CN105861457A - Enzyme-activity-improved ethanol dehydrogenase mutant and preparing method and application thereof - Google Patents
Enzyme-activity-improved ethanol dehydrogenase mutant and preparing method and application thereof Download PDFInfo
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Abstract
The invention discloses an enzyme-activity-improved ethanol dehydrogenase mutant and a preparing method and application thereof, and belongs to the field of biological medicine and the field of chemical industry. According to the enzyme-activity-improved ethanol dehydrogenase mutant and the preparing method and application thereof, the amino acid sequence nearby the active site of ethanol dehydrogenase is optimized, the ethanol dehydrogenase mutant is obtained, and the activity of the ethanol dehydrogenase mutant can be mostly improved 36%. When the enzyme-activity-improved ethanol dehydrogenase mutant is applied, the dosage of enzymes can be greatly decreased in the conversion process, and when S-N-t-butyloxycarboryl-3-hydroxypiper is prepared in a large-scale mode, production cost can be effectively reduced, and remarkable economic benefits are generated.
Description
Technical field
The present invention relates to a kind of enzyme alcohol dehydrogenase enzyme mutant improved alive and preparation method and application, belong to biological medicine and change
Work field.
Background technology
Ethanol dehydrogenase (alcohol dehydrogenase, ADH, E.C.1.1.1.1) is also referred to as ketoreductase (keto-reductase),
It is widely present in plant tissue, microorganism, people and mammal, with NAD+、NADP+Or PQQ is coenzyme, reversible
Catalysis oxidation short chain alcohol, aromatic alcohol etc. be corresponding aldehydes or ketone, be the important oxidoreductase of a class.At present to many
Plant the aerobic archeobacteria of microorganism such as superhigh temperature (Hyperthermophilic), fusobacterium (Clostridium beijerincki), sulfur
Change leaf Pseudomonas (Sulfolobus solfataricus), pseudomonas (Pseudomonas aeruginosa), acetobacter (Acetobacter
Pasteurianus), the ethanol that lactic acid bacteria (Lactobacillus brevis), Rhod (Rhodococcus ruber) are originated takes off
Hydrogen enzyme conducts in-depth research.Research finds, compared with the ethanol dehydrogenase that other are originated, derives from R.ruber DSM 44541
Ethanol dehydrogenase there is stronger organic solvent-resistant especially acetone and the characteristic of isopropanol, this characteristic makes substrate dissolubility
It is greatly improved.Further, acetone and isopropanol can be as the cosubstrate of the aldehyde ketone change of alcohol dehydrogenase enzyme catalysis for cofactors
Regeneration.
A lot of piperidine derivatives have antibacterial, anesthesia, antitumor, treatment senile dementia, diabetes and virus to be infected and (wraps
Include AIDS) etc. multiple pharmacologically active, wherein S-1-tertbutyloxycarbonyl-3-hydroxy piperidine be a kind of have chiral structure important in
Mesosome, is widely used in the synthesis of the medicines such as analgesia, psychosis and antitumor.This compound is used for as key intermediate
The inhibitor of synthesis of selective suppression bruton's tyrosine kinase (BTK) replaces Buddhist nun (Ibrutinib) according to Shandong.Synthesis S-1-tertiary fourth oxygen at present
The method of carbonyl-3-hydroxy piperidine is divided into chemical transformation and biological catalysis.Chemical transformation includes the fractionation of raceme and entirely closes
Become two kinds of methods, but all exist expensive, the shortcomings such as yield is low, prepare the most now (S)-1-tertbutyloxycarbonyl-3-hydroxyl
The method of the commonly used living things catalysis of method of phenylpiperidines.Living things catalysis rule needs the enzyme of high vigor.
Summary of the invention
The technical problem that the invention solves the problems that is to provide a kind of enzyme alcohol dehydrogenase enzyme mutant improved alive, is to aminoacid sequence
The tyrosine of the 54th of row ethanol dehydrogenase as shown in SEQ ID NO.1, or the phenylalanine of the 286th, or the
One or more in the tyrosine of 294 suddenlys change.
In one embodiment of the invention, described mutant is that the tyrosine of the 54th is sported tryptophan W.
In one embodiment of the invention, described mutant is that the phenylalanine of the 286th is sported leucine L.
In one embodiment of the invention, described mutant is that the tyrosine of 294 is sported phenylalanine F.
In one embodiment of the invention, the aminoacid sequence of described mutant is respectively as shown in SEQ ID NO.3,4,5.
Another technical problem that the invention solves the problems that is to provide the method obtaining above-mentioned alcohol dehydrogenase enzyme mutant, including walking as follows
Rapid:
Obtained the complete sequence of the gene of encoding wild type ethanol dehydrogenase by the method for PCR or gene chemical synthesis, arrange as mould with total order
Plate, is suddenlyd change to ethanol dehydrogenase wild type gene by rite-directed mutagenesis, it is thus achieved that alcohol dehydrogenase enzyme mutant.
Another that the invention solves the problems that technical problem is that the arbitrary described alcohol dehydrogenase enzyme mutant of application SEQ ID NO.3 to 5
Or the cell containing the arbitrary described alcohol dehydrogenase enzyme mutant of SEQ ID NO.3 to 5 prepares S-N-tertbutyloxycarbonyl-3-hydroxyl piperazine
The method of pyridine, comprises the steps:
With the arbitrary described alcohol dehydrogenase enzyme mutant of SEQ ID NO.3 to 5 as catalyst, with N-tertbutyloxycarbonyl-3-piperidones it is
Substrate, prepares S-N-tertbutyloxycarbonyl-3-hydroxy piperidine in the presence of cofactor and hydrogen donor.
In one embodiment of the invention, described cofactor is NADP or NAD.
In one embodiment of the invention, described hydrogen donor is isopropanol.
In one embodiment of the invention, described preparation reaction is carried out in the water phase buffer solution of pH7.0～9.0, and temperature is
25 DEG C～45 DEG C.
Beneficial effects of the present invention: by optimizing alcohol dehydrogenase activity location proximate aminoacid sequence, obtain SEQ ID NO.3
To alcohol dehydrogenase enzyme mutant shown in 5, its activity is the highest improves 36% than wild type.This enzyme ethanol improved alive is applied to take off
During hydrogen enzyme mutant, in the conversion process consumption of enzyme can be greatly lowered, thus at extensive preparation S-N-tertbutyloxycarbonyl-3-
Can effectively reduce production cost during hydroxy piperidine, produce significant economic benefit.
Accompanying drawing explanation
Fig. 1 is wild type alcohol dehydrogenase enzymatic structure；
Fig. 2 is that in embodiment 3, the enzyme reaction of wild type alcohol dehydrogenase obtains product HPLC figure after 22 hours；
Fig. 3 is to obtain product HPLC after alcohol dehydrogenase enzyme mutant KRED (ADH) in embodiment 3 _ F286L reacts 22 hours
Figure；
Fig. 4 is that in embodiment 3, alcohol dehydrogenase enzyme mutant obtains product yield comparison diagram after reacting 22 hours；
Fig. 5 is embodiment 4 product HNMR figure.
Detailed description of the invention
Product HPLC quantitative detecting method, condition:
Table A
Product chirality detection method, condition:
Table B
The acquisition of embodiment 1 wild type ethanol dehydrogenase
Wild type alcohol dehydrogenase gene shown in full genome synthesis SEQ ID NO.2, and according to the complete genome sequence following primer of design:
PCR forward primer: CTTTAAGAAGGAGATATACATATGAAAGCCGTCCAGTACACC
PCR downstream primer: GGCTTTGTTAGCAGCCGGATCTCA TCAGGGAACCACCACGCC
Table 1PCR reaction system
| Reagent | Consumption/μ l |
| PrimerStarPremix | 25 |
| PrimerFF | 1 |
| PrimerRR | 1 |
| DNAtemplate | 1 |
| ddH2O | 22 |
PCR reaction condition is as follows:
98 DEG C of 1min, 98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kbp, 30cycles, 16 DEG C.
PCR expands wild type alcohol dehydrogenase gene sequence；
PET21a carrier segments is carried out PCR process simultaneously:
PCR forward primer: ATGTATATCTCCTTCTTAAAG
PCR downstream primer: TGAGATCCGGCTGCTAACAAAGCCCGAAAGG
PCR system is with table 1；PCR reaction condition is as follows:
98 DEG C of 1min, 98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kbp, 30cycles, 16 DEG C
Glue reclaims genetic fragment and the pET21a carrier segments in the band mutational site that PCR obtains, according to gene sheet after mensuration concentration
Section than carrier segments 1:1 mass than cotransformation DH5a bacterial strain.Make it complete homologous recombination in somatic cells thus will carry wild
The nucleic acid fragment of type alcohol dehydrogenase gene inserts in pET-21a carrier and obtains pET-21a-KRED.Coated plate picking positive bacterium colony,
Incubated overnight in test tube, extraction plasmid preserves and samples and send order-checking.
By Plastid transformation BL21 (DE3) T1R bacterial strain correct for order-checking, picking converts correct bacterium colony test and expresses and preserve conduct
Produce strain.
Culture medium:
LB culture medium: yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L, pH7.0.
Solid medium adds 2% agar powder on this basis.
After obtaining engineered strain, inoculate 100ml triangle shaking flask incubated overnight, then according to 1:50 inoculates 1000ml culture bottle,
37 DEG C of 220rpm cultivate to OD600It is about 0.8, is cooled to 16 DEG C, add IPTG (ultimate density is 1mM), cultivate 10h,
Centrifuge washing collects thalline.
The acquisition of embodiment 2 alcohol dehydrogenase enzyme mutant
Three dimensional structure (PDB:2XAA) according to ethanol dehydrogenase, alcohol dehydrogenase activity immediate vicinity aminoacid include F43,
M47、Q51、A53、Y54、L119、A273、F281、F282、F286、Y294、W295.Owing to active center is attached
Near amino acid change may appreciable impact enzymatic activity, with Q51, A53, Y54, A273, F286, Y294 aminoacid position
As a example by Dian, wild type ethanol dehydrogenase is carried out rite-directed mutagenesis.Primer as listed in table 2 is designed according to mutational site.
Table 2 rite-directed mutagenesis primer sequence
| Primer | Primer sequence |
| KRED(ADH)_Q51M FF | CCGGCGGCGATGTACGCCTAC |
| KRED(ADH)_Q51M RR | GTAGGCGTACATCGCCGCCGG |
| KRED(ADH)_Q51L FF | CCGGCGGCGCTGTACGCCTAC |
| KRED(ADH)_Q51L RR | GTAGGCGTACAGCGCCGCCGG |
| KRED(ADH)_A53L FF | GCGCAGTACCTCTACGGCCTG |
| KRED(ADH)_A53L RR | CAGGCCGTAGAGGTACTGCGC |
| KRED(ADH)_A53M FF | GCGCAGTACATGTACGGCCTG |
| KRED(ADH)_A53M RR | CAGGCCGTACATGTACTGCGC |
| KRED(ADH)_Y54F FF | CAGTACGCCTTCGGCCTGCCG |
| KRED(ADH)_Y54F RR | CGGCAGGCCGAAGGCGTACTG |
| KRED(ADH)_Y54W FF | CAGTACGCCTGGGGCCTGCCG |
| KRED(ADH)_Y54W RR | CGGCAGGCCCCAGGCGTACTG |
| KRED(ADH)_A273L FF | GGCATCCACCTCGGCGCACAC |
| KRED(ADH)_A273L RR | GTGTGCGCCGAGGTGGATGCC |
| KRED(ADH)_F286L FF | ATGATCCCGTTAGGCGCCTCC |
| KRED(ADH)_F286L RR | GGAGGCGCCTAACGGGATCAT |
| KRED(ADH)_F286L FF | ATGATCCCGTTAGGCGCCTCC |
| KRED(ADH)_F286L RR | GGAGGCGCCTAACGGGATCAT |
| KRED(ADH)_Y294F FF | GTGACCCCGTTCTGGGGCACC |
| KRED(ADH)_Y294F RR | GGTGCCCCAGAACGGGGTCAC |
| KRED(ADH)_Y294L FF | GTGACCCCGTTATGGGGCACC |
| KRED(ADH)_Y294L RR | GGTGCCCCATAACGGGGTCAC |
Table 3PCR reaction system
| Reagent | Consumption/μ l |
| PrimerStar Premix | 25 |
| Primer FF | 1 |
| Primer RR | 1 |
| DNA template | 1 |
| ddH2O | 22 |
PCR reaction condition is as follows:
98 DEG C of 1min, 98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kbp, 30cycles, 16 DEG C,
With pET-21a-KRED as template, 5 ' ends (a) listed by PCR amplification table 4,5 and 3 ' end (b) two fragments:
Table 4a fragment PCR
Table 5b fragment PCR
Glue reclaims 5 ' end and 3 ' end fragments.
Hold with 5 ' and 3 ' end fragments are as template, the genetic fragment in band mutational site listed by PCR amplification table 6:
The genetic fragment in table 6 band mutational site
Table 7PCR reaction system
| Reagent | Consumption/μ l |
| PrimerStar Premix | 25 |
| Primer FF | 1 |
| Primer RR | 1 |
| DNA template | A fragment 0.5ul+B fragment 0.5 μ l |
| ddH2O | 22 |
PCR reaction condition is as follows:
98℃1min；98℃10s、55℃5s、72℃5s/kbp,30cycles；16℃.
PET21a carrier segments is carried out PCR process simultaneously:
PCR forward primer: ATGTATATCTCCTTCTTAAAG
PCR downstream primer: TGAGATCCGGCTGCTAACAAAGCCCGAAAGG
PCR system is with table 2；PCR reaction condition is as follows:
98 DEG C of 1min, 98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kbp, 30cycles, 16 DEG C.
Glue reclaims genetic fragment and the pET21a carrier segments in the band mutational site that PCR obtains, according to gene sheet after mensuration concentration
Section than carrier segments 1:1 mass than cotransformation DH5a bacterial strain.Make it complete homologous recombination in somatic cells thus band is suddenlyd change
The nucleic acid fragment in site inserts in pET-21a carrier.Coated plate picking positive bacterium colony, incubated overnight in test tube, extract plasmid and protect
Deposit and sample and send order-checking.
By Plastid transformation BL21 (DE3) T1R bacterial strain correct for order-checking, picking converts correct bacterium colony test and expresses and preserve conduct
Produce strain.
Culture medium:
LB culture medium: yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L, pH7.0.
Solid medium adds 2% agar powder on this basis.
After obtaining engineered strain, inoculate 100ml triangle shaking flask incubated overnight, then according to 1:50 inoculates 1000ml culture bottle,
37 DEG C of 220rpm cultivate to OD600It is about 0.8, is cooled to 16 DEG C, add IPTG (ultimate density is 1mM), cultivate 10h,
Centrifuge washing collects thalline.
The active testing of embodiment 3 ethanol dehydrogenase
By resuspended for above-mentioned collection thalline 50mM sodium phosphate buffer (pH7.0) to 3.5g/L, use high-pressure homogenization cell breakage
Instrument 15000Psi ice-water bath high-pressure homogenization crushes 2 times, obtains cell pyrolysis liquid.
Activity of enzyme reaction system (5ml reaction system) including: N-t-butoxycarbonyl-piperidin ketone 0.1g/ml, isopropanol 10% (v/v),
Na2HPO4·12H2O 24mg/ml, NaH2PO4·2H2O 6.6mg/ml, MgCl21mg/ml, coenzyme 0.4mg/ml, cell
Lysate 30% (v/v).
It is placed in 30 DEG C of 200rpm reactions of constant-temperature table；After reaction 15h, mend isopropanol 0.5ml；When continuing reaction to overall reaction
Between be 22h.After reaction terminates, sampling adds 2 times of volumes of acetonitrile extractings, and centrifugal 20min takes supernatant, 0.45 μm membrane filtration,
HPLC detects, and calculates conversion ratio.After enzymatic activity is defined as 30 degrees Celsius of reaction 22h, HPLC detects product N-tertiary butyloxycarbonyl
The peak area of base-piperidones accounts for the percentage ratio of substrate peak area.Alcohol dehydrogenase activity after sudden change improves 36% than wild type,
Optical purity e.e% is 100%.
Embodiment 4 applies alcohol dehydrogenase enzyme mutant to convert N-t-butoxycarbonyl-piperidin ketone
By resuspended to 50g/L for KRED (ADH)-F286L sudden change thalline 50mM sodium phosphate buffer (pH7.0), use height
Pressure homogenate cell crushing instrument 15000Psi ice-water bath high-pressure homogenization crushes 2 times, obtains cell pyrolysis liquid.
N-t-butoxycarbonyl-piperidin ketone (5.0g, 0.025mol) is dissolved in 5mL isopropanol 30 DEG C standing, weighs
Na2HPO4.12H2O (1.2g, 0.003mol), NaH2PO4.2H2O (0.33g, 0.002mol) adds 29ml water dissolution,
It is made into phosphate buffer.Measure 15mL cell pyrolysis liquid enzyme liquid, add 50mg MgCl2.6H2O, 40mg NADP.Will
Phosphate buffer, enzyme liquid is sequentially added in substrate, 2N/L NaOH solution regulation pH to 7-8, is heated to about 30 DEG C, and
Within every four hours, monitor and regulate pH.Reaction 54h.HPLC monitoring N-t-butoxycarbonyl-piperidin ketone is less than 0.5%.By reactant liquor
Filtering with kieselguhr, filter cake EA (25mL × 3) washes three times, and filtrate same volume EA extracts three times.Merge organic facies, anhydrous sulfur
Acid sodium is dried, and is spin-dried for obtaining yellow oil 4.5g, yield 89.1%.The Structural Identification data of product are
H-NMR (400MHz, DMSO): δ=1.27-1.30 [m, 2H ,-CH2], 1.40 [s, 9H ,-(CH) 3], 1.61-1.82 [m, 2H ,-CH2]
2.64-2.78[m,2H,-CH2],3.37-3.39[m,1H,-CH],3.59-3.77[m,2H,-CH2],4.84-4.85[d,1H,-OH]。
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (10)
1. the enzyme alcohol dehydrogenase enzyme mutant improved alive, it is characterised in that be by the ammonia of alcohol dehydrogenase activity location proximate
Base acid mutation obtains.
A kind of enzyme the most according to claim 1 alcohol dehydrogenase enzyme mutant improved alive, it is characterised in that be to aminoacid
The tyrosine of the 54th of sequence ethanol dehydrogenase as shown in SEQ ID NO.1, or the phenylalanine of the 286th, or
One or more in the tyrosine of the 294th suddenlys change.
A kind of enzyme the most according to claim 1 and 2 alcohol dehydrogenase enzyme mutant improved alive, it is characterised in that described prominent
Variant is that the tyrosine of the 54th is sported tryptophan W.
A kind of enzyme the most according to claim 1 and 2 alcohol dehydrogenase enzyme mutant improved alive, it is characterised in that described prominent
Variant is that the phenylalanine of the 286th is sported leucine L.
A kind of enzyme the most according to claim 1 and 2 alcohol dehydrogenase enzyme mutant improved alive, it is characterised in that described prominent
Variant is that the tyrosine of 294 is sported phenylalanine F.
6. prepare the method for alcohol dehydrogenase enzyme mutant described in claim 1 or 2 for one kind, it is characterised in that comprise the steps:
Obtained the complete sequence of the gene of encoding wild type ethanol dehydrogenase by the method for PCR or gene chemical synthesis, arrange as mould with total order
Plate, is suddenlyd change to ethanol dehydrogenase wild type gene by rite-directed mutagenesis, it is thus achieved that alcohol dehydrogenase enzyme mutant.
7. the method preparing S-N-tertbutyloxycarbonyl-3-hydroxy piperidine, it is characterised in that described in claim 1 or 2
Alcohol dehydrogenase enzyme mutant or the cell containing described alcohol dehydrogenase enzyme mutant are catalyst, with N-tertbutyloxycarbonyl-3-piperidones
For substrate, in the presence of cofactor and hydrogen donor, prepare S-N-tertbutyloxycarbonyl-3-hydroxy piperidine.
Method the most according to claim 7, it is characterised in that described cofactor is NADP or NAD.
Method the most according to claim 7, it is characterised in that described hydrogen donor is isopropanol.
Method the most according to claim 7, it is characterised in that preparation reaction is in the water phase buffer solution of pH7.0～9.0
Carrying out, temperature is 25 DEG C～45 DEG C.
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Cited By (9)
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| CN106497996A (en) * | 2016-10-11 | 2017-03-15 | 凯莱英医药集团（天津）股份有限公司 | The enzyme catalysiss preparation method of chiral alcohol |
| CN108949707A (en) * | 2017-05-24 | 2018-12-07 | 武汉大学 | A kind of Alcohol dehydrogenase mutant that thermal stability improves |
| CN109295019A (en) * | 2018-10-29 | 2019-02-01 | 浙江大学 | A kind of Alcohol dehydrogenase mutant and its application |
| CN110129307A (en) * | 2019-05-23 | 2019-08-16 | 南京趣酶生物科技有限公司 | Immobilization Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN110229796A (en) * | 2019-05-23 | 2019-09-13 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN110551700A (en) * | 2018-05-31 | 2019-12-10 | 康码（上海）生物科技有限公司 | ADH protein family mutant and application thereof |
| CN110777125A (en) * | 2019-11-15 | 2020-02-11 | 江南大学 | Efficient preparation method of heterocyclic drug intermediate |
| CN115011574A (en) * | 2022-06-24 | 2022-09-06 | 杭州师范大学 | Preparation method and application of site-controllable and ordered cross-linked double-enzyme aggregate |
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| CN108949707B (en) * | 2017-05-24 | 2020-07-10 | 武汉大学 | Alcohol dehydrogenase mutant with improved thermal stability |
| CN108949707A (en) * | 2017-05-24 | 2018-12-07 | 武汉大学 | A kind of Alcohol dehydrogenase mutant that thermal stability improves |
| CN110551700B (en) * | 2018-05-31 | 2022-06-21 | 康码（上海）生物科技有限公司 | ADH protein family mutant and application thereof |
| CN110551700A (en) * | 2018-05-31 | 2019-12-10 | 康码（上海）生物科技有限公司 | ADH protein family mutant and application thereof |
| CN109295019A (en) * | 2018-10-29 | 2019-02-01 | 浙江大学 | A kind of Alcohol dehydrogenase mutant and its application |
| CN109295019B (en) * | 2018-10-29 | 2021-01-12 | 浙江大学 | Alcohol dehydrogenase mutant and application thereof |
| CN110129307A (en) * | 2019-05-23 | 2019-08-16 | 南京趣酶生物科技有限公司 | Immobilization Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN110229796A (en) * | 2019-05-23 | 2019-09-13 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN110229796B (en) * | 2019-05-23 | 2023-09-05 | 安徽趣酶生物科技有限公司 | Ketoreductase mutant and application thereof in preparation of duloxetine chiral alcohol intermediate and analogue thereof |
| CN110129307B (en) * | 2019-05-23 | 2023-09-05 | 安徽趣酶生物科技有限公司 | Immobilized ketoreductase mutant and application thereof in preparation of duloxetine chiral alcohol intermediate and analogue thereof |
| CN110777125A (en) * | 2019-11-15 | 2020-02-11 | 江南大学 | Efficient preparation method of heterocyclic drug intermediate |
| CN115011574A (en) * | 2022-06-24 | 2022-09-06 | 杭州师范大学 | Preparation method and application of site-controllable and ordered cross-linked double-enzyme aggregate |
| CN116042555A (en) * | 2022-08-17 | 2023-05-02 | 无锡佰翱得生物科学有限公司 | Alcohol dehydrogenase mutant and application thereof |
| CN116042555B (en) * | 2022-08-17 | 2023-10-27 | 无锡佰翱得生物科学股份有限公司 | Alcohol dehydrogenase mutant and application thereof |
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