CN106520716A - Thermophilic ketone reductase mutant and application thereof - Google Patents
Thermophilic ketone reductase mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a thermophilic ketone reductase mutant. Tryptophan at the 21 position of thermophilic ketone reductase with an amino acid sequence being SEQ ID NO:1 is mutated into cysteine or glutamic acid, and/or tryptophan at the 86 position is mutated into alanine, leucine and valine, and the thermophilic ketone reductase mutant is obtained. Compared with wild type enzyme, thermophilic ketone reductase is used for catalyzing N-Boc-3-piperidone and enzyme activity of obtained (S)-N-Boc-3-pipradrol is multiplied, especially when double mutant is conducted, the enzyme activity can increase by about 10 fold, enantioselectivity of the catalyzed product is also improved, the enantioselectivity can reach as high as 99.8%, and the thermophilic ketone reductase mutant has a good industrial application prospect.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of thermophilic Ketoreductase mutant and its application.
Background technology
(S)-N-Boc-3- piperidine alcohols can be used for synthesizing a kind of non-natural medicine congestion DHF medicine Ka Morui
The different white thorn of woods, BTK inhibitor ibrurinib, natural materials drenches amine and the little gingko thorn prodrug such as alkali, market demand compared with
Height, therefore such compound is with a wide range of applications.
(S)-N-Boc-3- piperidine alcohols can be prepared by N-Boc-3- piperidones asymmetric reduction, introduce chiral centre.Hand
The prior synthesizing method of property alcohol has electronation and biological reducing.This process is capable of achieving using chemical catalysis method, but instead
Should be less efficient, condition is harsh, and the three wastes for producing are also more, do not have to realize industrial applications.Biocatalysis method is anti-
Mild condition is answered, green non-pollution, stereoselectivity are single-minded, with the potentiality promoted.
Biologically, generating for chiral product can pass through the chemical catalysis with enzyme, asymmetric reduction, multistep reaction synthesis.
Wherein, the asymmetric reduction of living things catalysis reaches can product enantioselectivity theoretically, under mild conditions
99%.At present increasing medicine or its intermediate are rather than the chemical syntheses by way of a kind of biological enzyme synthesizes.
It is obvious that enzymatic has great advantage compared to traditional chemical synthesis：1, poisonous reaction can be avoided in biological respinse to try
Agent or solvent；2, the features such as enzyme has high efficiency, substrate specificity；3, it is to avoid the side reaction including including racemization etc..
Realize reducing the technology for producing chiral alcohol comparative maturity using bioconversion, but literature search understands, only
One example using the carrot intact cell catalysis reduction reaction, used catalyst account for about the 20% of substrate quality, and source is unstable
Determine and heat endurance is not high.Contain various complicated ingredients in plant cell reaction, difficulty is increased to downstream extraction.It is prior
It is, the reaction substrate concentration relatively low (3.3mM), the extremely low only 0.05U/mg of enzyme activity, product optical purity has 98%, and does not have
Standby practical application in industry is worth.
All report in many documents and patent hydroxylated with regard to ketoreductase catalysis reduction ketone group containing substrate generation
Product, makes reaction become high efficiency, with the advantage such as substrate specificity or the pure selectivity of optically-active.For example, some researchs are from reddish brown
Color shadow yeast extracts a kind of ketoreductase catalysis to replacing acetophenone (Cl-, Br-, Me-, OMe-, MeC3, CF3 -Deng), optically-active
It is selective extremely low, need by design and rational mutational site, rite-directed mutagenesis by the methionine of 242 in its amino acid sequence with
Leucine replaces, and in amino acid sequence, the glutamine of 245 is replaced with proline, constitutes effective double-mutant enzyme.This is dashed forward
Variant enzyme is catalyzed with various substituents under old terms to replacing acetophenone, the product configuration of generation is changed into by R-
S-, while making the ee values of former S- products improve to 99%.This achievement has high value.
Also there is correlative study such as：Ketone fat reductase (ker) of Penicillium citrinum is extracted from, for being catalyzed the bromo- 3- epoxides butyric acid of 4-
The bromo- 3-hydroxybutyrates of highly purified (S-) -4- are generated, enzyme activity stability is low, and enzyme activity is reduced to just typically after 30 DEG C of heating bath 6h
The 15% of beginning enzyme activity.In order to improve stability, then error-prone PCR random mutation carries out high flux to mutant
Screening, final to obtain the single mutant that heat endurance is improved, they are 54 bright in its specific amino acid sequence respectively
The single mutant that propylhomoserin is replaced to by glutamine, in amino acid sequence, the arginine of 245 is dashed forward by the list that lysine is replaced to
The single-mutant enzyme that the asparagine of 271 positions is replaced to by aspartic acid in variant, and amino acid sequence, especially amino acid
After 30 DEG C of heating bath 6h, activity brings up to initial enzyme activity to the mutant enzyme that the leucine of 54 is replaced by glutamine in sequence
62%, the effect highly significant after mutation.
The content of the invention
The invention provides a kind of thermophilic Ketoreductase mutant and its application, Ketoreductase mutant that this is thermophilic is with very
The vigor of (S)-N-Boc-3- piperidine alcohols is catalyzed and synthesized well, with good prospects for commercial application.
Wild-type thermophilic ketoreductase (Gene Bank is WP_004082270.1) is from Thermotoga maritima
(Thermotoga maritime), but wild-type thermophilic ketoreductase catalysis N-Boc-3- piperidones obtains (S)-N-Boc-3-
The activity of piperidine alcohols is weaker, and S types N-Boc-3- piperidine alcohols enantiomeric excess (ee values) is 98.5% in product, also a small amount of
R type N-Boc-3- piperidine alcohols, so also not possessing practical application in industry value.If can be improved by genetic engineering modified
Its enzymatic activity and product optical purity (ee values), then which possesses corresponding industrial application value.
A kind of thermophilic Ketoreductase mutant, by amino acid sequence such as SEQ ID NO：Thermophilic ketoreductase shown in 1
21 tryptophans sport cysteine or glutamic acid, and/or the 86th tryptophan sports alanine, leucine or valine
Obtain.
By the active region design and rational to wild-type thermophilic ketoreductase, multiple sites are carried out with fixed point saturation and is dashed forward
Become, then after expression and purification, carry out the detection of enzyme activity and product ee values, final screening obtains the 21st tryptophan and sports half Guang ammonia
Acid or glutamic acid, or the 86th tryptophan sport alanine, leucine or valine, the enzyme of gained simple point mutation, enzyme activity are obtained
Growth at double is obtained, and the product ee values of enzymic catalytic reaction also increase.
Further, while the mutation of the mutation of the 21st and the 86th is introduced thermophilic ketoreductase, gained is double prominent
In change, the 21st tryptophan sports cysteine or glutamic acid, at the same the 86th tryptophan sport alanine, leucine or
Valine, the double mutant enzyme in 6 produced after combination kind, after testing enzyme activity can improve 10 times or so, and the product of enzymic catalytic reaction
Ee values also all reach more than 99%, and highest is even up to 99.8%.Amino acid sequence after the mutation such as SEQ ID NO：
3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：Shown in 8.The present invention is carried again
The gene of the coding thermophilic Ketoreductase mutant is supplied.
Invention further provides the expression cassette comprising the gene.
Present invention also offers the recombinant expression carrier comprising the gene.
Present invention also offers the genetic engineering bacterium comprising the recombinant expression carrier.
Present invention also offers the thermophilic Ketoreductase mutant obtains (S)-N- in catalysis N-Boc-3- piperidones
Application in Boc-3- piperidine alcohols.
Compared with wild-type enzyme, catalysis N-Boc-3- piperidones obtains (S)-N- to Ketoreductase mutant that the present invention is thermophilic
When the enzyme activity of Boc-3- piperidine alcohols has obtained a large amount of raisings, particularly double mutation, it is possible to increase 10 times or so, and catalytic reaction is produced
The optically-active of thing has selectively also increased, and highest can reach 99.8%, possess good prospects for commercial application.
Description of the drawings
Fig. 1 is the reaction that thermophilic Ketoreductase mutant is catalyzed that N-Boc-3- piperidones obtains (S)-N-Boc-3- piperidine alcohols
Schematic diagram；
Fig. 2 is the HPLC testing result figures of the catalytic reaction products of thermophilic Ketoreductase mutant W86A.
Specific embodiment
Embodiment 1
Wild-type thermophilic ketoreductase (Gene Bank is WP_004082270.1), but wild-type thermophilic ketoreductase
Enzyme activity it is relatively low, and ee (S) value is relatively low, it is impossible to meet the requirement of commercial Application well.
Wild-type thermophilic ketoreductase sequence such as SEQ ID NO：Shown in 1, with containing the wild-type thermophilic ketoreductase base
Because of (nucleotide sequence such as SEQ ID NO：Shown in 2) pET-28a recombinant plasmids be template, will point W21, it is full that W86 carries out fixed point
And mutation.Hence with the flat end primer of oligo7 Software for Design, laggard performing PCR rite-directed mutagenesis, primer are as shown in table 1, wherein
W21-R and W86-R is reverse primer, and remaining is forward primer, and the reverse primer in wherein each site is identical, in forward primer
First three base is mutational site.Using KOD-Plus-Neo archaeal dna polymerase kits, (science and technology is spun by Japan to be had for this PCR mutation
Limit company, Shanghai).
1 primer sequence of table.
Reaction system such as following table：
PCR cycle process：
PCR primer is reclaimed, Dpn I (1 μ l) is added in the system of 50 μ l, is kept for 37 DEG C, 1.5h, to digest template
DNA, then with agarose (1%) detected through gel electrophoresis its whether rite-directed mutagenesis is completed.Run out of on Ago-Gel is ensured bright
After aobvious band, after PCR cleaning kits (buying in JaRa bioengineering Co., Ltd, Shanghai) cleaning, PNK companies are carried out
Connect.
PNK kinases (JaRa bioengineering Co., Ltd, Shanghai) linked system is：
Connection 5 μ l of buffer solution
PNK 1μl
10 μ l of PCR cleaning,
After being well mixed, it is positioned over 16 DEG C in PCR instrument, after 1.5h, connection can be completed.Connection product converts Escherichia coli,
After flat board culture, select the monoclonal for growing and send sequencing, checking sequence is correct, has obtained mutant plasmid.
Embodiment 2
1st, the expression of thermophilic ketoreductase and its mutant
The mutant plasmid that embodiment 1 is obtained is transformed in e. coli jm109 (DE3) competent cell, is then applied
Plate, culture, observe bacteria growing situation, the son activation of picking recombinant conversion, and Amplification Culture after 10h.
(1) by recombinant conversion be inoculated in equipped with 5ml containing kanamycins (50 μ g/ml) LB culture mediums in, be placed in 37 DEG C,
Concussion and cultivate in 200rpm shaking tables, reaches 0.4～0.5 or so to OD600, obtains seed liquor.
(2) seed liquor is inoculated in self-induction culture medium with 1% inoculum concentration, 25 DEG C are cultivated 48 hours.
Self-induction culture medium prescription is：Arabinose 3g/L, glucose 0.5g/L, glycerine 5g/L, peptone 10g/L, phosphorus
Hydrochlorate 6.8g/L, sulfate 1.2g/L, NH4Cl 2.65g/L, MgSO40.98g/L, CaCl2 0.1g/L。
By bacterium solution with ultrasonic disruption, supernatant after centrifugation, is obtained, the corresponding enzyme liquid of as each mutant.
Embodiment 3
Protein quantification is detected and each mutant Enzyme activity assay.
According to document (Woong Kim, Eric J.Bennett, Edward L.Huttlin, Ailan Guo, Jing Li,
Anthony Possemato (2011) .44,2,325 340.) each body variant enzyme liquid protein concentration of method measure.
Thermophilic Ketoreductase mutant catalysis N-Boc-3- piperidones obtains the reaction principle of (S)-N-Boc-3- piperidine alcohols
As shown in Figure 1.Design the enzyme activity that following reaction system surveys each mutant.
Reaction system (2ml)：In the PBS of 1.9ml 50mM pH 7.0, Boc-3- piperidones 20mg are added, thermophilic ketone is also
Protoenzyme or mutant 0.5mg, NADPH 10mg.37 DEG C, after shaking table reacts 0.5 hour, add after the extraction of 2ml ethyl acetate from
The heart, carries out HPLC liquid phase analysis.Each sample do three it is parallel.
Liquid-phase condition：OD-H posts (25cm), n-hexane：Isopropanol=92: 8,1ml/min, Detection wavelength：210nm.
The screening criteria of beneficial mutant is：1, mutant catalytic capability is higher than wild type；2, ee values are not less than 98%.
Each mutant enzyme vigor is as follows：
2 single mutant of table is screened.
It is suitable in the ee values and wild type for ensureing mutant enzyme, and on the premise of catalytic capability is higher than wild type, select as
Lower single mutant：W21E, W21C, W86L, W86V, W86A.
Wherein catalytic reaction products of mutant W86A, HPLC testing results are as shown in Figure 2.
Embodiment 4
On the basis of the screening of first round simple point mutation, the double mutation of further optimum organization make more effectively to be mutated
Body.
(point mutation process is with embodiment 1) is mutated by W21C, W21E double with W86L, W86V, W86A composition respectively, is built double
Mutant library, is transformed into culture in e. coli jm109 (DE3), chooses bacterium, activate, expressing protein, carries out liquid phase detection (side
Method is ibid), the vigor of filtering out increase and ee value stabilizations double-mutant, step is performed as described above described in a 1-3.Measure product ee values
It is such as following table with enzyme activity：
3 double-mutant of table is screened.
| Mutant | Enzyme activity (U/mg) | Ee (S) value (%) |
| WT | 3.1 | 98.5 |
| W21E/W86L | 23.5 | 99.5 |
| W21E/W86A | 28.4 | 99.3 |
| W21E/W86V | 32.7 | 99.7 |
| W21C/W86L | 29.6 | 99.8 |
| W21C/W86A | 33.1 | 99.4 |
| W21C/W86V | 30.9 | 99.0 |
Shown in experimental result, double-mutant enzyme W21E/W86L, W21E/W86A, W21E/W86V, W21C/W86L, W21C/
The catalytic capability of W86A, W21C/W86V is higher than about 10 times of wild type, and ee values are not less than 99%.
SEQUENCE LISTING
<110>Hangzhou Mei Yi Bioisystech Co., Ltd
<120>A kind of thermophilic Ketoreductase mutant and its application
<130>
<160> 48
<170> PatentIn version 3.3
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Arg Asp Glu Glu Met Val Glu Leu Leu Lys Thr Ala Ile Lys Met Gly
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Leu Gly
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Ala Thr Glu Ile Lys Leu Ser Glu Glu Glu Met Lys Leu Leu Asp Ser
260 265 270
Leu Gly
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Met Leu Tyr Lys Glu Leu Gly Arg Thr Gly Glu Glu Ile Pro Ala Leu
1 5 10 15
Gly Leu Gly Thr Cys Gly Ile Gly Gly Phe Glu Thr Pro Asp Tyr Ser
20 25 30
Arg Asp Glu Glu Met Val Glu Leu Leu Lys Thr Ala Ile Lys Met Gly
35 40 45
Tyr Thr His Ile Asp Thr Ala Glu Tyr Tyr Gly Gly Gly His Thr Glu
50 55 60
Glu Leu Ile Gly Lys Ala Ile Lys Asp Phe Arg Arg Glu Asp Leu Phe
65 70 75 80
Ile Val Ser Lys Val Ala Pro Thr His Leu Arg Arg Asp Asp Leu Leu
85 90 95
Arg Ser Leu Glu Asn Thr Leu Lys Arg Leu Asp Thr Asp Tyr Val Asp
100 105 110
Leu Tyr Leu Ile His Trp Pro Asn Pro Glu Ile Pro Leu Glu Glu Thr
115 120 125
Leu Ser Ala Met Ala Glu Gly Val Arg Gln Gly Leu Ile Arg Tyr Ile
130 135 140
Gly Val Ser Asn Phe Asp Arg Arg Leu Leu Glu Glu Ala Ile Ser Lys
145 150 155 160
Ser Gln Glu Pro Ile Val Cys Asp Gln Val Lys Tyr Asn Ile Glu Asp
165 170 175
Arg Asp Pro Glu Arg Asp Gly Leu Leu Glu Phe Cys Gln Lys Asn Gly
180 185 190
Val Thr Leu Val Ala Tyr Ser Pro Leu Arg Arg Thr Leu Leu Ser Glu
195 200 205
Lys Thr Lys Arg Thr Leu Glu Glu Ile Ala Lys Asn His Gly Ala Thr
210 215 220
Ile Tyr Gln Ile Met Leu Ala Trp Leu Leu Ala Lys Pro Asn Val Val
225 230 235 240
Ala Ile Pro Lys Ala Gly Arg Val Glu His Leu Arg Glu Asn Leu Lys
245 250 255
Ala Thr Glu Ile Lys Leu Ser Glu Glu Glu Met Lys Leu Leu Asp Ser
260 265 270
Leu Gly
<210> 8
<211> 274
<212> PRT
<213>Artificial sequence
<400> 8
Met Leu Tyr Lys Glu Leu Gly Arg Thr Gly Glu Glu Ile Pro Ala Leu
1 5 10 15
Gly Leu Gly Thr Cys Gly Ile Gly Gly Phe Glu Thr Pro Asp Tyr Ser
20 25 30
Arg Asp Glu Glu Met Val Glu Leu Leu Lys Thr Ala Ile Lys Met Gly
35 40 45
Tyr Thr His Ile Asp Thr Ala Glu Tyr Tyr Gly Gly Gly His Thr Glu
50 55 60
Glu Leu Ile Gly Lys Ala Ile Lys Asp Phe Arg Arg Glu Asp Leu Phe
65 70 75 80
Ile Val Ser Lys Val Val Pro Thr His Leu Arg Arg Asp Asp Leu Leu
85 90 95
Arg Ser Leu Glu Asn Thr Leu Lys Arg Leu Asp Thr Asp Tyr Val Asp
100 105 110
Leu Tyr Leu Ile His Trp Pro Asn Pro Glu Ile Pro Leu Glu Glu Thr
115 120 125
Leu Ser Ala Met Ala Glu Gly Val Arg Gln Gly Leu Ile Arg Tyr Ile
130 135 140
Gly Val Ser Asn Phe Asp Arg Arg Leu Leu Glu Glu Ala Ile Ser Lys
145 150 155 160
Ser Gln Glu Pro Ile Val Cys Asp Gln Val Lys Tyr Asn Ile Glu Asp
165 170 175
Arg Asp Pro Glu Arg Asp Gly Leu Leu Glu Phe Cys Gln Lys Asn Gly
180 185 190
Val Thr Leu Val Ala Tyr Ser Pro Leu Arg Arg Thr Leu Leu Ser Glu
195 200 205
Lys Thr Lys Arg Thr Leu Glu Glu Ile Ala Lys Asn His Gly Ala Thr
210 215 220
Ile Tyr Gln Ile Met Leu Ala Trp Leu Leu Ala Lys Pro Asn Val Val
225 230 235 240
Ala Ile Pro Lys Ala Gly Arg Val Glu His Leu Arg Glu Asn Leu Lys
245 250 255
Ala Thr Glu Ile Lys Leu Ser Glu Glu Glu Met Lys Leu Leu Asp Ser
260 265 270
Leu Gly
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ttaggtatcg gcggttttga 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
gctggtatcg gcggttttga 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<400> 11
tttggtatcg gcggttttga 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
atgggtatcg gcggttttga 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
tgcggtatcg gcggttttga 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
gacggtatcg gcggttttga 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
gaaggtatcg gcggttttga 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
ggaggtatcg gcggttttga 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
cacggtatcg gcggttttga 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
attggtatcg gcggttttga 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<400> 19
aaaggtatcg gcggttttga 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
aatggtatcg gcggttttga 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence
<400> 21
cccggtatcg gcggttttga 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
caaggtatcg gcggttttga 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence
<400> 23
agaggtatcg gcggttttga 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
tccggtatcg gcggttttga 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
gttggtatcg gcggttttga 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
actggtatcg gcggttttga 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
tatggtatcg gcggttttga 20
<210> 28
<211> 24
<212> DNA
<213>Artificial sequence
<400> 28
catccgaccc atctgcgtcg cgat 24
<210> 29
<211> 24
<212> DNA
<213>Artificial sequence
<400> 29
cgtccgaccc atctgcgtcg cgat 24
<210> 30
<211> 24
<212> DNA
<213>Artificial sequence
<400> 30
aaaccgaccc atctgcgtcg cgat 24
<210> 31
<211> 24
<212> DNA
<213>Artificial sequence
<400> 31
gatccgaccc atctgcgtcg cgat 24
<210> 32
<211> 24
<212> DNA
<213>Artificial sequence
<400> 32
tgtccgaccc atctgcgtcg cgat 24
<210> 33
<211> 24
<212> DNA
<213>Artificial sequence
<400> 33
ggtccgaccc atctgcgtcg cgat 24
<210> 34
<211> 24
<212> DNA
<213>Artificial sequence
<400> 34
gaaccgaccc atctgcgtcg cgat 24
<210> 35
<211> 24
<212> DNA
<213>Artificial sequence
<400> 35
agtccgaccc atctgcgtcg cgat 24
<210> 36
<211> 24
<212> DNA
<213>Artificial sequence
<400> 36
gctccgaccc atctgcgtcg cgat 24
<210> 37
<211> 24
<212> DNA
<213>Artificial sequence
<400> 37
tttccgaccc atctgcgtcg cgat 24
<210> 38
<211> 24
<212> DNA
<213>Artificial sequence
<400> 38
attccgaccc atctgcgtcg cgat 24
<210> 39
<211> 24
<212> DNA
<213>Artificial sequence
<400> 39
ttaccgaccc atctgcgtcg cgat 24
<210> 40
<211> 24
<212> DNA
<213>Artificial sequence
<400> 40
atgccgaccc atctgcgtcg cgat 24
<210> 41
<211> 24
<212> DNA
<213>Artificial sequence
<400> 41
aatccgaccc atctgcgtcg cgat 24
<210> 42
<211> 24
<212> DNA
<213>Artificial sequence
<400> 42
ccaccgaccc atctgcgtcg cgat 24
<210> 43
<211> 24
<212> DNA
<213>Artificial sequence
<400> 43
caaccgaccc atctgcgtcg cgat 24
<210> 44
<211> 24
<212> DNA
<213>Artificial sequence
<400> 44
actccgaccc atctgcgtcg cgat 24
<210> 45
<211> 24
<212> DNA
<213>Artificial sequence
<400> 45
gttccgaccc atctgcgtcg cgat 24
<210> 46
<211> 24
<212> DNA
<213>Artificial sequence
<400> 46
tatccgaccc atctgcgtcg cgat 24
<210> 47
<211> 22
<212> DNA
<213>Artificial sequence
<400> 47
ggtacccaga cccagtgccg ga 22
<210> 48
<211> 24
<212> DNA
<213>Artificial sequence
<400> 48
aactttgctc acaatgaaca gatc 24
Claims (6)
1. a kind of thermophilic Ketoreductase mutant, it is characterised in that by amino acid sequence such as SEQ ID NO：Thermophilic ketone shown in 1
The 21st tryptophan of reductase sports cysteine or glutamic acid, and/or the 86th tryptophan sports alanine, leucine
Or valine is obtained.
2. the gene of thermophilic Ketoreductase mutant as claimed in claim 1 is encoded.
3. the expression cassette of gene as claimed in claim 2 is included.
4. the recombinant expression carrier of gene as claimed in claim 2 is included.
5. the genetic engineering bacterium of recombinant expression carrier as claimed in claim 4 is included.
6. thermophilic Ketoreductase mutant obtains (S)-N-Boc-3- piperazines in catalysis N-Boc-3- piperidones as claimed in claim 1
Application in pyridine alcohol.
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| CN201610963639.9A CN106520716A (en) | 2016-10-28 | 2016-10-28 | Thermophilic ketone reductase mutant and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048416A (en) * | 2017-12-25 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | Improved Ketoreductase mutant and its preparation method and application |
| CN111363732A (en) * | 2020-03-12 | 2020-07-03 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus NIH2624 and application thereof |
| CN115537406A (en) * | 2021-06-30 | 2022-12-30 | 尚科生物医药（上海）有限公司 | Ketoreductase and application thereof in preparation of (S) -1- (4-pyridyl) -1,3-propylene glycol |
| CN116790569A (en) * | 2022-04-07 | 2023-09-22 | 杭州酶易生物技术有限公司 | Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048416A (en) * | 2017-12-25 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | Improved Ketoreductase mutant and its preparation method and application |
| CN108048416B (en) * | 2017-12-25 | 2021-05-18 | 吉林凯莱英医药化学有限公司 | Improved ketoreductase mutant and preparation method and application thereof |
| CN111363732A (en) * | 2020-03-12 | 2020-07-03 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus NIH2624 and application thereof |
| CN111363732B (en) * | 2020-03-12 | 2023-05-23 | 卡柔恩赛生物技术湖北有限公司 | Transaminase mutant from aspergillus terreus NIH2624 and application thereof |
| CN115537406A (en) * | 2021-06-30 | 2022-12-30 | 尚科生物医药（上海）有限公司 | Ketoreductase and application thereof in preparation of (S) -1- (4-pyridyl) -1,3-propylene glycol |
| CN115537406B (en) * | 2021-06-30 | 2024-04-12 | 尚科生物医药（上海）有限公司 | Ketoreductase and application thereof in preparation of (S) -1- (4-pyridyl) -1, 3-propanediol |
| CN116790569A (en) * | 2022-04-07 | 2023-09-22 | 杭州酶易生物技术有限公司 | Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound |
| CN116790569B (en) * | 2022-04-07 | 2024-04-26 | 杭州酶易生物技术有限公司 | Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound |
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