CN105671010B - A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application - Google Patents
A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application Download PDFInfo
- Publication number
- CN105671010B CN105671010B CN201610124451.5A CN201610124451A CN105671010B CN 105671010 B CN105671010 B CN 105671010B CN 201610124451 A CN201610124451 A CN 201610124451A CN 105671010 B CN105671010 B CN 105671010B
- Authority
- CN
- China
- Prior art keywords
- cyano
- butyl ester
- aldehyde
- mutant
- gene
- 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.)
- Active
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.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/002—Nitriles (-CN)
Abstract
The invention discloses a kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and preparing 6- cyano-(3R, application in 5R)-dihydroxy hecanoic acid t-butyl ester, the aldehyde Ketoreductase mutant are that the 295th of amino acid sequence shown in SEQ ID NO.1, the 296th are carried out to single mutation or double mutation acquisitions；Compared with wild type aldehyde ketone reductase, Kluyveromyces lactis aldehyde Ketoreductase mutant provided by the invention, asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester catalysis specific enzyme activity increases substantially, 7.95U/mg is increased to from 0.56U/mg, improve 14.2 times, and product 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester non-enantiomer selectivity maintains 99.5% or more；The present invention multiple aldehyde Ketoreductase mutants obtained prepare 6- cyano-(3R particularly suitable for catalysis 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction, 5R)-dihydroxy hecanoic acid t-butyl ester has preferable prospects for commercial application.
Description
(1) technical field
The present invention relates to a kind of preparation method of the double chiral intermediates of Atorvastatin, in particular to a kind of Kluyveromyces Lactis dimension
Yeast aldehyde Ketoreductase mutant and encoding gene, carrier, recombination engineering and the aldehyde ketone reductase are in asymmetric reduction 6-
Cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester prepares double chiral intermediate 6- cyano-(3R, the 5R)-dihydroxies of Atorvastatin
Application in base hecanoic acid t-butyl ester.
(2) background technique
6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester is the important chirality in Atorvastatin synthesis route
Intermediate, and crucial pharmacophoric group.Since harsh limitation (e.e. value is arranged to drug chiral purity in various countries' drug administration department
> 99.5%, d.e. value > 99%), 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester synthetic technology becomes Atorvastatin
The key core technologies of synthesis.Traditional 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester chemical synthesis process is from ketone acid
(ester) sets out, and constructs chiral centre by asymmetric syntheses, reaction route is complicated, needs using inflammable and explosive borine, positive fourth
Base lithium etc. and the particular surroundings such as deep cooling, lead to that product d.e. value is low, yield is low, energy consumption is high.Moreover, the boride that reaction generates is useless
Object processing is difficult, needs to be quenched by cumbersome methanol repeatedly, vacuum distillation processing.Enzyme has excellent selectivity, reaction item
Part is mild, generally carries out under room temperature, normal pressure and near-neutral sulfite deinking, the unfavorable pair such as decomposition, isomerization, racemization, rearrangement
Reaction is preferably minimized limit, the base that biocatalysis technology has raising process Atom economy, realizes process green environment close friend
This condition.Therefore, it develops biology asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester and synthesizes 6- cyano -
(3R, 5R)-dihydroxy hecanoic acid t-butyl ester technology has huge economic benefit and social benefit.
Aldehyde ketone reductase superfamily is a kind of NAD (P) H dependent form oxidoreducing enzyme, is distributed widely in animal, plant and micro-
In biological cell, endocellular metabolism reaction is participated in, adverse effect of the harmful substance to cell in environment is eliminated.It has now been found that
Aldehyde ketone reductase is distributed in 16 families more than 190 kinds.Aldehyde ketone reductase is typically about 320 amino acid and forms single subunit
Albumen, size 34-37kDa have (α/β)8Tubular structure, catalysis tetrad by tyrosine (Tyr), histidine (His),
Aspartic acid (Asp) and lysine (Lys) are constituted, substrate-function spectrum width, including aliphatic and fragrance group aldehyde and ketone, monosaccharide, class
Sterol and prostaglandin etc..It is cloned and is expressed in foreign host there are many aldehyde ketone reductase gene at present and be widely used in not
Symmetrical synthesis of chiral intermediate, some of them be used for asymmetric reduction synthesis Atorvastatin intermediate 6- cyano-(3R,
5R)-dihydroxy hecanoic acid t-butyl ester.
We obtain from the clone of Kluyveromyces lactis Kluyveromyces lactis CCTCC M 2014380
Aldehyde ketone reductase KlAKR, and realize heterologous overexpression (Enzyme and at Escherichia coli (Escherichia coli)
Microbial Technology 2015,77:68-77).The enzyme can be catalyzed 6- cyano-(5R)-hydroxyl -3- carbonyl caproic acid uncle
Butyl ester asymmetric reduction synthesizes 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, and product de value is greater than 99%.But the enzyme is to 6-
Cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester vigor is not high, to limit its industrial applications.By having reported
Aldehyde ketone reductase crystal structure determine the space structure of the enzyme and possible relevant to activity using molecular simulation means
Amino acid sites improve aldehyde ketone reductase to 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester by site-directed mutagenesis technique
Catalysis activity, will have stronger industrial application value.
(3) summary of the invention
Object of the present invention is to for before report aldehyde ketone reductase KlAKR to 6- cyano-(5R)-hydroxyl -3- carbonyl oneself
The lower problem of tert-butyl acrylate asymmetric reduction activity provides a kind of aldehyde ketone reductase mutein and its encoding gene,
Recombinant expression carrier and recombination engineering containing the gene, the recombination engineering for expressing the aldehyde Ketoreductase mutant is crushed
Crude enzyme liquid later as catalyst 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction reaction,
Prepare optical voidness 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester.It is provided by the invention compared with wild type aldehyde ketone reductase
Aldehyde Ketoreductase mutant has higher catalytic activity.
The technical solution adopted by the present invention is that:
The present invention provides a kind of aldehyde Ketoreductase mutant, and the aldehyde Ketoreductase mutant is by SEQ ID NO.1 institute
Show that the 295th of amino acid sequence, the 296th carries out single mutation or double being mutated acquisition, the preferably described aldehyde ketone restores enzyme mutant
Body is to carry out the 295th progress single mutation of amino acid sequence shown in SEQ ID NO.1 or to the 295th and the 296th double
What mutation obtained.
Further, the preferably described single mutation is to be mutated the 295th tyrosine of amino acid sequence shown in SEQ ID NO.1
For tryptophan, amino acid sequence is shown in SEQ ID NO.2, and nucleotides sequence is classified as shown in SEQ ID NO.5；Described pair sports
295th tyrosine of amino acid sequence shown in SEQ ID NO.1 is sported into tryptophan, and the 296th tryptophan is mutated
For leucine, amino acid sequence is shown in SEQ ID NO 3, and nucleotides sequence is classified as shown in SEQ ID NO.6.
The present invention is mutated aldehyde ketone reductase KlAKR encoding gene using fixed point saturation mutation technology, connection expression
Host e. coli is converted after carrier, is examined again by the direct mutation that efficient liquid phase detection method improves activity after inducing expression
Out.The mutation physical efficiency of acquisition is catalyzed (1~50g/L) 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction, system
Standby optical voidness 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, the specific method is as follows: deriving from K.lactis CCTCC M
The aldehyde ketone reductase KlAKR (GenBank accession number:KU145407) that 2014380 clones obtain is by 309 ammonia
Base acid residue composition, succeeded construction of expression vector pET28b-klakr, and normally functioning zymoprotein is in e. coli bl21
(DE3) overexpression is realized in.Then pass through homologous modeling and molecular docking, select potentially influence according to Optimum configuration
The site of enzymatic activity.Setting fixed point saturation mutation site, redesigns and synthesizes primer appropriate, also with the aldehyde ketone containing parent
The recombinant expression plasmid of nitroreductase gene is template, the plasmid of PCR amplification overall length mutated gene.Pass through the matter that will be mutated containing overall length
Grain is transformed into host cell appropriate, through culture, inducing expression, filters out Positive mutants with high activity.Finally from sun
Property muton in extract Plasmid DNA, DNA sequencing analysis is carried out, to determine the mutation of primer.It is prominent in aldehyde ketone reductase of the present invention
In the preparation of variant, any suitable carrier can be used.
The present invention also provides a kind of aldehyde ketone reductase mutant code gene, the aldehyde ketone reductase mutant codes
Gene constructed recombinant vector and recombination engineering bacteria, the preferably described recombinant plasmid is pET28b；The host cell is big
Enterobacteria E.coli BL21 (DE3).
It is described the invention further relates to application of the aldehyde ketone reductase mutant code gene in preparation and reorganization aldehyde ketone reductase
Using are as follows: the recombinant vector for constructing the reduction enzyme mutant gene containing the aldehyde ketone converts the recombinant vector (excellent to host strain
Select Escherichia coli) in, the recombination engineering bacteria of acquisition carries out Fiber differentiation, and culture solution is isolated to contain recombination aldehyde ketone reduction
The somatic cells of enzyme have higher catalytic activity compared with wild type aldehyde ketone reductase.
The invention further relates to a kind of aldehyde Ketoreductase mutants in preparation 6- cyano-(3R, 5R)-dihydroxy caproic acid uncle
Application in butyl ester, the specific application is with the fermented culture of recombination engineering bacteria of the reduction enzyme mutant gene containing aldehyde ketone
The wet thallus of acquisition is catalyst, using 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester as substrate, with dehydrogenation containing glucose
The glucose dehydrogenase wet thallus that the engineering bacterium fermentation culture of enzyme gene obtains is coenzyme regeneration enzyme, is auxiliary bottom with glucose
Catalyst and the reaction of glucose dehydrogenase wet thallus are situated between by object using pH 7.0,100mM phosphate buffer as reaction medium
Matter suspends, and ultrasonication adds substrate and cosubstrate, at 30 DEG C, reacts under the conditions of 200r/min, after fully reacting, obtains
Obtain 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester；The glucose dehydrogenase is with the engineering containing glucose dehydrogenase gene
Bacterium (Escherichia Coli BL21 (DE3)/pET28b-esgdh) fermented and cultured obtains wet thallus, the specific recombination base
Because engineering bacteria Escherichia Coli BL21 (DE3)/pET28b-esgdh is will to derive from Exiguobacterium
Glucose dehydrogenase gene (GenBank No.KM817194.1) the insertion pET-28b building recombinant expression matter of sibiricum
Grain, and the recombinant plasmid is imported made from Escherichia coli；The glucose dehydrogenase wet thallus dosage is 10-250g/L buffering
The dosage of liquid (preferably 25g/L), the catalyst is calculated as 10-250g/L buffer (preferably 75g/L) with wet thallus weight, described
Final substrate concentrations are 1-100g/L buffer (preferably 50g/L), and the final concentration of 5-300g/L buffer of the glucose is (preferably
200g/L)。
Further, the catalyst is prepared as follows: by the recombination engineering of the reduction enzyme mutant gene containing aldehyde ketone
It is seeded in the LB liquid medium of the kanamycins of 50mg/L containing final concentration, 37 DEG C of culture 10h, then with the inoculation of volumetric concentration 4%
Into the LB liquid medium of the fresh kanamycins of 50mg/L containing final concentration, 37 DEG C of cultures to cell concentration OD600For 0.6-
0.8, then be added into culture solution the lactose of final concentration of 9g/L, after 28 DEG C of culture 12h, 4 DEG C, 8000g be centrifuged 10min, collect
Somatic cells.The wet thallus preparation method that the engineering bacterium fermentation culture containing glucose dehydrogenase gene obtains is same to contain aldehyde ketone also
The recombination engineering of protoenzyme mutant gene.Further, the ultrasonication condition are as follows: ice bath is placed in Ultrasonic Cell Disruptor, with
400W power is crushed 20min, and broken 1s stops 1s.
Glucose dehydrogenase wet thallus of the present invention will be the preparation method comprises the following steps: Exiguobacterium will be derived from
Glucose dehydrogenase gene (GenBank No.KM817194.1) the insertion pET-28b building recombinant expression matter of sibiricum
Grain, and the recombinant plasmid is imported Escherichia coli Escherichia Coli BL21 (DE3) and obtained and contains glucose dehydrogenase gene
Recombination engineering bacteria；Recombination engineering containing glucose dehydrogenase gene is seeded to the kanamycins of 50mg/L containing final concentration
LB liquid medium in, 37 DEG C of culture 10h, then the fresh card of 50mg/L containing final concentration is inoculated into volumetric concentration 4% that is mould
In the LB liquid medium of element, 37 DEG C of cultures to cell concentration OD600For 0.6-0.8, then it is added into culture solution final concentration of
The lactose of 9g/L, after 28 DEG C of culture 12h, 4 DEG C, 8000g be centrifuged 10min, collect somatic cells.
Aldehyde Ketoreductase mutant of the present invention can be used with the full cellular forms of engineering bacteria, be also possible to without pure
The use form of the enzyme of the thick enzyme or purifying changed uses.If desired, immobilization technology known in the art can also be used
Immobilised enzymes or immobilized cell is made in aldehyde Ketoreductase mutant of the invention.
Compared with prior art, beneficial achievement of the invention is mainly reflected in: compared with wild type aldehyde ketone reductase, this hair
The Kluyveromyces lactis aldehyde Ketoreductase mutant of bright offer, asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl
The catalysis specific enzyme activity of base hecanoic acid t-butyl ester increases substantially and (is increased to 7.95U/mg from 0.56U/mg, improve 14.2 times), and
Product 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester non-enantiomer selectivity maintains 99.5% or more.The present invention is obtained
The multiple aldehyde Ketoreductase mutants obtained are asymmetric particularly suitable for catalysis 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
Reduction preparation 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, has preferable prospects for commercial application.
(4) Detailed description of the invention
Fig. 1 is aldehyde ketone reductase and the coupling catalysed 6- cyano-(5R) of glucose dehydrogenase-tertiary fourth of hydroxyl -3- carbonyl caproic acid
The reaction schematic diagram of ester asymmetric reduction preparation 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester.
Fig. 2 is aldehyde Ketoreductase mutant Y295W/W296L and recombinant glucose dehydrogenase thallus mass ratio to asymmetry
Restore the influence of 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester.
Fig. 3 is concentration of glucose to the influence of asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester.
Fig. 4 is aldehyde Ketoreductase mutant KlAKR-Y295W/W296K and the coupling catalysed 50g/L6- cyanogen of glucose dehydrogenase
Base-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction preparation 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester
Reaction process.
(5) specific embodiment
According to following embodiments, the present invention may be better understood.As it will be easily appreciated by one skilled in the art that embodiment is said
Specific material proportion, process conditions and its result of description are merely to illustrate the present invention, want without that should will not limit right
Seek the present invention described in detail in book.
Recombination aldehyde ketone reductase gene of the present invention Kluyveromyces isolated in environment
Lactis is preserved in China typical culture collection center, the deposit date is on August 14th, 2014, deposit number CCTCC
NO:M 2014380, preservation address are Wuhan, China, Wuhan University, postcode 430072, in patent application
(201510004669.2) it is disclosed in.
Embodiment 1: the preparation of aldehyde Ketoreductase mutant
Obtained aldehyde ketone reductase KlAKR gene is cloned from Kluyveromyces lactis CCTCC M 2014380
(GenBank accession number:KU145407) (nucleotides sequence is classified as shown in SEQ ID NO.4, encodes histone amino
Acid sequence is shown in SEQ ID NO.1), succeeded construction of expression vector pET28b-klakr.
The preparation of aldehyde Ketoreductase mutant pinpoints saturation mutation by two-wheeled to realize.The first round is by SEQ ID NO.1 institute
Show that the 295th tyrosine of aldehyde ketone reductase KlAKR amino acid sequence sports tryptophan, is with recombinant plasmid pET28b-klakr
Template carries out full plasmid amplification, and primer pair is designed as Y295-F and Y295-R (as shown in table 1), obtains aldehyde Ketoreductase mutant
Y295W (amino acid sequence is shown in SEQ ID NO.2, and nucleotides sequence is classified as shown in SEQ ID NO.5).Second wheel is to contain 295
Position tyrosine sports the weight of aldehyde ketone reduction enzyme mutant gene (i.e. nucleotides sequence is classified as shown in SEQ ID NO.5) of tryptophan
Group plasmid is that template carries out full plasmid amplification, and primer pair is designed as Y295W/W296-F and Y295W/W296-R (as shown in table 1),
It obtains and the 295th tyrosine of amino acid sequence shown in SEQ ID NO.1 is sported into tryptophan, and by the 296th
Tryptophan sport leucine aldehyde Ketoreductase mutant Y295W/W296L (amino acid sequence as shown in SEQ ID NO.3,
Nucleotide sequence is as shown in SEQ ID NO.6).PCR system are as follows: 5 × PS buffer, 10 μ L, d NTP (every kind of nucleotide 2.5mM)
4 μ L, each 0.5 μ L of mutant primer, 0.5 μ L, PrimeSTAR archaeal dna polymerase of template (recombinant plasmid), 0.5 μ L, moisturizing to 50 μ L.
PCR condition are as follows: 95 DEG C of initial denaturation 5min, recycle through 27: 95 DEG C of 15s, 56 DEG C of 15s, 72 DEG C of 7min, last 72 DEG C re-extend
10min.After 0.9% agarose gel electrophoresis analysis PCR is positive, 20 μ L of PCR solution is taken, 1 μ L Dpn I, 37 DEG C of enzymes are added
3h removal template plasmid, 65 DEG C of inactivations 10min, transformed competence colibacillus cell E.coli BL21 (DE3) are cut, coating contains kanamycins
The LB plate of (50 μ g/mL), 37 DEG C of overnight incubations.Through Shanghai, Sani Bioisystech Co., Ltd carries out sequencing confirmation, obtains
Aldehyde ketone reductase mutant strain E.coli BL21 (DE3)/pET28b-klakr-Y295W (Y295W) and E.coli BL21
(DE3)/pET28b-klakr-Y295W/W295L(Y295W/W295L)。
Recombinate wild type aldehyde ketone reductase bacterial strain E.coli BL21 (DE3)/pET28b-klakr and recombination glucose dehydrogenation
Enzyme bacterial strain E.coli BL21 (DE3)/pET28b-esgdh is respectively from Kluyveromyces lactis CCTCC
Aldehyde ketone reductase KlAKR gene (GenBank accession number:KU145407) that M2014380 is cloned and from
The glucose dehydrogenase gene (GenBank No.KM817194.1) of Exiguobacterium sibiricum is inserted into respectively
PET-28b constructs recombinant expression plasmid, and the recombinant plasmid is imported Escherichia coli Escherichia Coli BL21 (DE3)
Middle acquisition.
1 aldehyde ketone reductase mutant primer of table
Embodiment 2: the inducing expression of aldehyde ketone reductase parent and mutant and glucose dehydrogenase
By the aldehyde ketone reductase in 1 starting strain E.coli BL21 (DE3) of embodiment/pET28b-klakr and embodiment 1
Mutant strain E.coli BL21 (DE3)/pET28b-klakr-Y295W (Y295W) and E.coli BL21 (DE3)/pET28b-
Klakr-Y295W/W295L (Y295W/W295L) and recombinant glucose dehydrogenase bacterial strain E.coli BL21 (DE3)/
PET28b-esgdh is inoculated into respectively in the LB liquid medium containing final concentration 50mg/L kanamycins, 37 DEG C of culture 10h, then
It is inoculated into the fresh LB liquid medium containing final concentration 50mg/L kanamycins with volumetric concentration 4%, 37 DEG C of cultures are extremely
Cell concentration OD600It is 0.6~0.8, after the lactose that final concentration of 9g/L is added into culture solution, 28 DEG C of culture 12h, 4 DEG C,
8000g is centrifuged 10min, collects somatic cells.It can be used for purifying and biocatalysis preparation 6- cyano-(3R, the 5R)-dihydroxy of enzyme
Hecanoic acid t-butyl ester.
Embodiment 3: the purifying of aldehyde ketone reductase parent and its mutant
Aldehyde ketone reductase somatic cells described in embodiment 2 are used into the buffer A (sodium phosphate buffer of 20mM, pH 8.0 respectively
Liquid NaCl containing 500mM and 20mM imidazoles) it suspends, ultrasonication 20min (ice bath, power 400W, broken 1s stop 1s), 4 DEG C,
12000rpm is centrifuged 20min, takes supernatant.Also using Ni affinity column (1.6cm × 10cm, Bio-Rad company, the U.S.) purifying aldehyde ketone
Protoenzyme parent and its mutant protein, concrete operations are as follows:
(1) Ni column is balanced with above-mentioned buffer A；
(2) supernatant being centrifuged after above-mentioned ultrasound is passed through into Ni column with the flow velocity of 1mL/min, is adsorbed onto target enzyme
On Ni column packing；
(3) Ni column is rinsed with the flow velocity of 1mL/min with the buffer A of above-mentioned 5 times of column volumes, elutes unadsorbed albumen；
(4) with buffer B (the sodium phosphate buffer and 500mM of NaCl containing the 500mM imidazoles of 20mM, pH 8.0) with
The flow velocity of 1mL/min elutes destination protein.Merge the collection liquid containing targeted enzymatic activity, and in sodium phosphate buffer (20mM, pH
8.0) dialysed overnight collects trapped fluid, obtains wild type aldehyde ketone reductase KLAKR and its mutant Y295W and Y295W/ respectively
The enzyme solution of W296L.All purification steps all carry out in 4 DEG C.
Embodiment 4: the measurement of aldehyde ketone reductase and its mutant enzyme specific enzyme activity
Enzyme-activity unit (U) is defined as: under the conditions of 30 DEG C, 7.0 pH, required for every 1 minute 1 micromole NADH of oxidation
Enzyme amount is defined as 1U.Specific enzyme activity is defined as unit of activity number possessed by every milligram of albumen.
Protein concentration is with bicinchoninic acid protein determination kit (Nanjing KaiJi Biology Science Development Co., Ltd, Nanjing)
Measurement.
Specific enzyme activity measuring method: using 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester as substrate, NADH is continuously monitored
The reduction of absorbance at 340nm measures the catalysis activity of aldehyde ketone reductase and its mutant.Enzyme activity assay system is by phosphorus
Sour potassium buffer (100mM, pH 7.0), 0.5mM NADH, 0.5mM 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester and
0.5 μ g/ μ L of aldehyde ketone reductase or its mutant enzyme solution obtained in embodiment 3, total volume are 200 μ L.
The specific enzyme activity of aldehyde ketone reductase and its mutant is as shown in table 2.
Embodiment 5: aldehyde ketone reductase and its mutant are diastereomeric to 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
The measurement of body
Asymmetric reduction reaction carries out in 1.5mL centrifuge tube, and reaction system is by 20mM 6- cyano-(5R)-hydroxyl -3-
It is slow to add potassium phosphate for carbonyl hecanoic acid t-butyl ester, aldehyde ketone reductase or its mutant obtained in 15mM NADH and 5U embodiment 3
Fliud flushing (100mM, pH 7.0) to total volume is 1mL composition.30 DEG C, 200rpm reaction 2h, sampling are raw using liquid chromatographic detection
At 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester de value.
Liquid phase testing conditions: ODS-2C18Column (4.6 × 250mm, 5 μm), mobile phase acetonitrile: water=1:3 (v/v), column temperature 40
DEG C, flow velocity 1mL/min, ultraviolet detection wavelength 210nm, 20 μ L of sample volume.6- cyano-(3S, 5R)-dihydroxy hecanoic acid t-butyl ester,
6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester retention time difference
For 7.4min, 8.1min, 12.5min.
The selectivity of aldehyde ketone reductase and its mutant is as shown in table 2.
2 wild type KlAKR of table and its mutant to 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester Rate activity and
Non-enantiomer selectivity
Embodiment 6: aldehyde ketone reductase and its mutant Y295W/W296L asymmetric reduction 6- cyano-(5R)-hydroxyl -3-
The comparison of carbonyl hecanoic acid t-butyl ester
The preparation of glucose dehydrogenase thallus: from the glucose dehydrogenase of Exiguobacterium sibiricum
Gene (GenBank No.KM817194.1), insertion pET-28b constructs recombinant expression plasmid, and the recombinant plasmid is imported greatly
Enterobacteria Escherichia Coli BL21 (DE3) obtains the recombination engineering bacteria containing glucose dehydrogenase gene；Portugal will be contained
The recombination engineering of grape glucocorticoid dehydrogenase gene is seeded in the LB liquid medium of the kanamycins of 50mg/L containing final concentration, 37 DEG C of trainings
10h is supported, then is inoculated into the LB liquid medium of the fresh kanamycins of 50mg/L containing final concentration with volumetric concentration 4%, 37 DEG C
It cultivates to cell concentration OD600For 0.6-0.8, then it is added into culture solution the lactose of final concentration of 9g/L, after 28 DEG C of culture 12h,
4 DEG C, 8000g centrifugation 10min, collect somatic cells.
By aldehyde ketone reductase obtained in embodiment 2 (starting strain or its mutant Y295W/W296L) wet thallus 3.75g
It mixes with glucose dehydrogenase thallus 1.25g, is suspended with 50ml kaliumphosphate buffer (100mM, pH 7.0), thallus total concentration is
20g DCW/L.Bacteria suspension press 3 method ultrasonication of embodiment, gained be crushed liquid for 6- cyano-(5R)-hydroxyl -3- carbonyl oneself
The asymmetric reduction reaction of tert-butyl acrylate, as shown in Figure 1.Reduction reaction carries out in 100mL round-bottomed flask, and reaction system is not by
With 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester (0.5g or 1.5g or 2.5g) of concentration and 1.5 times of quality (relative to
6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester) glucose above-mentioned broken liquid 50mL be added constitute.Reaction 30 DEG C,
Magnetic agitation rotating speed is to carry out under 300rpm, stream plus 1M Na2CO3Solution makes reaction solution pH maintain 7.0.Sampling, with embodiment 5
Shown liquid phase process is detected.The substrate transformation rate and product de value are as shown in table 3.
3 wild type KlAKR of table and its mutant Y295W/W296L asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl
Hecanoic acid t-butyl ester
Embodiment 7: whether aldehyde Ketoreductase mutant Y295W/W296L with recombinant glucose dehydrogenase thallus mass ratio to or not
Claim the influence of reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
By aldehyde Ketoreductase mutant Y295W/W296L wet thallus obtained in embodiment 2 and glucose dehydrogenase thallus
(preparation method is with embodiment 6) respectively by thallus mass ratio be 2:1,1:1,1.5:1,2:1,2.5:1,3:1,3.5:1,4:1 and
4.5:1 mixing, 50ml kaliumphosphate buffer (100mM, pH 7.0) suspend, and thallus total concentration is calculated as 100g/L with wet thallus.Bacterium
Suspension presses 3 method ultrasonication of embodiment, and gained is crushed liquid and is used for 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester not
Asymmetric reduction reaction, as shown in Figure 1.Reaction carries out in 10mL shaking flask, and reaction system is by 6- cyano-(5R)-hydroxyl -3- carbonyl
Hecanoic acid t-butyl ester 0.1g and 0.1g glucose is added above-mentioned broken liquid 10mL and constitutes.30 DEG C, react 10min under 200rpm, sample,
Product 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester yield and de value are detected with liquid phase process shown in embodiment 5.Such as figure
Shown in 2, efficiency of pcr product when aldehyde Ketoreductase mutant Y295W/W296L and recombinant glucose dehydrogenase thallus mass ratio are 4:1
Reach peak 56.8%.
Embodiment 8: concentration of glucose is to the influence of asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
By aldehyde Ketoreductase mutant Y295W/W296L wet thallus 3.75g obtained in embodiment 2 and 6 method of embodiment
The glucose dehydrogenase thallus 1.25g of preparation is mixed, and 50ml kaliumphosphate buffer (100mM, pH 7.0) suspends, thallus total concentration
For 20g DCW/L.Bacteria suspension presses 3 method ultrasonication of embodiment, and gained is crushed liquid and is used for 6- cyano-(5R)-hydroxyl -3- carbonyl
The asymmetric reduction reaction of hecanoic acid t-butyl ester.Reaction carried out in 10mL shaking flask, addition 6- cyano-(5R)-hydroxyl -3- carbonyl oneself
Tert-butyl acrylate 0.1g, glucose are respectively 0.05g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g and 0.45g,
With above-mentioned broken liquid 10mL.It reacts 10min respectively at 30 DEG C, 200rpm, samples, with the detection of liquid phase process shown in embodiment 5
Product 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester yield and de value.As shown in figure 3, concentration of glucose is 15g/L
Efficiency of pcr product reaches peak 68% when (as 1.5 times of concentration of substrate), and de value is greater than 99.5%.
Embodiment 9: aldehyde Ketoreductase mutant Y295W/W296L asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl
Application in hecanoic acid t-butyl ester.
By aldehyde Ketoreductase mutant Y295W/W296L wet thallus 3.75g and glucose dehydrogenase obtained in embodiment 2
Thallus 1.25g mixing, 50ml kaliumphosphate buffer (100mM, pH 7.0) suspend, and thallus total concentration is 20g DCW/L.Bacteria suspension
By 3 method ultrasonication of embodiment, gained is crushed the asymmetry that liquid is used for 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
Reduction reaction.Reduction reaction carries out in 100mL round-bottomed flask, and reaction system is by 6- cyano-(5R)-hydroxyl -3- carbonyl caproic acid
Tert-butyl ester 2.5g and 3.75g glucose is added above-mentioned broken liquid 50mL and constitutes.Reaction is in 30 DEG C, magnetic agitation rotating speed 300rpm
Lower progress, stream plus 1M Na2CO3Solution makes reaction solution pH maintain 7.0.With liquid phase process monitoring reaction course shown in embodiment 5
Middle product 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester generates and the variation of de value, reaction process curve such as Fig. 4 institute
Show.The figure shows, production concentration passage at any time and gradually rise, it is final to obtain 6- cyano-until 80min completes reaction
(3R, 5R)-dihydroxy hecanoic acid t-butyl ester 162.7mM, product de value maintain 99.5% or more always.
Claims (8)
1. a kind of aldehyde Ketoreductase mutant, it is characterised in that the aldehyde Ketoreductase mutant is one of following:
(1) the 295th tyrosine of amino acid sequence shown in SEQ ID NO.1 is sported into tryptophan, amino acid sequence SEQ
Shown in ID NO 2；(2) the 295th tyrosine of amino acid sequence shown in SEQ ID NO.1 is sported into tryptophan, and by
296 tryptophans sport leucine, and amino acid sequence is shown in SEQ ID NO 3.
2. aldehyde ketone reductase mutant code gene described in a kind of claim 1.
3. a kind of recombinant vector of the building of encoding gene described in claim 2.
4. a kind of recombination engineering bacteria of construction of recombinant vector described in claim 3.
5. aldehyde Ketoreductase mutant described in a kind of claim 1 is in preparation 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester
In application.
6. application as claimed in claim 5, it is characterised in that the application is with the weight of the reduction enzyme mutant gene containing aldehyde ketone
The wet thallus that the fermented culture of group genetic engineering bacterium obtains is catalyst, with 6- cyano-(5R)-tertiary fourth of hydroxyl -3- carbonyl caproic acid
Ester is substrate, using containing glucose dehydrogenase gene engineering bacterium fermentation culture obtain glucose dehydrogenase wet thallus as coenzyme again
Raw enzyme, using glucose as cosubstrate, using pH 7.0,100mM phosphate buffer as reaction medium, by catalyst and glucose
Dehydrogenase wet thallus is suspended with reaction medium, and ultrasonication adds substrate and cosubstrate, at 30 DEG C, 200r/min condition
Lower reaction after fully reacting, obtains 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester；The glucose dehydrogenase wet thallus
Dosage is 10-250g/L buffer, and the dosage of the catalyst is calculated as 10-250g/L buffer, the bottom with wet thallus weight
The final concentration of 1-100g/L buffer of object, the final concentration of 5-300g/L buffer of the glucose.
7. application as claimed in claim 6, it is characterised in that the catalyst is prepared as follows: will contain aldehyde ketone reductase
The recombination engineering of mutant gene is seeded in the LB liquid medium of the kanamycins of 50mg/L containing final concentration, 37 DEG C of cultures
10h, then be inoculated into the LB liquid medium of the fresh kanamycins of 50mg/L containing final concentration with volumetric concentration 4%, 37 DEG C of trainings
It supports to cell concentration OD600For 0.6-0.8, then it is added into culture solution the lactose of final concentration of 9g/L, after 28 DEG C of culture 12h, 4
DEG C, 8000g be centrifuged 10min, collect somatic cells.
8. application as claimed in claim 6, it is characterised in that the ultrasonication condition are as follows: ice bath is placed in Ultrasonic Cell Disruptor
In, 20min is crushed with 400W power, broken 1s stops 1s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610124451.5A CN105671010B (en) | 2016-03-04 | 2016-03-04 | A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610124451.5A CN105671010B (en) | 2016-03-04 | 2016-03-04 | A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671010A CN105671010A (en) | 2016-06-15 |
| CN105671010B true CN105671010B (en) | 2019-01-08 |
Family
ID=56306783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610124451.5A Active CN105671010B (en) | 2016-03-04 | 2016-03-04 | A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671010B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107794270B (en) * | 2016-08-31 | 2021-02-02 | 安琪酵母股份有限公司 | Ketone reductase DNA molecule, recombinant vector, strain and application |
| CN106929521B (en) * | 2017-01-21 | 2020-08-21 | 浙江工业大学 | Aldehyde ketone reductase gene recombination co-expression vector, engineering bacterium and application thereof |
| CN107058248B (en) * | 2017-04-26 | 2020-10-09 | 浙江工业大学 | Recombinant aldone reductase mutant, gene, vector, engineering bacterium and application thereof |
| CN108048416B (en) * | 2017-12-25 | 2021-05-18 | 吉林凯莱英医药化学有限公司 | Improved ketoreductase mutant and preparation method and application thereof |
| CN108192879B (en) * | 2018-01-12 | 2021-04-13 | 长江大学 | Preparation and application of finless eel aldone reductase mutant protein |
| CN109055324B (en) * | 2018-07-10 | 2021-05-04 | 天津迪沙医药技术开发有限公司 | Improved ketoreductase and application thereof |
| CN109055327B (en) * | 2018-07-23 | 2021-04-06 | 浙江工业大学 | Aldehyde ketone reductase mutant and application thereof |
| CN110004120B (en) * | 2019-01-25 | 2021-02-02 | 浙江工业大学 | Recombinant aldone reductase mutant and application thereof |
| CN109913429A (en) * | 2019-04-10 | 2019-06-21 | 南京普瑞特生物科技有限公司 | A kind of novel keto reductase and its it is used for chiral alcohol synthetic method |
| CN110643556A (en) * | 2019-08-23 | 2020-01-03 | 浙江工业大学 | Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof |
| CN110577940B (en) * | 2019-09-29 | 2021-06-08 | 浙江工业大学 | Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof |
| CN110938608A (en) * | 2019-12-20 | 2020-03-31 | 台州酶易生物技术有限公司 | Aldehyde ketone reductase mutant, encoding gene and application of aldehyde ketone reductase mutant in synthesis of (S) -TCPE |
| CN117126823B (en) * | 2023-09-01 | 2024-03-29 | 华南理工大学 | Ketone reductase mutant and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101528917A (en) * | 2006-10-02 | 2009-09-09 | 科德克希思公司 | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
| 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 |
| CN104357502A (en) * | 2014-10-16 | 2015-02-18 | 尚科生物医药（上海）有限公司 | Biological preparation method of (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoate |
| CN104498510A (en) * | 2014-11-13 | 2015-04-08 | 浙江工业大学 | Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof |
| CN104630241A (en) * | 2015-01-05 | 2015-05-20 | 浙江工业大学 | Aldehyde ketone production reductase strain, gene, enzyme, vector, engineering bacteria and application |
| 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 |
-
2016
- 2016-03-04 CN CN201610124451.5A patent/CN105671010B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101528917A (en) * | 2006-10-02 | 2009-09-09 | 科德克希思公司 | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
| 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 |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
| CN104357502A (en) * | 2014-10-16 | 2015-02-18 | 尚科生物医药（上海）有限公司 | Biological preparation method of (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoate |
| CN104498510A (en) * | 2014-11-13 | 2015-04-08 | 浙江工业大学 | Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof |
| CN104630241A (en) * | 2015-01-05 | 2015-05-20 | 浙江工业大学 | Aldehyde ketone production reductase strain, gene, enzyme, vector, engineering bacteria and application |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461;Xi Luo et al;《Enzyme and Microbial Technology》;20150612;第77卷;68页摘要，69页2.3，2.5，第70-71页 结果与讨论，图1；第73-75页结果部分，表3-4，第76页结论部分 * |
| Structural and mutational studies on an aldo-keto reductase AKR5C3 from Gluconobacter oxydans;Liu Xu et al;《Protein Science》;20140811;第23卷(第11期);第1540-1549页 * |
| Xuri Wu et al.Enzymatic preparation of t-butyl-6-cyano-(3R, 5R)-dihydroxyhexanoate by a whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase.《Process Biochemistry》.2014,第50卷(第1期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671010A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105671010B (en) | A kind of aldehyde Ketoreductase mutant, gene, engineering bacteria and its application | |
| CN108467860B (en) | Method for high yield of gamma-aminobutyric acid | |
| CN109055327A (en) | Aldehyde Ketoreductase mutant and its application | |
| CN109609474A (en) | A kind of amino acid dehydrogenase mutant and its application in synthesis L-glufosinate-ammonium | |
| CN109609475A (en) | Glufosinate-ammonium dehydrogenase mutant and its application for synthesizing L-glufosinate-ammonium | |
| CN107475281B (en) | Bioconversion methanol metabolic pathway | |
| CN103497911B (en) | Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate | |
| CN101501202A (en) | Process for production of optically active alcohol | |
| 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 | |
| CN101857887B (en) | Method for preparing optically pure aryl alcohol with cell-free extracts of recombinant strains by catalytic asymmetric conversion | |
| CN103421823B (en) | Come from the short-chain dehydrogenase of Lei Fusong Salmonella, encoding gene, carrier, engineering bacteria and application | |
| CN101691560B (en) | Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof | |
| CN109852644A (en) | A method of preparing Bu Waxitan intermediate | |
| CN102206686B (en) | Preparation method of methyl (R)-o-chloromandelate utilizing biocatalytic asymmetric reduction | |
| CN109055324A (en) | A kind of improved ketoreductase and its application | |
| CN112831488B (en) | Glutamic acid decarboxylase and gamma-aminobutyric acid high-yield strain | |
| CN109929822A (en) | A kind of Aspergillus oryzae lipase mutant and its application | |
| CN107653259A (en) | A kind of method of external enzyme reaction production D () 3-hydroxy-2-butanone | |
| CN110423740A (en) | It is a kind of improve enantioselectivity halide alcohol dehalogenase mutant and its application | |
| CN110004120A (en) | A kind of recombination aldehyde Ketoreductase mutant and application | |
| CN104830744A (en) | Method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase | |
| CN105602913B (en) | Recombinate carbonyl reduction enzyme mutant ReCR-Mut, encoding gene, engineering bacteria and application | |
| CN103966185A (en) | Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof | |
| CN108277216A (en) | High activity S- cyanalcohols lyases and its application | |
| CN108441433B (en) | Rhodotorula mucilaginosa NQ1 and application thereof in preparation of chiral alcohol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |