CN109055327A - Aldehyde Ketoreductase mutant and its application - Google Patents
Aldehyde Ketoreductase mutant and its application Download PDFInfo
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Abstract
6- cyano-(3R is prepared the invention discloses a kind of aldehyde Ketoreductase mutant and its in asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester, application in 5R)-dihydroxy hecanoic acid t-butyl ester, the aldehyde Ketoreductase mutant are to obtain the 125th, amino acid shown in SEQ ID No.2,30,212,63 progress single mutation or multimutation.The more maternal aldehyde ketone reductase of specific enzyme activity of aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R-I63W prepared by the present invention improves 1.31 times, maximum substrate 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester feeds intake and reaches 80g/L, which has more prospects for commercial application.
Description
Technical field
The present invention relates to aldehyde ketone reductase KlAKR mutation construction, develops aldehyde ketone reductase recombinant bacterium and enzyme is cut down in atropic
Application in terms of statin side chain 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester chirality biosynthesis.
Background technique
Cardiovascular and cerebrovascular disease, which has become, endangers one of principal disease of human health, cardiovascular and cerebrovascular disease morbidity and human body
Blood Cholesterol level has substantial connection.The crucial speed limit of Atorvastatin calcium energy Reverse transcriptase liver inner cholesterol synthesis
The activity of enzyme -3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase reduces the synthesis of cholesterol, and it is low to reduce blood
Density lipoprotein-cholesterol (LDL-C) and triglyceride concentration are the lists that global unique accumulated sales revenue is more than 100,000,000,000 dollars
One lipid-lowering medicine kind.
6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester is among important double chiral diols of Atorvastatin calcium synthesis
Body, and crucial pharmacophoric group.Due to various countries' drug administration department to drug chiral purity be arranged harsh limitation (e.e. value >
99%, d.e. value > 99%), 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester synthetic technology is closed as Atorvastatin calcium
At key core technologies.Traditional 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester chemical synthesis process is from ketone acid (ester)
It sets out, building chiral centre is restored by borine asymmetric hydrogenation, reaction route is complicated, needs using inflammable and explosive borine etc.
The reaction condition of reagent and harshness, product optical purity is low, yield is low, and energy consumption is high for process, reaction offal treatment is difficult, belongs to
In not environment friendly type production method.There is selectivity using oxidoreducing enzyme asymmetric reduction prochiral ketone synthesis of chiral alcohol
It is high, reaction condition is mild, the technical advantages such as environmental-friendly, the economy of manufacturing process is high, and meets the requirement of Green Chemistry.Cause
This, exploitation biology asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester synthesizes 6- cyano-(3R, 5R)-dihydroxy
Base hecanoic acid t-butyl ester technology has huge economic benefit and social benefit.
Aldehyde ketone reductase superfamily is the oxidoreducing enzyme of a kind of NAD (P) H dependent form, and aldehyde ketone reductase is typically about 320
A amino acid forms monomeric protein, and size 34-37kDa has (α/β)8Tubular structure is catalyzed tetrad by junket ammonia
Acid, histidine, aspartic acid and lysine composition, substrate spectrum width, including aliphatic, aromatic aldehyde, ketone, monosaccharide etc..
We, which clone from Kluyveromyces lactis Kluyveromyces lactis CCTCC M 2014380, obtains
Aldehyde ketone reductase KlAKR, and realize that heterologous overexpression, the enzyme can be catalyzed 6- at Escherichia coli (Escherichia coli)
Cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction synthesizes 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester,
And de value is greater than 99%, but the enzyme is not high enough to 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester vigor, passes through fixed point
It is public in patent application (201610124451.5) that mutating technology obtains one plant of predominant mutation strain KlAKR-Y295W-W296L
It opens, the substrate of 50g/L can be converted completely in 80 minutes, but find the enzyme in the case where not adding external source type coenzyme NADP 11
Vigor, stability, substrate inventory there are also room for promotion, this patent uses for reference reported aldehyde ketone reductase crystal structure letter
Breath determines the space structure of KlAKR, the amino acid near enzymatic activity pocket with homologous modeling and Molecular Simulation Technique means
Aldehyde ketone reductase is improved to 6- cyanogen by protein engineering in site and the entrance of possible substrate, product release channel etc.
Base-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester catalysis activity and substrate load, have industrial application value.
Summary of the invention
Object of the present invention is to not right to 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester for existing aldehyde ketone reductase
Claim reduction activation is not high and concentration of substrate is low problem, a kind of stereoselectivity aldehyde Ketoreductase mutant is provided and utilizes the aldehyde
Ketoreductase mutant gene recombination bacterium and its crude enzyme liquid as biocatalyst, for 6- cyano-(3R, 5R)-dihydroxy oneself
Tert-butyl acrylate chirality biosynthesis, the activity of catalyst improve 1.31 times, and concentration of substrate improves 60%.
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.2 institute
Show the 125th, amino acid, 30,212,63 carry out what single mutation or multimutation obtained.Amino acid shown in SEQ ID No.2
For maternal aldehyde ketone reductase KlAKR, nucleotides sequence is classified as shown in SEQ ID No.1.
Further, the preferably described aldehyde Ketoreductase mutant is one of following: (1) by amino acid shown in SEQ ID No.2
125th isoleucine mutation is leucine (pKlAKR-I125L) or valine (pKlAKR-I125V)；(2) by SEQ ID
The 125th isoleucine mutation of amino acid shown in No.2 is valine, while the 30th mutant serine is alanine
(pKlAKR-I125V-S30A), histidine (pKlAKR-I125V-S30H) or proline (pKlAKR-I125V-S30P)；(3)
It is valine by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2, and the 212nd glutamine sports essence
Propylhomoserin (pKlAKR-I125V-Q212R) or asparagine (pKlAKR-I125V-Q212N)；(4) by ammonia shown in SEQ ID No.2
Base the 125th isoleucine mutation of acid is valine, while the 30th mutant serine is proline, and the 212nd glutamy
Amine sports arginine (pKlAKR-I125V-S30P-Q212R) or asparagine (pKlAKR-I125V-S30P-Q212N)；
It (5) is valine by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2, while the 30th mutant serine is
Alanine, and the 212nd glutamine sports arginine (pKlAKR-I125V-S30A-Q212R)；(6) by SEQ ID
The 125th isoleucine mutation of amino acid shown in No.2 is valine, while the 30th mutant serine is proline, and the
212 glutamine sport arginine, and the 63rd isoleucine mutation is tryptophan (pKlAKR-I125V-S30P-Q212R-
I63W)。
Further, the aldehyde Ketoreductase mutant is by the 125th isoleucine of amino acid shown in SEQ ID No.2
Sport valine, while the 30th mutant serine is proline, and the 212nd glutamine sports arginine, the 63rd
Position isoleucine mutation is tryptophan.
The invention further relates to the encoding gene of aldehyde Ketoreductase mutant, recombinant vector and engineering bacterias.It is preferred that recombinantly expressing
Carrier pET-28b (+)；The preferred E. coli BL21 (DE3) of host cell, passes through protein induced expression, clasmatosis
Crude enzyme liquid is obtained, catalysis characteristics are superior to maternal aldehyde ketone reductase.
The present invention also provides a kind of aldehyde Ketoreductase mutants in asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl
Base hecanoic acid t-butyl ester prepares the application in 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, the specific application method are as follows:
The wet thallus that the recombination engineering bacteria of the reduction enzyme mutant gene containing aldehyde ketone is obtained through Fiber differentiation and dehydrogenation containing glucose
After being resuspended after the wet thallus mixing that the engineering bacteria of enzyme gene is obtained through Fiber differentiation with the phosphate buffer of pH 7.5,100mM
Ultrasonication, taking broken mixed liquor is catalyst, using 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester as substrate, with Portugal
Substrate constitutes reaction system supplemented by grape sugar, is reacted under the conditions of 30 DEG C, 150-300 revs/min, reaction terminates, reaction solution
It isolates and purifies, obtains 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester；The glucose dehydrogenase gene nucleotides sequence is classified as
SEQ ID No.3 (shown coding protein amino acid sequence is SEQ ID No.4).
Further, in the reaction system, 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester final concentration 50~
100g/L, glucose 75~150g/L of final concentration, catalyst amount are calculated as 50-100g/L (preferably with broken preceding wet thallus total amount
75-80g/L), the wet thallus and contain Portugal that the recombination engineering bacteria of the reduction enzyme mutant gene containing aldehyde ketone is obtained through Fiber differentiation
The wet thallus that the engineering bacteria of grape glucocorticoid dehydrogenase gene is obtained through Fiber differentiation is with mass ratio 3:1 mixing.
Further, the wet thallus is prepared as follows: by the recombination work of the reduction enzyme mutant gene containing aldehyde ketone
Journey bacterium is inoculated into the LB liquid medium of the 50 μ g/mL kanamycins containing final concentration, and 37 DEG C are cultivated 9 hours, with volumetric concentration 2%
Inoculum concentration be inoculated into the LB liquid medium of the fresh 50 μ g/mL kanamycins containing final concentration, 37 DEG C, 180 revs/min training
It supports 1.5 hours, then final concentration of 0.1mM IPTG is added into culture solution, after 28 DEG C are cultivated 10 hours, 4 DEG C, 8000 revs/min
Centrifugation 10 minutes obtains the wet thallus of the reductase containing aldehyde ketone；The engineering bacteria containing glucose dehydrogenase gene is through Fiber differentiation
Wet thallus of the wet thallus preparation method of acquisition with the reductase gene containing aldehyde ketone.
Further, the ultrasonication condition are as follows: pass through the recombination engineering bacteria of the reduction enzyme mutant gene containing aldehyde ketone
After the wet thallus mixing that the wet thallus that Fiber differentiation obtains and the engineering bacteria containing glucose dehydrogenase gene are obtained through Fiber differentiation
It is resuspended with pH 7.5,100mM phosphate buffer, ultrasonication 10 minutes on mixture of ice and water, ultrasonication condition: power
For 400W, it is crushed 1 second, pause 1 second.
Aldehyde ketone reductase female parent KlAKR and aldehyde Ketoreductase mutant base sequence overall length of the present invention is 930bp, from
One base, which rises to the 930th base, stops, initiation codon ATG, terminator codon TAA.
The acquisition of aldehyde Ketoreductase mutant of the present invention is using fixed point saturation mutation technology, using the technology to institute
It states SEQ ID No.1 aldehyde ketone reductase gene to be mutated, the mutant plasmid of acquisition is transferred to E.coliBL21 in a manner of thermal shock
(DE3) competent cell is inoculated with acquisition bacterial strain, is transferred, being induced, thallus recycling, crude enzyme liquid is utilized to be catalyzed 6- cyano-
(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction, prepares optical voidness 6- cyano-tertiary fourth of (3R, 5R)-dihydroxy caproic acid
Ester, the specific method is as follows: the first step activates original bacteria, obtains maternal E.coli BL21 (DE3) pET-28b (+)-
Klakr is extracted plasmid pET-28b (+)-klakr (pET-28b (+)-pklakr), and is saved stand-by.Second step passes through SWISS-
MODEL obtains the template protein crystal structure of homologous modeling compared with pKlAKR, using the homologous modeling of Modeller 9.14, and
Molecular docking is carried out, suitable mutational site is selected, reconnaissance is mainly the amino that active channel nearby obtains active pocket attachment
Sour residue designs the primer of mutation, using pET-28b (+)-pklakr as template plasmid, carries out mutation PCR, obtains mutant plasmid,
And convert, the screening of predominant mutation bacterium is carried out, send sequence to detect and save predominant mutation body.
The inoculation of aldehyde Ketoreductase mutant of the present invention and glucose dehydrogenase gene engineering bacteria, switching, induction, thallus return
It receives, culture medium can make thalli growth and generate culture medium of the invention for this field is any, preferably LB culture medium: peptone
10g/L, yeast powder 5g/L, NaCl 10g/L, distilled water dissolution, pH 7.0.Cultural method and condition of culture do not have special limit
System, cultural method and condition can be fitted according to the difference of the factors such as host type and cultural method by this field general knowledge
When selection.The glucose dehydrogenase derives from Exiguobacterium sibiricum, glucose dehydrogenase gene sequence
Number (GenBank:No.KM817194.1), used carrier pET-28b (+) are constructed recombinant expression carrier pET-28b (+)-
esgdh。
Compared with prior art, the main beneficial effect of the present invention is mainly reflected in: aldehyde ketone reductase prepared by the present invention
The more maternal aldehyde ketone reductase of the specific enzyme activity of mutant pKlAKR-I125V-S30P-Q212R-I63W improves 1.31 times, most outsole
Object 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester feeds intake and reaches 80g/L, which answers
Use prospect.
Detailed description of the invention
Fig. 1 is that aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R-I63W is urged with glucose dehydrogenase coupling
Change 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction and prepares 6- cyano-(3R, 5R)-dihydroxy caproic acid uncle
The reaction schematic diagram of butyl ester.
Fig. 2 is the nucleic acid electrophoresis figure of aldehyde ketone reductase fixed point saturation mutation.M: standard nucleic acid molecules amount；Swimming lane 1:pET-
28b(+)-pklakr；Swimming lane 2:pET-28b (+)-pklakr-I125V；Swimming lane 3:pET-28b (+)-pklakr-S30P；Swimming lane
4:pET-28b (+)-pklakr-Q212R；Swimming lane 5:pET-28b (+)-pklakr-I63W；Swimming lane 6:pET-28b (+)-
pklakr-I125V-S30P；Swimming lane 7:pET-28b (+)-pklakr-I125V-S30P-Q212R；Swimming lane 8:pET-28b (+)-
pklakr-I125V-S30P-Q212R-I63W。
The SDS-PAGE electrophoresis of Fig. 3 aldehyde Ketoreductase mutant crude enzyme liquid (A) and pure enzyme solution (B).M: standard protein point
Son amount；Swimming lane 1: maternal aldehyde ketone reductase；Swimming lane 2:pKlAKR-I125V；Swimming lane 3:pKlAKR-S30P；Swimming lane 4:pKlAKR-
Q212R；Swimming lane 5:pKlAKR-I63W；Swimming lane 6:pKlAKR-I125V-S30P；Swimming lane 7:pKlAKR-I125V-S30P-
Q212R；Swimming lane 8:pKlAKR-I125V-S30P-Q212R-I63W.
Fig. 4 is asymmetric using aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R-I63W coupling EsGDH
It restores 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester and prepares 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester time
Fate map.
Specific embodiment
The present invention is described further combined with specific embodiments below.
Embodiment 1: the building and screening of aldehyde ketone reductase mutant library
By aldehyde ketone reductase gene, (shown in amino acid sequence SEQ ID No.2, nucleotides sequence is classified as SEQ ID No.1 institute
Show) construction of expression vector pET-28b (+)-klakr, conversion Escherichia coli, acquisition starting strain E.coliBL21 (DE3)/
pET28b-pklakr。
The preparation of aldehyde ketone reductase mutant library realized by 4 wheel fixed point saturation mutations, design of primers such as table 1, the
One wheel, will through saturation mutation PCR using I125F and I125R in table 1 as primer using carrier pET-28b (+)-klakr as template
The 125th isoleucine mutation that aldehyde ketone shown in SEQ ID No.2 restores enzyme amino acid sequence is remaining 19 kinds of amino acid, and is turned
Change, apply plate, is screened by dominant strain and obtain aldehyde Ketoreductase mutant pKlAKR-I125V.Second wheel is with amino acid sequence
The corresponding mutant pKlAKR-I125V of SEQ ID No.3 is template, using S30F and S30R in table 1 as primer, through saturation mutation
PCR, conversion apply plate, are screened by dominant strain and obtain aldehyde Ketoreductase mutant pKlAKR-I125V-S30P.Third round
Using mutant pKlAKR-I125V-S30P as template, using Q212F and Q212R in table 1 as primer, through saturation mutation PCR, convert,
Plate is applied, is screened by dominant strain and obtains aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R.Fourth round is with prominent
Variant pKlAKR-I125V-S30P-Q212R is template, using I63F and I63R in table 1 as primer, through saturation mutation PCR, is converted,
Plate is applied, is screened by dominant strain and obtains aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R-I63W, later period
Remaining advantage single mutant pKlAKR-S30P, pKlAKR-Q212R, pKlAKR-I63W are constructed by same method in experiment.
It is mutated PCR system (100 μ L) are as follows: it is each to be mutated primer up and down by 2 times of Phanta Max buffers, 25 1 μ L of μ L, dNTPs
1 μ L, 1 μ L, Phanta Super-Fidelity archaeal dna polymerase of template, 0.5 μ L mend ddH2O to 50 μ L.PCR condition are as follows: 95 DEG C
Initial denaturation 5 minutes, through 25 recycle: 95 DEG C 15 seconds, 56 DEG C 15 seconds, 72 DEG C 6 minutes, it is last 72 DEG C eventually extend 10 minutes.PCR knot
Fruit carries out DNA agarose gel electrophoresis positive verification respectively, by PCR product carry out Dpn I enzymic digestion template, 37 DEG C, 3 hours,
It 160 revs/min, 65 DEG C, inactivates within 1 minute, PCR product thermal shock is converted, and E. coli BL21 (DE3) is activated,
37 DEG C, 160 revs/min are placed in, cultivates 1 hour, is coated on the LB plate containing 50 μ g/mL kalamycin resistances, 37 DEG C of inversions
Overnight incubation carries out the screening of predominant mutation body to the mutant of acquisition, and screening conditions are as follows: (aldehyde ketone is also for 10g DCW/L cell
Protoenzyme mutant and glucose dehydrogenase thallus mass ratio 3:1), cell is resuspended in the PBS (100mM) that pH 7.0 is added, in ice water
It is crushed 10min (ultrasonication condition: power 400W breaks 1s, stops 1s) on mixture, obtains crude enzyme liquid, final concentration 75g/L is added
6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester, the glucose of 112.5g/L carry out under the conditions of 30 DEG C, 150 revs/min
Reaction, reaction terminate, sample detection 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester concentration, and screening obtains dominant strain.It will
Obtaining dominant strain send Hangzhou Qing Ke Bioisystech Co., Ltd to be sequenced, and saves.The preparation of glucose dehydrogenase thallus is the same as implementation
Example 2.
1 aldehyde ketone reductase of table pinpoints saturation mutation design of primers
Embodiment 2: the inducing expression of aldehyde ketone reductase female parent, mutant and glucose dehydrogenase
(nucleotides sequence is classified as shown in SEQ ID No.3 glucose dehydrogenase gene esgdh, and amino acid sequence is SEQ ID
Shown in No.4) it clones and obtains from Exiguobacterium sibiricum, and pET-28b (+) carrier is connected to by double digestion
On, which is imported in E. coli BL21 (DE3), recombinant glucose dehydrogenase bacterial strain E.coli is obtained
BL21(DE3)/pET28b-esgdh。
By 1 starting strain E.coli BL21 (DE3) of embodiment/pET28b-pklakr and aldehyde ketone reductase mutant strain with
And recombinant glucose dehydrogenase bacterial strain E.coli BL21 (DE3)/pET28b-esgdh is inoculated into respectively containing 50 μ g/ of final concentration
In the LB liquid medium of mL kanamycins, 37 DEG C are cultivated 9 hours, are inoculated into fresh contain eventually with volume fraction 2% (v/v)
In the LB liquid medium of 50 μ g/mL kanamycins of concentration, 37 DEG C, 180 revs/min are cultivated 1.5 hours, then added into culture solution
Enter final concentration of 0.1mM IPTG, after 28 DEG C are cultivated 10 hours, 4 DEG C, 8000 revs/min are centrifuged 10 minutes, are obtained corresponding wet
Somatic cells.Cell production achieved above has corresponding albumen, can be used for the preparation of the pure enzyme solution of albumen, it can also be used to which crude enzyme liquid is urged
Change asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester and prepares 6- cyano-(3R, 5R)-dihydroxy caproic acid uncle
Butyl ester.
Embodiment 3: mutated library screening
The mutant strain wet thallus of 2 inducing expression of embodiment and glucose dehydrogenase wet thallus are mixed with mass ratio 3:1, pressed
It pH 7.5 is added, is resuspended in 100mM phosphate buffer, the ultrasonication on mixture of ice and water according to the amount of thallus total amount 50g/L
10 minutes, ultrasonication condition: power 400W was crushed 1 second, pause 1 second, obtains mutant strain crude enzyme liquid.Under similarity condition, use
Starting strain E.coli BL21 (DE3)/pET28b-pklakr Substitution bacterial strain wet thallus prepares starting strain crude enzyme liquid.
Using mutant strain crude enzyme liquid or starting strain crude enzyme liquid as catalyst, with 6- cyano-(5R)-hydroxyl -3- carbonyl caproic acid
The tert-butyl ester is substrate, using glucose as cosubstrate, does not add exogenous NADPH or NADP+, with thallus Endogenous Type NADPH,
Set up the coenzyme circulatory system.Reaction system is selected as 10mL, catalyst amount 50g/L in terms of broken preceding wet thallus total concentration,
Final substrate concentrations 75g/L, glucose final concentration 112.5g/L, 30 DEG C, 150 revs/min sample for reaction 1 hour, extract reaction solution 100
μ L, 900 μ L dehydrated alcohol protein precipitations, i.e. reaction solution dilute 10 times, and -20 DEG C overnight, and 12000 revs/min are centrifuged 3 minutes, take
Supernatant crosses 0.22 μM of microfiltration membranes, and as liquid phase sample, HPLC detects 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester, 6-
Cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, 6- cyano-(3S, 5R)-dihydroxy hecanoic acid t-butyl ester and depValue.With product 6-
Cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester and depFor index, predominant mutation body is screened, experimental result is shown in table 2.
Liquid phase testing conditions: chromatographic columnC18 (4.6 × 250mm, Acchrom, China) column, mobile phase acetonitrile:
Water volume ratio is 1:3, flow velocity 1.0mL/min, Detection wavelength 210nm, sample volume 20 μ L, 40 DEG C of column temperature.6- cyano-(5R)-hydroxyl
Base -3- carbonyl hecanoic acid t-butyl ester, 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, 6- cyano-(3S, 5R)-dihydroxy caproic acid
Tert-butyl ester retention time is respectively as follows: 15.6 minutes, 10.1 minutes, 9.3 minutes.
The catalytic performance and stereoselectivity of 2 pKlAKR of table and its mutant
Embodiment 4: the purifying of aldehyde ketone reductase female parent and its mutant
Embodiment 3 is obtained predominant mutation body (pKlAKR-I125V, pKlAKR-S30P, pKlAKR-Q212R in table 2,
PKlAKR-I63W, pKlAKR-I125V-S30P, pKlAKR-I125V-S30P-Q212R,
PKlAKR-I125V-S30P-Q212R-I63W), aldehyde Ketoreductase mutant is obtained according to 2 the method for embodiment
Wet thallus is suspended with buffer solution A (NaCl containing 0.3M, the pH 8.0 of 30mM imidazoles, 50mM sodium phosphate buffer) respectively, ultrasound
20 minutes broken (ice bath, power 400W are crushed 1 second, suspend 1 second), 4 DEG C, 12000 revs/min of centrifugation 20min take supernatant.
Using Ni affinity column (1.6 × 10cm, Bio-Rad company, the U.S.) purified mutant body protein, concrete operations are as follows:
1. Ni column is balanced with the combination buffer (pH 8.0, the 50mM sodium phosphate buffer of the NaCl containing 0.3M) of 5 times of column volumes, until base
Line is stablized；2. sample loading, flow velocity 1mL/min, applied sample amount are adsorbed in target protein on Ni column in 25-40mg/mL albumen；
3. rinsing miscellaneous egg with the buffer solution A (NaCl containing 0.3M, the pH 8.0 of 30mM imidazoles, 50mM sodium phosphate buffer) of 6 times of column volumes
It is white, flow velocity 1mL/min, until baseline stability；4. with buffer solution B (NaCl containing 0.3M, the pH 8.0 of 500mM imidazoles, 50mM phosphoric acid
Sodium buffer) elution, flow velocity 1mL/min, collection destination protein.Destination protein is placed in pH 7.5,20mM phosphate buffer
Middle dialysed overnight obtains purifying enzyme；5. combination buffer (pH 8.0, the 50mM sodium phosphate of the NaCl containing 0.3M of 5 times of column volumes
Buffer) Ni column is rinsed until baseline stability, the ultrapure water with 5 times of column volumes containing 20% ethyl alcohol save Ni column.
The pure enzyme of starting strain E.coli BL21 (DE3)/pET28b-pklakr aldehyde ketone reductase is received using the same terms
Collection.
Embodiment 5: the measurement of maternal aldehyde ketone reductase and its mutant enzyme specific enzyme activity
Enzyme-activity unit (U) is defined as: under the conditions of 30 DEG C, 7.5 pH, per minute it is every generate 1 μm of ol 6- cyano-(3R,
Enzyme amount needed for 5R)-dihydroxy hecanoic acid t-butyl ester is defined as an enzyme-activity unit, U.Specific enzyme activity is defined as every milligram of zymoprotein institute
The unit of activity number having, U/mg.
Enzyme activity assay standard conditions: 30mM 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester, 3mM NADPH are fitted
Enzyme solution is measured, is reacted 5 minutes under the conditions of 30 DEG C, 7.5,300 revs/min of pH, sample treatment simultaneously carries out HPLC detection and analysis.
Protein concentration is with bicinchoninic acid protein determination kit (Nanjing KaiJi Biology Science Development Co., Ltd, Nanjing)
Measurement.
The specific enzyme activity of maternal aldehyde ketone reductase and its mutant is as shown in table 3.
The opposite enzyme activity of 3 mutant of table is with poor to corresponding isomer selective (dep) value
A: at the standard conditions, the initial enzyme activity of pKlAKR is appointed as 100%
Embodiment 6: the measurement of maternal aldehyde ketone reductase and its mutant kinetic parameter
The kinetic parameter of aldehyde ketone reductase and its mutation is investigated, with 6- cyano-(5R)-tertiary fourth of hydroxyl -3- carbonyl caproic acid
As substrate, concentration is set as 2-10mM (2,4,6,8,10mM) ester, exogenous coenzyme NADP 11 concentration be set as 1-5mM (1,2,
3,4,5mM), a certain amount of pure enzyme solution is added (referring to embodiment 3).
Reaction system is selected as 500 μ L, and substrate and exogenous coenzyme is added in the 100 μ L of pure enzyme solution that embodiment 3 is collected
NADPH, pH 7.5,100mM phosphate buffer are reaction medium, and 30 DEG C, 150 revs/min sample for reaction 1 hour, negate and answer
Liquid HPLC detects 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester concentration (referring to embodiment 3).
Sequence is followed according to aldehyde ketone reductase catalytic reaction mechanism and forces reaction mechanism, can be calculated by double-reciprocal plot
V outmax、Km A、Km B, the results are shown in Table 4, by comparing kcatAnd Km, it is found that pKlAKR is to 6- cyano-(5R)-hydroxyl-
The K of 3- carbonyl hecanoic acid t-butyl ester and NADPHmValue is 3.45mM, 0.11mM respectively, except mutant pKlAKR-S30P is promoted,
Remaining mutant has certain decline, has to the affinity of 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester and NADPH
Increase trend.Mutant pKlAKR-I125V-S30P-Q212R-I63W is to 6- cyano-(5R)-tertiary fourth of hydroxyl -3- carbonyl caproic acid
The catalytic efficiency k of estercat/Km BReach 36.31s-1·mM-1, more maternal (kcat/Km B=12.41s-1·mM-1) 2.93 times are improved,
1438.23s is reached to the catalytic efficiency of coenzyme NADP 11-1·mM-1, more maternal (kcat/Km A=385.67s-1·mM-1) improve
3.73 again.
4 female parent pKlAKR of table and its mutant kinetic parameter compare
Embodiment 7: aldehyde Ketoreductase mutant pKlAKR-I125V-S30P-Q212R-I63W asymmetric reduction 6- cyano-
(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester
According to the description of embodiment 2, aldehyde Ketoreductase mutant pKlAKR-I125V-S30P- is obtained by fermentation
Q212R-I63W thallus 3g and glucose dehydrogenase EsGDH thallus 1g mixing, and with 40mL, pH 7.5, phosphate buffer
(100mM) is resuspended, and is crushed (ultrasonication condition: power 400W breaks 1s, stops 1s) on ice, takes whole broken mixed liquors (i.e. thick
Enzyme solution), reduction 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester of final concentration 80g/L, the Portugal of final concentration 120g/L is added
Grape sugar constitutes reaction system, is reacted under being 300rpm in 30 DEG C, magnetic agitation rotating speed, stream plus 2M Na2CO3Aqueous solution makes instead
Liquid pH is answered to maintain 7.5.With product 6- cyano-(3R, 5R)-dihydroxy in the detection reaction process of liquid phase process shown in embodiment 3
The generation of hecanoic acid t-butyl ester and the variation of de value, reaction process curve are as shown in Figure 4.The figure shows, production concentration is at any time
Elapse and gradually rise, in 90 minutes react complete, the substrate transformation rate be greater than 99%, product de value remain at 99.5% with
On.
Sequence table
<110>Zhejiang Polytechnical University
<120>aldehyde Ketoreductase mutant and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 930
<212> DNA
<213>unknown (Unknown)
<400> 1
atgaccaccc agaaattctt caccctgtct aacggtaaca aaatcccggc tgttgctatc 60
gttggtaccg gtaccgcttg gtacaaatct gaagaaaccg acgctacctt ctctcagccg 120
ctggttgaca tcgttaaaaa aaccctggac accgttccgg gtgttgttca catcgacgct 180
gctgaaatct accgtaccta cccggaactg ggtgctgctc tgaaagacac caaaaaaccg 240
cgtgacgaaa tcttcatcac cgacaaatac tctaccctga aacagctgtc tgaaaacccg 300
aaagttgctc tggaaacctc tctgaaaaaa ctgggtgttg actacgttga cctgtacctg 360
ctgcactctc cgatcatcaa agaatctaaa ctggacgttg aagctaactg gaaatacctg 420
gaagaactgt acaaatctgg taaagctaaa aacatcggtg tttctaactt cgctgttaaa 480
gacctggaaa aactgctggc tgttgctgaa gttaaaccgc aggttaacca gatcgaattc 540
tctccgttcc tgcagaacca gaccccgggt atcgttgaat tctctcagaa aaacggtatc 600
ctgctggaag cttactctcc gctgggtccg ctgcagcgtc gtccggaaga cgctgacaaa 660
ctgccgttct accagtacat cgctgaactg tctaaaaaat acaacaaatc tgaagctcag 720
atcctgctgt cttgggttta cgaacgtggt atcctgccgg ttaccacctc ttctaaaatc 780
gaacgtatcc agcaggctca ggacatcttc tctttctctc tggctaacga agaagttcag 840
aaaatcaccc agctgggtct gcagcacccg gctctgcgtc tctggctgac tgacgtgtac 900
agcaaatacg actctgaatc tcagaaataa 930
<210> 2
<211> 309
<212> PRT
<213>unknown (Unknown)
<400> 2
Met Thr Thr Gln Lys Phe Phe Thr Leu Ser Asn Gly Asn Lys Ile Pro
1 5 10 15
Ala Val Ala Ile Val Gly Thr Gly Thr Ala Trp Tyr Lys Ser Glu Glu
20 25 30
Thr Asp Ala Thr Phe Ser Gln Pro Leu Val Asp Ile Val Lys Lys Thr
35 40 45
Leu Asp Thr Val Pro Gly Val Val His Ile Asp Ala Ala Glu Ile Tyr
50 55 60
Arg Thr Tyr Pro Glu Leu Gly Ala Ala Leu Lys Asp Thr Lys Lys Pro
65 70 75 80
Arg Asp Glu Ile Phe Ile Thr Asp Lys Tyr Ser Thr Leu Lys Gln Leu
85 90 95
Ser Glu Asn Pro Lys Val Ala Leu Glu Thr Ser Leu Lys Lys Leu Gly
100 105 110
Val Asp Tyr Val Asp Leu Tyr Leu Leu His Ser Pro Ile Ile Lys Glu
115 120 125
Ser Lys Leu Asp Val Glu Ala Asn Trp Lys Tyr Leu Glu Glu Leu Tyr
130 135 140
Lys Ser Gly Lys Ala Lys Asn Ile Gly Val Ser Asn Phe Ala Val Lys
145 150 155 160
Asp Leu Glu Lys Leu Leu Ala Val Ala Glu Val Lys Pro Gln Val Asn
165 170 175
Gln Ile Glu Phe Ser Pro Phe Leu Gln Asn Gln Thr Pro Gly Ile Val
180 185 190
Glu Phe Ser Gln Lys Asn Gly Ile Leu Leu Glu Ala Tyr Ser Pro Leu
195 200 205
Gly Pro Leu Gln Arg Arg Pro Glu Asp Ala Asp Lys Leu Pro Phe Tyr
210 215 220
Gln Tyr Ile Ala Glu Leu Ser Lys Lys Tyr Asn Lys Ser Glu Ala Gln
225 230 235 240
Ile Leu Leu Ser Trp Val Tyr Glu Arg Gly Ile Leu Pro Val Thr Thr
245 250 255
Ser Ser Lys Ile Glu Arg Ile Gln Gln Ala Gln Asp Ile Phe Ser Phe
260 265 270
Ser Leu Ala Asn Glu Glu Val Gln Lys Ile Thr Gln Leu Gly Leu Gln
275 280 285
His Pro Ala Leu Arg Leu Trp Leu Thr Asp Val Tyr Ser Lys Tyr Asp
290 295 300
Ser Glu Ser Gln Lys
305
<210> 3
<211> 738
<212> DNA
<213>unknown (Unknown)
<400> 3
atgggcattg gcgaagcgat catccgtcgc tatgcagaag aaggcatgcg cgttgttatc 60
aactatcgta gccatccgga ggaagccaaa aagatcgccg aagatattaa acaggcaggt 120
ggtgaagccc tgaccgtcca gggtgacgtt tctaaagagg aagacatgat caacctggtg 180
aaacagactg ttgatcactt cggtcagctg gacgtctttg tgaacaacgc tggcgttgag 240
atgccttctc cgtcccacga aatgtccctg gaagactggc agaaagtgat cgatgttaat 300
ctgacgggtg cgttcctggg cgctcgtgaa gctctgaaat acttcgttga acataacgtg 360
aaaggcaaca ttatcaatat gtctagcgtc cacgaaatca tcccgtggcc tactttcgta 420
cattacgctg cttctaaggg tggcgttaaa ctgatgaccc agactctggc tatggaatat 480
gcaccgaaag gtatccgcat taacgctatc ggtccaggcg cgatcaacac tccaattaat 540
gcagaaaaat tcgaggatcc gaaacagcgt gcagacgtgg aaagcatgat cccgatgggc 600
aacatcggca agccagagga gatttccgct gtcgcggcat ggctggcttc tgacgaagcg 660
tcttacgtta ccggcatcac cctgttcgca gatggtggca tgaccctgta cccgagcttt 720
caggctggcc gtggttaa 738
<210> 4
<211> 245
<212> PRT
<213>unknown (Unknown)
<400> 4
Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly Met
1 5 10 15
Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys Ile
20 25 30
Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln Gly
35 40 45
Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Thr Val
50 55 60
Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val Glu
65 70 75 80
Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys Val
85 90 95
Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala Leu
100 105 110
Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met Ser
115 120 125
Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala Ala
130 135 140
Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu Tyr
145 150 155 160
Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile Asn
165 170 175
Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala Asp
180 185 190
Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu Ile
195 200 205
Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val Thr
210 215 220
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
225 230 235 240
Gln Ala Gly Arg Gly
245
Claims (8)
1. a kind of aldehyde Ketoreductase mutant, it is characterised in that the aldehyde Ketoreductase mutant is will be shown in SEQ ID No.2
What the 125th, amino acid, 30,212,63 progress single mutation or multimutation obtained.
2. aldehyde Ketoreductase mutant as described in claim 1, it is characterised in that the aldehyde Ketoreductase mutant be it is following it
One: (1) being leucine or valine by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2；(2) by SEQ ID
The 125th isoleucine mutation of amino acid shown in No.2 is valine, while the 30th mutant serine is alanine, histidine
Or proline；It (3) is valine, and the 212nd paddy ammonia by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2
Amide sports arginine or asparagine；It (4) is figured silk fabrics by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2
Propylhomoserin, while the 30th mutant serine is proline, and the 212nd glutamine sports arginine or asparagine；(5)
It is valine by the 125th isoleucine mutation of amino acid shown in SEQ ID No.2, while the 30th mutant serine is the third ammonia
Acid, and the 212nd glutamine sports arginine；(6) the 125th isoleucine of amino acid shown in SEQ ID No.2 is dashed forward
Become valine, while the 30th mutant serine is proline, and the 212nd glutamine sports arginine, the 63rd
Isoleucine mutation is tryptophan.
3. aldehyde Ketoreductase mutant as described in claim 1, it is characterised in that the aldehyde Ketoreductase mutant is by SEQ
The 125th isoleucine mutation of amino acid shown in ID No.2 is valine, while the 30th mutant serine is proline, and
212nd glutamine sports arginine, and the 63rd isoleucine mutation is tryptophan.
4. aldehyde Ketoreductase mutant described in a kind of claim 1 asymmetric reduction 6- cyano-(5R)-hydroxyl -3- carbonyl oneself
Tert-butyl acrylate prepares the application in 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester.
5. application as claimed in claim 4, it is characterised in that the application method are as follows: aldehyde Ketoreductase mutant base will be contained
The wet thallus that the recombination engineering bacteria of cause is obtained through Fiber differentiation and the engineering bacteria containing glucose dehydrogenase gene are trained through induction
Ultrasonication after being resuspended after the wet thallus mixing obtained with the phosphate buffer of pH 7.5,100mM is supported, takes the broken mixed liquor to be
Catalyst, using 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester as substrate, substrate is constituted reactant supplemented by glucose
System, is reacted under the conditions of 30 DEG C, 150-300 revs/min, and reaction terminates, and reaction solution isolates and purifies, and obtains 6- cyano-
(3R, 5R)-dihydroxy hecanoic acid t-butyl ester；The glucose dehydrogenase gene nucleotides sequence is classified as shown in SEQ ID No.3.
6. application as claimed in claim 5, it is characterised in that in the reaction system, 6- cyano-(5R)-hydroxyl -3- carbonyl
Hecanoic acid t-butyl ester 50~100g/L of final concentration, glucose 75~150g/L of final concentration, catalyst amount are total with broken preceding wet thallus
Amount is calculated as 50-100g/L, the wet thallus that the recombination engineering bacteria of the reduction enzyme mutant gene containing aldehyde ketone is obtained through Fiber differentiation
The wet thallus obtained with the engineering bacteria containing glucose dehydrogenase gene through Fiber differentiation is with mass ratio 3:1 mixing.
7. application as claimed in claim 5, it is characterised in that the wet thallus is prepared as follows: will contain aldehyde ketone reductase
The recombination engineering bacteria of mutant gene is inoculated into the LB liquid medium of the 50 μ g/mL kanamycins containing final concentration, and 37 DEG C
Culture 9 hours is inoculated into the LB liquid training of the fresh 50 μ g/mL kanamycins containing final concentration with the inoculum concentration of volumetric concentration 2%
It supports in base, 37 DEG C, 180 revs/min are cultivated 1.5 hours, then final concentration of 0.1mM IPTG is added into culture solution, 28 DEG C of cultures
After 10 hours, 4 DEG C, 8000 revs/min are centrifuged 10 minutes, obtain the wet thallus of the Ketoreductase mutant containing aldehyde；It is described to contain grape
The wet thallus preparation method that the engineering bacteria of glucocorticoid dehydrogenase gene is obtained through Fiber differentiation is the same as the reduction enzyme mutant gene containing aldehyde ketone
Wet thallus.
8. application as claimed in claim 5, it is characterised in that the ultrasonication condition are as follows: aldehyde Ketoreductase mutant will be contained
The wet thallus that the recombination engineering bacteria of gene is obtained through Fiber differentiation and the engineering bacteria containing glucose dehydrogenase gene are through inducing
It is resuspended after the wet thallus mixing that culture obtains with pH 7.5,100mM phosphate buffer, ultrasonication 10 on mixture of ice and water
Minute, ultrasonication condition: power 400W is crushed 1 second, pause 1 second.
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