 | 沈阳药科大学 | Short-chain dehydrogenase mutant and application thereof |
| CN111041008B (en) * | 2018-10-11 | 2022-02-01 | 沈阳药科大学 | Short-chain dehydrogenase mutant and application thereof |
| CN110129382A (en) * | 2019-01-23 | 2019-08-16 | 西北工业大学 | A kind of carbonyl reductase catalytically synthesizing chiral ortho position is halogenated-method of alpha-phenyl ethyl alcohol |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636020A (en) | Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application | |
| CN102876734B (en) | Carbonyl reductase, gene and applications of carbonyl reductase in asymmetric reduction of prochiral carbonyl compound | |
| CN107586763A (en) | Carbonyl reduction enzyme mutant, carrier, engineering bacteria and its application | |
| CN103497911B (en) | Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate | |
| CN105671010A (en) | Aldehyde ketone reductase mutant, gene, engineering bacterium and application of mutant | |
| CN112359036B (en) | Nitrilase mutant with improved catalytic activity and reaction specificity and application thereof | |
| CN110396505A (en) | Ketone group pantoic acid lactone reductase and its application | |
| CN109468291B (en) | Carbonyl reductase EbSDR8 mutant and construction method and application thereof | |
| CN106520715B (en) | A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate | |
| CN106497895A (en) | Leucine dehydrogenase mutant, encoding gene, carrier, engineering bacteria and its application | |
| CN103898177B (en) | Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof | |
| CN107858340A (en) | The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity | |
| CN112143764A (en) | Method for preparing intermediate compound of brivaracetam by using biological enzyme catalysis | |
| CN104152506A (en) | Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system | |
| CN105154381A (en) | Novel succinic acid-producing mutant microorganism simultaneously using sucrose and glycerol and method for producing succinic acid using the same | |
| CN113801859A (en) | Carbonyl reductase mutant for preparing chiral alcohol compound and application thereof | |
| CN106701723A (en) | D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof | |
| CN105200076A (en) | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application | |
| WO2010050231A1 (en) | Scyllo-inositol-producing cell and scyllo-inositol production method using said cells | |
| CN115975964A (en) | High-activity ketopantolactone reductase mutant and coding gene and application thereof | |
| CN105602913B (en) | Recombinate carbonyl reduction enzyme mutant ReCR-Mut, encoding gene, engineering bacteria and application | |
| CN111394396B (en) | Method for producing 1, 3-propylene glycol by using glycerol fermentation by microorganisms | |
| CN113913399A (en) | Ketopantolactone reductase derived from Candida maltosa Xu316 | |
| CN114085820A (en) | Ketopantolactone reductase derived from Candida viswanathii | |
| JP5403498B2 (en) | Method for producing (R) -3-quinuclidinol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |