CN105062985A - Carbonyl reductase mutant and application thereof - Google Patents
Carbonyl reductase mutant and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
Abstract
Genes of a female parent carbonyl reductase ChKRED20 are mutated randomly with an error-prone PCR (polymerase chain reaction) technique, a mutation library is established, and five mutation sites with improved thermal stability are screened through a high-throughput screening system. Two or more of the five beneficial amino acid mutation sites are integrated, and a combination mutant with improved thermal stability is obtained. The reaction speed of enzyme of the combination mutant is higher at high temperature in comparison with the female parent, the reaction cycle can be shortened, the space-time efficiency of catalyzation of substrates such as ethyl 4-chloroacetoacetate, 3,5-bis(trifluoromethyl)acetophenone and the like is improved and can be improved by four times at most, and the carbonyl reductase mutant has a good industrial application prospect.
Description
Technical field
The invention belongs to genetically engineered and technical field of enzyme engineering, particular content relates to carbonyl reduction enzyme mutant of thermotolerance raising and uses thereof.
Background technology
Carbonyl reductase (EC1.1.1.184), be a member of redox enzyme family, catalysis depends on coenzyme NAD H or NADPH, can specific catalytic ketone (aldehyde), conversion between alcohol.Chiral alcohol is the important intermediate of synthesis of chiral medicine, and carbonyl reductase can the reduction of catalysis prochiral ketone efficiently, is one of important method preparing chiral alcohol.The application of carbonyl reductase in pharmaceutical industry, Fine Chemical Industry and information industry is very extensive.In order to promote carbonyl reductase range of application in the industry further, its existing performance is had higher requirement, in long-time, keep activity stabilized, in extreme environment, keep high activity (extreme temperature or pH value etc.) or different substrates (comprising non-natural substrates) can be accepted.Wherein the thermostability of enzyme is for very important industrial application, and under hot conditions, the speed of response of enzyme is faster, can shorten reaction time, improve space-time yield, cost-saving, is also conducive to avoiding in reaction process by other microbial contamination.
Along with protein engineering and molecular biological development, the means of orthogenesis and rationality/half design and rational are used to carry out artificial evolution to enzyme molecule and transform the focus having become current enzyme engineering area research.Up to the present, existing many scholars various enzyme that used this technology successfully to transform, achieves the progress (Zhao, 2007, BiotechnogyandBioengineering98 (2), 271-275) attracted people's attention.Wherein fallibility PCR (error-pronePCR), DNA reorganize the means that (DNAshuffling), half design and rational etc. have become conventional in enzyme molecular modification, greatly accelerate the evolutionary process (LehmannandWyss of protein, 2001, CurrentOpinioninBiotechnology12 (4), 371-375).
Summary of the invention
The present invention utilizes fallibility round pcr to carry out molecular improvement to the carbonyl reductase ChKRED20 (SEQIDNO.2) deriving from Chryseobacterium sp CA49 (Chryseobacteriumsp.CA49), one or more amino acid is replaced, thus obtains the mutant of thermostability raising.
Described carbonyl reduction enzyme mutant is with SEQIDNO.2 for sequence of setting out, and is Threonine, the Aspartic acid mutations of the 102nd is L-glutamic acid, the glutamic acid mutation of the 128th is Methionin, the glutamic acid mutation of the 131st is glycine, the mutant serine of the 154th is the mutant that obtains of proline(Pro) or its arbitrary combination or increase the mutant that neutral mutation site obtains on this basis by the alanine mutation of the 100th.
According to this area common knowledge, the carrier expressing said mutation body, genetic engineering bacterium etc. of structure also belong to protection scope of the present invention.
In order to reach above object, the present invention to maternal carbonyl reductase ChKRED20 gene random mutation, sets up mutated library, by high flux screening System For Screening mutant with fallibility round pcr.The mutant that first run screening acquisition 5 thermostabilitys improve.Wherein will carry out unit point fractionation by 2 multisite mutants with site-directed mutagenesis technique, obtain again the mutant that 2 thermotolerances improve.Therefore, obtain 5 beneficial mutation sites altogether, two or more of this 5 beneficial mutation sites are integrated, obtain the combination mutant that thermostability improves.
Specific embodiment of the invention method is:
(1) structure of libraries of random mutants and screening: we have disclosed from Chryseobacterium sp CA49 (Chryseobacteriumsp.CA49, on November 27th, 2012 is in China typical culture collection center preservation, deposit number is NO:CCTCCM2012484) in gene order (the NCBI accession number: KC342020 of carbonyl reductase ChKRED20 of coding 250 amino-acid residues of clone, gene order is shown in SEQIDNO.1, and aminoacid sequence is shown in SEQIDNO.2).Adopt fallibility PCR method to carry out random mutation to it, connect PCR fragment with pET28a (+) carrier, electric shock proceeds to intestinal bacteria DH10B, obtains more than 2 × 10
4the mutant library of individual clone, extracts plasmid by after mutation library clone collection, proceeds to E. coli expression strains BL21-DE3, selects mono-clonal expressing protein in 96 orifice plates.Then that thalline is centrifugal, add N,O-Diacetylmuramidase smudge cells, and centrifugal, get part supernatant liquor (crude enzyme liquid) with 2-chloroacetyl acetacetic ester for substrate, reaction appropriate time measures enzyme and lives (control group).Meanwhile, separately get the supernatant liquor thermal treatment certain hour of same volume, measure residual activity in the same way.Residual activity is transferred in 96 new well culture plates higher than the bacterial strain contrasted and carries out repeating screening.Residual activity screening obtained, higher than the bacterial strain of contrast, serves the order-checking of Hai Ying fine horse Bioisystech Co., Ltd, obtains mutant DNA sequence dna information.Detailed protocol is shown in example 1.
Build and screening through above-mentioned random mutation storehouse, obtain the mutant that 5 thermostabilitys improve, be respectively 36F7,123D9,22E10,122F11,32B11, its feature is as follows:
The alanine mutation of 36F7: the 100 is Threonine (DNA sequence dna becomes ACA from GCA).
The Aspartic acid mutations of 123D9: the 102 is L-glutamic acid (DNA sequence dna becomes GAG from GAT).
The glutamic acid mutation of 22E10: the 128 is Methionin (DNA sequence dna becomes AAA from GAA).
The glutamic acid mutation of 122F11: the 131 is glycine (DNA sequence dna becomes GGA from GAA), the valine mutation of the 137th is Isoleucine (DNA sequence dna becomes ATT from GTT).
The glutamic acid mutation of 32B11: the 71 is aspartic acid (DNA sequence dna becomes GAT from GAA), the lysine mutation of the 110th is arginine (DNA sequence dna becomes AGA from AAA), the mutant serine of the 154th is proline(Pro) (DNA sequence dna becomes CCA from TCA).
(2) above-mentioned 2 multisite mutation are split as single site mutation:
Many site amino acid residues muton 122F11,32B11 comprised by rite-directed mutagenesis are split, and build and obtain simple point mutation E131G, V137I, E71D, K110R, S154P, its feature is as follows:
The glutamic acid mutation of E131G: the 131 is glycine (DNA sequence dna becomes GGA from GAA).
The valine mutation of V137I: the 137 is Isoleucine (DNA sequence dna becomes ATT from GTT).
The glutamic acid mutation of E71D: the 71 is aspartic acid (DNA sequence dna becomes GAT from GAA).
The lysine mutation of K110R: the 110 is arginine (DNA sequence dna becomes AGA from AAA).
The mutant serine of S154P: the 154 is proline(Pro) (DNA sequence dna becomes CCA from TCA).
Wherein, E131G and S154P is the mutational site that thermotolerance improves, and V137I, E71D, K110R are neutral mutation site.
The integration in (3) 5 sites:
By above-mentioned (1) and (2) screen mutation, obtain 5 beneficial mutation sites, i.e. 36F7,123D9,22E10, E131G and S154P, build Sites Combination mutant further by rite-directed mutagenesis.The following combination mutant of preferred structure: MC35, MC23, MC235, MC135, MC134, its feature is as follows:
The glutamic acid mutation of MC35: the 128 is Methionin (DNA sequence dna becomes AAA from GAA), the mutant serine of the 154th is proline(Pro) (DNA sequence dna becomes CCA from TCA).
The Aspartic acid mutations of MC23: the 102 is L-glutamic acid (DNA sequence dna becomes GAG from GAT), the glutamic acid mutation of the 128th is Methionin (DNA sequence dna becomes AAA from GAA).
The Aspartic acid mutations of MC235: the 102 is L-glutamic acid (DNA sequence dna becomes GAG from GAT), the glutamic acid mutation of the 128th is Methionin (DNA sequence dna becomes AAA from GAA), the mutant serine of the 154th is proline(Pro) (DNA sequence dna becomes CCA from TCA).
The alanine mutation of MC135: the 100 is Threonine (DNA sequence dna becomes ACA from GCA), the glutamic acid mutation of the 128th is Methionin (DNA sequence dna becomes AAA from GAA), the mutant serine of the 154th is proline(Pro) (DNA sequence dna becomes CCA from TCA).
The alanine mutation of MC1345: the 100 is Threonine (DNA sequence dna becomes ACA from GCA), the glutamic acid mutation of the 128th is Methionin (DNA sequence dna becomes AAA from GAA), the glutamic acid mutation of the 131st is glycine (DNA sequence dna becomes GGA from GAA), the mutant serine of the 154th is proline(Pro) (DNA sequence dna becomes CCA from TCA).
These combination mutants MC35, MC23, MC235, MC135 and MC1345 thermostability all have and improve in various degree compared with female parent.MC135 stability lifting amplitude is maximum, and reactive behavior is uninfluenced, and optimal reactive temperature is increased to 65 DEG C, and highly stable at 65 DEG C, and after processing 15 days continuously, residual activity is greater than 95%, and the wild-type heat inactivation transformation period is with this understanding only 12min.
With the sub-M135 of combinatorial mutagenesis for biological catalyst, conversion of substrate 4-chloroacetyl acetacetic ester, temperature of reaction 65 DEG C, space-time yield has significant improvement than female parent, the crude enzyme liquid of 3g/L can transform 300g/L substrate completely and generate (S)-CHBE, ee value >99% in 1h.
Beneficial effect of the present invention: the muton that above-mentioned all thermotolerances improve is compared with female parent, the speed of response of enzyme is faster, reaction time can be shortened, the spatiotemporal efficiency of catalytic substrate is improved, wherein the spatiotemporal efficiency of muton M135 conversion of substrate 4-chloroacetyl acetacetic ester is up to 333g/ (L.h), has good prospects for commercial application.
Accompanying drawing explanation
Fig. 1 is that the single-point mutants that 5 thermostabilitys improve compares with maternal with the remaining vigor of combination mutant, and the thermostability of enzyme processes 3 hours at 70 DEG C of temperature.
Fig. 2 is that the optimal reactive temperature mensuration curve of the optimal reactive temperature mensuration figure of maternal ChKRED20 and mutant MC135, maternal ChKRED20 represents with zero, and the optimal reactive temperature of mutant MC135 measures curve and uses ● represent.
Fig. 3 is the t of maternal ChKRED20 and combination mutant
1/2measure figure, represent with ◇ 65 DEG C of maternal ChKRED20 heat inactivation transformation period of surveying; Use 65 DEG C of mutant MC235 heat inactivation transformation period of surveying ◆ represent; 65 DEG C of mutant MC1345 heat inactivation transformation period of surveying with ▲ represent; Use 65 DEG C of mutant MC135 heat inactivation transformation period of surveying ● represent; Represent with zero 80 DEG C of mutant MC135 heat inactivation transformation period of surveying.
Fig. 4 is the time curve of maternal ChKRED20 and mutant MC135 conversion of substrate 4-chloroacetyl acetacetic ester under 65 DEG C of conditions, the reaction times curve of maternal ChKRED20 and mutant MC135 represents with dotted line and solid line respectively, concentration of substrate is 100g/L (zero), 150g/L (◆), 200g/L (◇), and300g/L (■ &).
Specific implementation method
Below in conjunction with embodiment, the present invention will be further described, it is pointed out that the present embodiment only for explaining the present invention, but not limitation of the scope of the invention.
Embodiment 1 fallibility PCR (error – pronePCR) method builds carbonyl reductase libraries of random mutants
Utilize
iIRandomMutagenesiskit carries out random mutation to carbonyl reductase ChKRED20 gene (see SEQIDNO.1).
The primer is: T7:5 ′ – TAATACGACTCACTATAGGG – 3 '
T7ter：5′–TGCTAGTTATTGCTCAGCGG–3′
Reaction conditions is: 95 DEG C of denaturation 2min, 95 DEG C of sex change 30s, and 55 DEG C of annealing 30s and 72 DEG C of extension 1min30s, totally 25 circulations, reclaim test kit with glue after electrophoresis and reclaim gene fragment.
To reclaim fragment EcoRI and SalI double digested after, ligation is carried out with pET28a (+) carrier (kalamycin resistance gene) cut through same enzyme, reaction conditions is: carrier and fragment in molar ratio 1:3 ratio mixing, add the T4 ligase enzyme of 400 units, 16 DEG C are spent the night.Electric shocking method proceeds to intestinal bacteria DH10B, obtains more than 2 × 10
4the mutant library of individual clone.
The screening of embodiment 2 carbonyl reductase ChKRED20 mutant library
Extract plasmid by embodiment 1 after mutation library clone collection, proceed to E. coli expression strains BL21-DE3, the LB of coating containing kantlex is dull and stereotyped, cultivates 12h.Picking mono-clonal is in 96 orifice plates, and 200 μ LTB substratum (containing 50 μ g/mL kantlex, 0.5mMIPTG) are contained in every hole, 30 DEG C, 180rpm, and 18h is cultivated in concussion.It is dull and stereotyped in LB solid medium that 96 orifice plate reproducers copy each mono-clonal, after 37 DEG C of cultivation 12h, and 4 DEG C of Refrigerator stores.By the centrifugal 10min under 4000rpm of 96 orifice plates after thalline abduction delivering, supernatant discarded, each hole adds the lysis buffer re-suspended cell (configuration of lysis buffer: 0.1M of 200 μ L, the potassium phosphate buffer of pH8.0,10mg/mL N,O-Diacetylmuramidase, 1 μ g/mLDNaseI, 10mMMgCl
2).After 96 orifice plates being added with lysate are placed 60min at 37 DEG C, under 4000rpm, centrifugal 10min, gets supernatant.With the supernatant liquor in the volley of rifle fire gently each hole of sucking-off 96 orifice plate, add 10 μ L supernatant liquors respectively in two piece of 96 orifice plate by corresponding position.One packagedly has 96 orifice plates of 10 μ L supernatant liquors to add the 190 μ L reaction solution (NADH of 1mM, 0.1M, the dimethyl sulphoxide solution containing 1mM4-chloroacetyl acetacetic ester of the potassium phosphate buffer of pH8.0 and 0.02 times of volume mixes), after 30 DEG C of reaction 50min, under 340nm, measure the absorbancy of NADH.Another is packaged has 96 plates of 10 μ L supernatant liquors to be placed in 70 DEG C of baking ovens, is placed on rapidly 4 DEG C of fast coolings after 1h process.If above-mentioned reaction is to measure mutant residual activity, get the residual activity bacterial strain higher than wild-type ChKRED20 in 96 new well culture plates, carry out repeating screening.Screen the mutant that 5 thermostabilitys improve, be respectively 36F7,22E10,123D9,122F11,32B11, residual activity is 30-100 times of wild type control.Picking mono-clonal serves the order-checking of Hai Ying fine horse biotech company respectively.
The mensuration of embodiment 3 crude enzyme liquid enzyme activity and remaining vigor
The preparation of 3.1 crude enzyme liquids
Picking mono-clonal is in LB (containing kantlex 50 μ g/mL) substratum, 37 DEG C of incubated overnight, the inoculum size with 1% is forwarded in TB (containing kantlex 50 μ g/mL) substratum, cultivates 3h for 37 DEG C, after adding 0.5mMIPTG induction, 30 DEG C are continued to be cultured to 18h.Bacterium liquid centrifugal collection bacterium, cell clarifixator is broken, and centrifuging and taking supernatant is crude enzyme liquid.
The mensuration of 3.2 thick enzyme activities
The pure enzyme liquid (total protein concentration) of thick enzyme activity determination reaction conditions: 0.03mg/mL, the NADH of 80mM, the substrate 4-chloroacetyl acetacetic ester stock solution (final concentration 120mM) of the potassium phosphate buffer of 0.1M, pH8.0 and 0.12 times of reaction volume.Substrate is dissolved in methyl-sulphoxide the stock solution being mixed with 1M.40 DEG C, 150rpm reacts 5min.After reaction terminates, equal-volume extraction into ethyl acetate, measures the growing amount of product.
The mensuration of 3.3 remaining vigor
The mensuration of remaining vigor: the PCR pipe crude enzyme liquid 100 μ L of total protein concentration 1mg/mL being placed in 250 μ L, the repetition of 3, each sample, at 65 DEG C of temperature, 8h is processed with PCR, or process 3h at 70 DEG C of temperature, place cooled on ice rapidly, 4 DEG C, 12000rpm, centrifugal 10min, gets appropriate crude enzyme liquid and measures residual activity, with reference to the thick enzyme activity determination of embodiment 3.2.Again with the crude enzyme liquid without pyroprocessing for reference, obtain remnant enzyme activity per-cent.
The fractionation of embodiment 4 multisite mutation, qualification beneficial mutation site
By sub-for screened multisite mutation 122F11,32B11 by rite-directed mutagenesis, be split as muton E131G, V137I, E71D, K110R, the S154P with single mutational site.Rite-directed mutagenesis all with carbonyl reductase ChKRED20 gene for template, the primer:
E131G-F：5′–GAGTTAGAACAAATGG
GAAAAAACGGGGGCGGC–3′
E131G-R：5′–GCCGCCCCCGTTTTTT
CCCATTTGTTCTAACTC–3′
V137I-F：5′–GAAAAAAACGGGGGCGGC
ATTATTGTGAATATGG–3′
V137I-R：5′–CCATATTCACAATAA
TGCCGCCCCCGTTTTTTTC–3′
E71D-F：5′–CCCTGAAGAAGTGGA
TGCTTTAGTAAAAAGAACAG–3′
E71D-R：5′–CTGTTCTTTTTACTAAAGC
ATCCACTTCTTCAGGG–3′
K110R-F：5′–CTCGACAGCTGGCGAA
GAGTATTAAGCATAAATC–3′
K110R-R：5′–GATTTATGCTTAATACT
CTTCGCCAGCTGTCGAG–3′
S154P-F：5′–CTGCACCGCTTTCC
CCAGCCTACACTTCTGCAAAG–3′
S154P-R：5′–CTTTGCAGAAGTGTAGGCTG
GGGAAAGCGGTGCAG–3′
PCR condition is: 10 × Buffer5 μ L, the each 6 μ L of primer (10mM), dNTP (2.5mM) 4 μ L, pfu (2.5U/mL) 1 μ L, plasmid 10ng, ultrapure water supplies 50 μ L, condition: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 68 DEG C extend 6min, totally 16 circulations.PCR primer 1 μ LDpnI37 DEG C of process 1h.PCR primer 10 μ L chemical method proceeds to E.coliDH5a.Send and check order in Shanghai Ying Jun Bioisystech Co., Ltd.After order-checking is correct, extracts plasmid and proceed to expression strain E.coliBL21-DE3.
The protein expression of muton and the preparation method of crude enzyme liquid are shown in embodiment 3.1.
The relative activity of 5 mutons 36F7,22E10,123D9,122F11,32B11 compared with female parent that 5 mutons E131G, V137I, E72D, K110R, S154P that epicycle sudden change obtains obtain with embodiment 2, and the remaining vigor after 8 hours that processes at 65 DEG C of temperature is in table 1.The remaining vigor comparing wild-type carbonyl reductase ChKRED20 is 1%, muton 36F7,123D9,22E10,122F11,32B11, E131G, V137I, E71D, K110R, S154P, residual activity is respectively 40%, 99%, 88%, 32%, 29%, 30%, 0%, 0%, 1%, 29%.Wherein, E131G, S154P are beneficial mutation point, and E71D, K110R, V137I are neutral mutation point.Therefore, we obtain 5 beneficial mutation sites, i.e. 36F7,123D9,22E10, E131G and S154P altogether.
The relative activity of table 1 mutant and female parent and remaining vigor
The all numerical value of a all surveyed with crude enzyme liquid, and measuring method is see embodiment 3.2.
The Rate activity of b wild-type is 105 ± 7U/mg.
The thermostability of c enzyme processes 8 hours at 65 DEG C of temperature.
Embodiment 5 mutational site construction and integration carbonyl reductase ChKRED20 new mutant
The structure of 5.1 mutant MC23
The Histidine mutations of the 102nd of mutant 22E10 the is propylhomoserin by directed mutagenesis method, and build mutant MC23, primer used is as follows:
D101E-F：5′–GAACAGGCGCTGGCAGGCGA
GTACGGTCTCGACAGCTG–3′
D101E-R：5′–CAGCTGTCGAGACCGTA
CTCGCCTGCCAGCGCCTGTTC–3′
PCR condition and operation, with embodiment 4, obtain new mutant MC23.
The structure of 5.2 mutant MC35
The Histidine mutations of the 154th of mutant 22E10 the is propylhomoserin by directed mutagenesis method, and build mutant MC35, primer used is as follows:
S154P-F：5′–CTGCACCGCTTTCC
CCAGCCTACACTTCTGCAAAG–3′
S154P-R：5′–CTTTGCAGAAGTGTAGGCTG
GGGAAAGCGGTGCAG–3′
PCR condition and operation, with embodiment 4, obtain new mutant MC35.
The structure of 5.3 mutant MC235
The Histidine mutations of the 154th of mutant MC23 the is propylhomoserin by directed mutagenesis method, and build mutant MC235, primer used is as follows:
S154P-F：5′–CTGCACCGCTTTCC
CCAGCCTACACTTCTGCAAAG–3′
S154P-R：5′–CTTTGCAGAAGTGTAGGCTG
GGGAAAGCGGTGCAG–3′
PCR condition and operation, with embodiment 4, obtain new mutant MC235.
The structure of 5.4 mutant MC135
The phenylalanine of the 100th of mutant MC35 the is sported tyrosine by directed mutagenesis method, and build mutant MC135, primer used is as follows:
Y1W2-F：5′–GAACAGGCGCTG
ACAGGCGATTACGGTCTCGACAGCTG–3′
Y1W2-R：5′–CAGCTGTCGAGACCGTAATCGCCTG
TCAGCGCCTGTTC–3′
PCR condition and operation, with embodiment 4, obtain new mutant MC135.
The structure of 5.5 mutant MC1345
The phenylalanine of the 131st of mutant MC135 the is sported tyrosine by directed mutagenesis method, and build mutant MC1345, primer used is as follows:
Y3Y4-F：5′–CGGGTGCAAATATGAGTTA
AAACAAATGG
GAAAAAACGGGG–3′
Y3Y4-R：5′–CCCCGTTTTTT
CCCATTTGTT
TTAACTCATATTTGCACCCG–3′
PCR condition and operation, with embodiment 4, obtain new mutant MC1345.
Comparing of 5.6 simple point mutations, the sub vigor remaining with female parent of combinatorial mutagenesis
Compared with female parent all, MC35, MC23, MC235, MC135 and MC1345 thermostability has and improves in various degree.Process 3 hours at 70 DEG C of temperature after, maternal ChKRED20 completely loses vigor, the remaining vigor of the sub-A100T of simple point mutation (that is: 36F7), D102E (that is: 123D9), E128K (that is: 22E10), E131G, S154R is respectively 5.8%, 11.8%, 7.4%, 4.7%, 4.5%, and the remaining vigor of combinatorial mutagenesis MC35, MC23, MC235, MC135, MC1345 is respectively 11.5%, 10.3%, 0%, 99.9%, 73.7%.Wherein, the thermostability increase rate of muton MC135 is maximum.Experimental result is shown in Figure of description 1.
It should be noted that, although the remaining vigor of combination mutant MC235 after 70 DEG C of process 3h is identical with female parent, its t
1/2but higher than female parent, see Figure of description 3.Therefore MC235 is also the combination mutant that thermotolerance improves.
The protein purification of embodiment 6 female parent and combinatorial mutagenesis type MC135 and thermostability
The purifying of 6.1 female parents and combination mutant MC135
The purifying of maternal carbonyl reductase ChKRED20 and combination mutant MC135 enzyme adopts affinity chromatography (Bio-Rad).Yeast culture is carried out with reference to example 3.1, thalline after thalline inducing culture 18h is with 13000rpm, 4 DEG C of collected by centrifugation, be resuspended in BufferA (50mM phosphate buffered saline buffer, pH8.0, 300mMNaCl, 10mM imidazoles), cell clarifixator is broken, afterwards with 13000rpm, 4 DEG C of centrifugal 20min, supernatant liquor is added in the post material by BufferA balance, slight mixing 30min, with the BufferA rinsing foreign protein containing 20mM imidazoles, again with the buffer solution elution target protein of the BufferA containing 250mM imidazoles, last electroresis appraisal purity, and measure protein concentration with BCAProteinAssaykit.
The mensuration of 6.2 female parents and combination mutant MC135 optimal reactive temperature
Under pH8 condition, measure carbonyl reductase wild-type and saltant type maximum speed of reaction at different temperatures, the concentration of enzyme is 0.05mg/mL, and temperature of reaction is respectively 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C.Measuring method is: the reaction solution of 1mL comprises the pure enzyme liquid of 0.05mg/mL, the NAD of 0.2g/L
+, the aqueous isopropanol containing 300mM4-chloroacetyl acetacetic ester of the potassium phosphate buffer of 0.1M, pH8.0 and 0.3 times of volume, reacts 20min at different temperatures.Equal-volume extraction into ethyl acetate, measures the growing amount of product.With activity during maximum speed of reaction for 100%, it is ordinate zou that the enzyme measured at other temperature is lived with its ratio per-cent, and temperature of reaction is X-coordinate, draws the curve that enzyme work changes with temperature of reaction.The results are shown in Figure of description 2.Mutant MC135 has maximum speed of reaction at 65 DEG C, improves 15 DEG C than wild-type.
The thermal stability determination of 6.3 female parents and combination mutant
The pure enzyme liquid 100 μ L of protein concentration 1mg/mL is placed in the PCR pipe of capacity 250 μ L, the repetition of 3, each sample, the different time is processed 65 DEG C and 80 DEG C by PCR instrument, then sample hose is positioned over cooled on ice, 4 DEG C, 12000rpm, centrifugal 10min, the enzyme liquid got after appropriate process measures enzyme activity, and measuring method is shown in 6.2.With the enzyme liquid without pyroprocessing for reference, obtain relative activity.Take treatment time as X-axis, the natural logarithm of remnant enzyme activity per-cent is Y-axis, scatter diagram is done with origin75 software, add Trendline, obtain the linear equation of enzyme heat treatment time and relative activity, be 3.912023 to calculate the corresponding time with the natural logarithm of relative activity 50%, this time is the heat inactivation transformation period t of mutant
1/2.The results are shown in Figure of description 4.Under 65 DEG C of conditions, the t of ChKRED20 female parent
1/2for only 12min, the t of mutant MC235
1/2for 13.4min, the t of mutant MC1345
1/2for 131.6min, mutant MC135 is in thermal treatment after 15 days, and residual activity is still greater than 95%; Under 80 DEG C of conditions, maternal inactivation instantaneously, and the t of MC135
1/2reach 91min.
The application of embodiment 7 muton in biocatalysis
The sign of 7.1 combinatorial mutagenesis type MC135 and application example analysis
Combinatorial mutagenesis type MC135 is replaced by 3 amino acid to produce on the basis of maternal carbonyl reductase ChKRED20.The optimal reactive temperature of wild-type ChKRED20 is 50 DEG C, but stability is undesirable at this temperature, and therefore real reaction temperature generally adopts 40 DEG C.And combinatorial mutagenesis type MC135 optimal reactive temperature is increased to 65 DEG C, and highly stable at 65 DEG C, and after processing 15 days continuously, residual activity is greater than 95%.The space-time yield of mutant conversion of substrate under 65 DEG C of conditions has significant improvement than female parent, the crude enzyme liquid of 3g/L can transform 100g/L4-chloroacetyl acetacetic ester completely and generate (S)-CHBE in 18min, ee value >99%, space-time yield 333g/ (L.h), exceeds more than 4 times than the maximum space-time yield of female parent.And can being completely converted in 30min, 40min, 1h respectively of 150g/L, 200g/L, 300g/L.The results are shown in Figure of description 4.The application example analysis of 7.2 combinatorial mutagenesises MC23, MC35, MC1345
Under 30 DEG C of conditions, the maternal carbonyl reductase ChKRED20 crude enzyme liquid of 6g/L can transform 3, the 5-bis trifluoromethyl methyl phenyl ketones generation R type alcohol of 200g/L, transformation efficiency >99%, ee value >99% in 12h.This product is a crucial chiral intermediate of synthesis Aprepitant.The space-time yield of mutant conversion of substrate under 50 DEG C of conditions improves greatly than female parent.Under 50 DEG C of conditions, 6g/L combinatorial mutagenesis MC23, MC35 and MC1345 reduce 3,5-bis trifluoromethyl methyl phenyl ketone completely and generate Aprepitant crucial chiral intermediate R type alcohol and need 7h, 6.3h and 5h respectively, space-time yield be the 1.7-2.4 of wild-type doubly.
SEQIDNO.1
ATGGGAATTTTAGACAACAAAGTAGCACTTGTTACAGGAGCAGGATCCGGAATCGGATTAGCTGTTGCTCATTCGTATGCAAAAGAAGGCGCCAAAGTTATTGTATCCGATATTAATGAAGATCACGGTAACAAAGCAGTCGAAGACATTAAAGCACAAGGCGGGGAAGCGTCTTTTGTAAAAGCAGATACTTCAAACCCTGAAGAAGTGGAAGCTTTAGTAAAAAGAACAGTAGAAATCTACGGAAGACTTGATATTGCATGTAATAATGCGGGAATCGGTGGCGAACAGGCGCTGGCAGGCGATTACGGTCTCGACAGCTGGCGAAAAGTATTAAGCATAAATCTTGATGGCGTATTCTACGGGTGCAAATATGAGTTAGAACAAATGGAAAAAAACGGGGGCGGCGTTATTGTGAATATGGCCTCTATTCATGGTATTGTTGCTGCACCGCTTTCCTCAGCCTACACTTCTGCAAAGCACGCAGTGGTAGGGCTTACTAAAAATATAGGAGCAGAATACGGACAGAAAAATATCCGTTGCAATGCGGTGGGGCCTGCTTATATTGAAACCCCGCTGTTGGAAAGCCTGACAAAGGAAATGAAGGAAGCACTGATTTCAAAACATCCGATGGGAAGACTGGGAAAACCTGAAGAAGTAGCAGAACTGGTGTTGTTCCTGAGTTCAGAAAAATCATCTTTTATGACGGGAGGCTATTATCTTGTAGATGGTGGCTACACGGCAGTTTAA
SEQIDNO.2
MGILDNKVALVTGAGSGIGLAVAHSYAKEGAKVIVSDINEDHGNKAVEDIKAQGGEASFVKADTSNPEEVEALVKRTVEIYGRLDIACNNAGIGGEQALAGDYGLDSWRKVLSINLDGVFYGCKYELEQMEKNGGGVIVNMASIHGIVAAPLSSAYTSAKHAVVGLTKNIGAEYGQKNIRCNAVGPAYIETPLLESLTKEMKEALISKHPMGRLGKPEEVAELVLFLSSEKSSFMTGGYYLVDGGYTAV
Claims (5)
1. the carbonyl reduction enzyme mutant of a thermostability raising, it is characterized in that with the sequence shown in SEQIDNO.2 for sequence of setting out, be Threonine by the alanine mutation of the 100th, the Aspartic acid mutations of the 102nd is L-glutamic acid, the glutamic acid mutation of the 128th is Methionin, the glutamic acid mutation of the 131st is glycine, the mutant serine of the 154th is the mutant that obtains of proline(Pro) or its arbitrary combination or increase the mutant that neutral mutation site obtains on this basis.
2. carbonyl reduction enzyme mutant according to claim 1, is characterized in that being Threonine by the alanine mutation of the 100th, the glutamic acid mutation of the 128th is Methionin, the mutant serine of the 154th is proline(Pro).
3. carbonyl reduction enzyme mutant according to claim 1, is characterized in that the glutamic acid mutation of the 131st is glycine, the valine mutation of the 137th is Isoleucine.
4. carbonyl reduction enzyme mutant according to claim 1, is characterized in that being aspartic acid by the glutamic acid mutation of the 71st, the lysine mutation of the 110th is arginine, the mutant serine of the 154th is proline(Pro).
5. the application of carbonyl reduction enzyme mutant according to claim 1 in catalysis of carbonyl compound.
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