CN109468290A - A kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application - Google Patents
A kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application Download PDFInfo
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Abstract
The invention discloses a kind of carbonyl reduction enzyme mutants, the 145th Histidine mutagenesis that the carbonyl reduction enzyme mutant is selected from amino acid sequence shown in SEQ ID NO.2 is alanine, and mutational site further includes following one or several: the 78th valine mutation is leucine, the 107th glycine mutation is alanine, the 204th glutamic acid mutation is arginine.Carbonyl reduction enzyme mutant provided by the present invention is compared with prototype, there is more excellent catalytic activity to 2 '-iodo- 5 '-fluoro acetophenones, the concentration of mutant catalytically synthesizing chiral alcohol is in 100~800mM/L, conversion ratio is up to 90%~99.9%, yield 70%~98%, optical purity is greater than 99%, has good application and development prospect.
Description
Technical field
The present invention relates to technical field of biochemical industry, more particularly to a kind of carbonyl reduction enzyme mutant, expression vector, work
Journey bacterium and its application.
Background technique
Often there is two kinds of enantiomters of chiral drug different effect or its function and effect to differ greatly, therefore,
The synthesis of single enantiomer is more and more concerned.As one of most important chiral building block, optical activity chirality alcohol is answered extensively
Among synthesis for chiral drug and fine chemicals.The asymmetric reduction of prochiral ketone prepares optical activity chirality alcohol
Important method can theoretically convert 100% substrate ketone to the chiral alcohol of single enantiomer, have very high industrial application valence
Value.
Compared with chemical method asymmetric reduction, biological catalysis is in chemo-selective, regioselectivity and stereoselectivity
Aspect has more advantage, and product optical purity is high；In addition, biocatalysis prochiral ketone asymmetric reduction synthesis of chiral alcohol is because having reason
It is high by yield, selectivity it is good, by-product is few and reaction condition is mild the advantages that and become chiral alcohol green syt optimization approach.
The encoding gene of carbonyl reductase is as shown in SEQ ID No.1, the amino acid sequence of carbonyl reductase such as SEQ ID
Shown in No.2, catalysis prochiral ketone reduction prepares high-optical-purity chiral alcohol, and there are many reports.Peng etc. is utilized and is derived from
Short-chain dehydrogenase asymmetric reduction 1- (3- hydroxy phenyl) -2- (methylamino) second of SerratiamarcescensBCRC10948
Ketone obtains (R)-phyenlephrinium (Journal of Biotechnology, 2014,170:6-9) of the ee value up to 99%.
Pennacchio etc. will be used for the not right of benzil derived from the short-chain dehydrogenase of SulfolobusacidocaldariusDSM639
Claim reduction, the ee value of conversion ratio and product reach 98% (Appl Microbiol Biotechnology, 2013,97 (9):
3949-3964).However, most of biocatalyst catalysis prochiral ketone asymmetric reduction processes follow Prelog rule, it then follows
The biocatalyst that anti-Prelog rule is catalyzed prochiral ketone reduction is relatively fewer.The catalysis of most of carbonyl reductases is living
Property far from industrialization production requirements are met, at present be applied to industrialized reduction enzyme catalyst proportion still deficiency total amount
20%, moreover, the carboxyl reduction enzymatic activity of prototype is lower, conversion ratio is relatively low.Therefore, how to be found for specific substrates
Biocatalyst with high catalytic activity and selectivity is the most important problem in the field.
Summary of the invention
Regarding the issue above, the present invention provides a kind of catalytic activity significantly improves, highly selective and high conversion
The carbonyl reduction enzyme mutant of rate.
To achieve the above object, according to the first aspect of the invention, a kind of carbonyl reduction enzyme mutant, the carbonyl are provided
It is alanine that base, which restores enzyme mutant and is selected from the 145th Histidine mutagenesis of amino acid sequence shown in SEQ ID NO.2, is dashed forward
Displacement point further includes following one or several: the 78th valine mutation is leucine, the 107th glycine mutation is third
Propylhomoserin, the 204th glutamic acid mutation are arginine.
Further, the multi-point combination mutation of the amino acid sequence of the mutation are as follows: the 78th valine mutation
It is alanine, the paddy that the 145th Histidine mutagenesis is alanine, the 204th for leucine, the 107th glycine mutation
Histidine mutations are arginine；The amino acid sequence of the carbonyl reduction enzyme mutant is amino acid sequence shown in SEQ ID NO.4
Column.
According to the second aspect of the invention, a kind of carbonyl reduction enzyme mutant gene, nucleotide sequence such as SEQ are provided
Shown in ID NO.3.
According to the third aspect of the invention we, a kind of nucleosides comprising carbonyl reduction enzyme mutant gene of the invention is provided
The recombinant expression carrier of acid sequence.These recombinant vectors can be by conventional method in that art by carbonyl reduction enzyme mutant of the invention
Body nucleotide sequence is connected to built-up on various carriers；The carrier is various plasmids, bacteriophage or viral vectors.
Further, the carrier is pET-30a.
According to the fourth aspect of the invention, provide a kind of genetic engineering bacterium of carbonyl reduction enzyme mutant, can pass through by
Recombinant expression carrier of the invention is converted to be obtained into host microorganism.The host microorganism can be each of this field routine
Kind host microorganism, as long as self-replacation can be stablized and entrained carbonyl reductase of the invention by meeting recombinant expression carrier
Mutant gene can be with effective expression.
Further, the host cell of the genetic engineering bacterium is Escherichia coli.
Further, the host cell of the genetic engineering bacterium is E. coli BL21 (DE3).
According to the fifth aspect of the invention, it provides a kind of carbonyl reduction enzyme mutant or its genetic engineering bacterium is urged in biology
Change the application in prochiral ketone asymmetric reduction synthesizing optical activity chiral alcohol.
Further, the mutant or genetic engineering bacterium of carbonyl reductase are added using prochiral ketone as substrate, 20~50 DEG C
Under, it reacts in the conversion reaction system of the buffer composition of pH 5.5~10.5, after fully reacting, reaction solution is isolated and purified
Obtain corresponding product.
Further, initial substrate concentration is 10~800mmol/L in the transformation system.
Further, the quality dosage of mutant or thallus is calculated as 100~300g/ with thallus weight in wet base in the reaction system
L。
It further, further include organic solvent in the system.
Further, the organic cosolvent is one of dimethyl sulfoxide, isopropanol, methanol or a variety of.
Further, the organic cosolvent is isopropanol, and the concentration of isopropanol is 30% in reaction system.
Further, the conversion reaction solution isolation and purification method are as follows: after reaction, by the second of reaction solution appropriate volume
Acetoacetic ester extraction, organic layer are the crude product containing corresponding chiral alcohol, and crude product purification is obtained corresponding chiral alcohol.The crude product mentions
Pure method is techniques well known, usually organic solvent extraction, chromatographic isolation and adsorbing separation etc..
Compared with prior art, the beneficial effects of the present invention are:
Carbonyl reduction enzyme mutant provided by the present invention has more 2 '-iodo- 5 '-fluoro acetophenones compared with prototype
Excellent catalytic activity, the concentration of mutant catalytically synthesizing chiral alcohol in 100~800mM/L, conversion ratio up to 90%~99.9%,
Yield 70%~98%, optical purity are greater than 99%, have good application and development prospect.
Specific embodiment
To better illustrate the content in the present invention, it is described further combined with specific embodiments below.
Embodiment 1: the building of mutant
Using the oligonucleotide fragment containing catastrophe point as primer (table 1), using QuickChangeTM method
(Stratagene, La Jolla, CA) expands the pET-30a recombinant plasmid containing carbonyl reduction enzyme gene.
1 mutation construction primer of table
A underscore is denoted as mutational site
PCR reaction system:
5 × PrimerSTAR buffer (Mg2+plus), 5 μ L；
DNTPs (each 2.5mM), 2.0 μ L；
Upstream primer (10 μM), 1.0 μ L；
Downstream primer (10 μM), 1.0 μ L；
Recombinant plasmid template, 15ng；
Primer STAR polymerase TM HS (2.5U/ μ L), 0.5 μ L；
Add ddH2O to total volume be 25 μ L.
PCR program:
(1) 98 DEG C, 1min；
(2) 98 DEG C, 10s；
(3) 55 DEG C, 10s；
(4) 72 DEG C, 7min.
Step (2)-(4) are cooled to 4 DEG C after recycling 20 times.
PCR product is once purged, using the restriction enzyme Dpn I of specific recognition methylation sites digested with
Degradation template plasmid.Endonuclease reaction system and condition: the PCR product of the 17 cleaned processing of μ L, 2.0 μ 10 × buffers of L, 1.0 μ
L restriction enzyme Dpn I, 37 DEG C of heat preservation 1h.
The above-mentioned PCR product through digestion processing is converted into e. coli bl21 (DE3), obtains recombinating large intestine accordingly
Bacillus, coating and the plate containing kanamycins, overnight incubation at 37 DEG C, random picked clones carry out bacterium colony PCR identification and sequencing
Verifying, the results showed that the recombinant expression carrier successful conversion containing carbonyl reduction enzyme mutant gene to expressive host E.coli
In BL21 (DE3).It is final to obtain mutant H145A.
Embodiment 2: the inducing expression of carbonyl reduction enzyme mutant
The engineering bacteria that embodiment 1 constructs is seeded in the LB culture medium of 50 μ g/mL kanamycins, and 37 DEG C, 200rpm culture
Overnight, then be seeded in the LB culture medium containing 50 μ g/mL kanamycins with 1% inoculum concentration (v/v), 37 DEG C, 200rpm cultivate to
Cell concentration OD600 to 0.6 or so, is added the IPTG of final concentration of 0.1mM, after 26 DEG C of Fiber differentiation 6h, 4 DEG C, 8000rpm from
Heart 10min collects thallus, and -80 DEG C store for future use.
Embodiment 3: the fermentation tank culture of carbonyl reduction enzyme mutant
The engineering bacteria that embodiment 1 constructs is seeded in the LB culture medium of 50 μ g/mL kanamycins, and 37 DEG C, 200rpm culture
Overnight, it then is seeded in the culture medium containing 50 μ g/mL kanamycins with 2% inoculum concentration (v/v), 37 DEG C, 200rpm culture, right
In the fermentor for the fermentation medium for being seeded to the 50 μ g/mL kanamycins containing 15L with 10% inoculum concentration (v/v) when number mid-term, 37
DEG C, culture 14h or so (logarithm middle and later periods) is added after lactose carries out induction 20h, and using tube centrifuge, that thalline were collected by centrifugation is standby
With.Fermentation medium used is known in the art to grow mutant and generate carbonyl reduction of the present invention in embodiment 3
The culture medium of enzyme mutant albumen.
Embodiment 4:EbSDR8 and its mutant H145A recombinant cell catalytic activity
Reaction system (10.0mL): the wet thallus cell in 2g embodiment 3, the iodo- 5 '-fluoro acetophenone of 100mM 2 '-,
3.0mL isopropanol, 5.0mL Na2HPO4-NaH2PO4Buffer (100mM, PH 7.0).In 37 DEG C, reacted under the conditions of 200rpm.
In 3h, the conversion ratio of EbSDR8 only has 2%, and H145A has 99.9% or more.
Embodiment 5: carbonyl reductase EbSDR8 mutant H145A converts 2 '-iodo- 5 '-fluoro acetophenone concentration expanding tests
Reaction system (10.0mL): the wet thallus cell in 2g embodiment 3, the iodo- 5 '-fluoro acetophenone of various concentration 2 '-,
3.0mL isopropanol, 5.0mL Na2HPO4-NaH2PO4 buffer (100mM, PH 7.0).It is anti-under the conditions of 200rpm in 37 DEG C
It answers.H145A remains to convert in 6h to 99% or more in the case where concentration of substrate is 800mM/L.
For those skilled in the art, it can make other each according to the above description of the technical scheme and ideas
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that all should belong to the claims in the present invention
Within enclosing.
Sequence table
<110>the fragrant marine growth Science and Technology Ltd. in Hangzhou
<120>a kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application
<130>the fragrant marine growth Science and Technology Ltd. in Hangzhou
<141> 2018-10-12
<150> 2018105589078
<151> 2018-06-01
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Claims (10)
1. a kind of carbonyl reduction enzyme mutant, which is characterized in that the carbonyl reduction enzyme mutant is selected from shown in SEQ ID NO.2
The 145th Histidine mutagenesis of amino acid sequence be alanine, mutational site further includes following one or several: the 78th
Valine mutation be leucine, the 107th glycine mutation is alanine, the 204th glutamic acid mutation is arginine.
2. carbonyl reduction enzyme mutant according to claim 1, which is characterized in that the amino acid sequence of the mutation
Multi-point combination mutation are as follows: the 78th valine mutation is leucine, the 107th glycine mutation is alanine, the 145th
The Histidine mutagenesis of position is alanine, the 204th glutamic acid mutation is arginine；The amino of the carbonyl reduction enzyme mutant
Acid sequence is amino acid sequence shown in SEQ ID NO.4.
3. a kind of carbonyl reduction enzyme mutant gene, which is characterized in that nucleotide sequence is as shown in SEQID NO.3.
4. a kind of recombinant expression carrier, which is characterized in that the nucleosides comprising encoding amino acid sequence of any of claims 1 or 2
Acid sequence.
5. recombinant expression carrier according to claim 4, which is characterized in that the carrier is pET-30a.
6. a kind of genetic engineering bacterium of carbonyl reduction enzyme mutant, which is characterized in that the genetic engineering bacterium includes claim 3
The carbonyl reduction enzyme mutant gene.
7. genetic engineering bacterium according to claim 6, which is characterized in that the host cell of the genetic engineering bacterium is large intestine
Bacillus E.coli BL21 (DE3).
8. the mutant of carbonyl reductase as claimed in claim 1 or 2 or genetic engineering bacterium as claimed in claims 6 or 7
Application in biocatalysis prochiral ketone asymmetric reduction synthesizing optical activity chiral alcohol.
9. application according to claim 8, which is characterized in that method are as follows:
Step 1: using prochiral ketone as substrate, initial substrate concentration is 10~800mmol/L, is added such as claims 1 or 2 institute
The mutant for the carbonyl reductase stated or genetic engineering bacterium as claimed in claims 6 or 7, the quality dosage of mutant or thallus
100~300g/L is calculated as with thallus weight in wet base；
Step 2: it at 20~50 DEG C, is reacted in the conversion reaction system of the buffer composition of pH 5.5~10.5, fully reacting
Afterwards, reaction solution is isolated and purified to obtain corresponding product.
10. application according to claim 9, which is characterized in that it also include organic solvent in the reaction system, it is described to have
Solvent is one of dimethyl sulfoxide, isopropanol, methanol or a variety of.
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| CN111411116A (en) * | 2020-04-01 | 2020-07-14 | 安徽联创生物医药股份有限公司 | Ketoreductase, nucleic acid, recombinant expression plasmid and strain, and application in synthesis of Laolatinib intermediate |
| CN111575258A (en) * | 2020-04-15 | 2020-08-25 | 杭州馨海生物科技有限公司 | Carbonyl reductase EbSDR8 mutant and construction method and application thereof |
| CN111575258B (en) * | 2020-04-15 | 2023-07-04 | 杭州馨海生物科技有限公司 | Carbonyl reductase EbSDR8 mutant and construction method and application thereof |
| CN113201511A (en) * | 2021-04-15 | 2021-08-03 | 华东理工大学 | (R) -5-carbonyl decanoate (ester) reductase mutant and application thereof in preparation of (R) -gamma/delta-lactone |
| CN113201511B (en) * | 2021-04-15 | 2022-08-30 | 华东理工大学 | (R) -5-carbonyl decanoate (ester) reductase mutant and application thereof in preparation of (R) -gamma/delta-lactone |
| CN113801859A (en) * | 2021-09-29 | 2021-12-17 | 山东寰酶生物制药有限公司 | Carbonyl reductase mutant for preparing chiral alcohol compound and application thereof |
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