CN109295021A

CN109295021A - Ketoreductase and its application in bis- (trifluoromethyl) benzyl carbinols of asymmetric syntheses R-3,5-

Info

Publication number: CN109295021A
Application number: CN201811283888.9A
Authority: CN
Inventors: 吴其华; 葛德培; 王海涛
Original assignee: ANHUI LIANCHUANG BIOLOGICAL MEDICINE Co Ltd
Current assignee: ANHUI LIANCHUANG BIOLOGICAL MEDICINE Co Ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2019-02-01

Abstract

The present invention provides provide a kind of ketoreductase, encode the nucleic acid of the ketoreductase, recombinant expression plasmid comprising the nucleic acid, recombinant strains and preparation method thereof comprising the recombinant expression plasmid, and the ketoreductase prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-.Relative to existing other preparation methods, using the R-3 prepared by ketoreductase that states of the invention, not only concentration and optical purity are high for bis- (trifluoromethyl) benzyl carbinols of 5-, and reaction condition is mild, and it is environmentally friendly, it is easy to operate, it is easy to industrial amplification, there is good prospects for commercial application.

Description

Ketoreductase and its in bis- (trifluoromethyl) benzyl carbinols of asymmetric syntheses R-3,5- Using

Technical field

The invention belongs to technical field of biochemical industry, more particularly to a kind of ketoreductase and its in asymmetric syntheses R-3, Application in bis- (trifluoromethyl) benzyl carbinols of 5-.

Background technique

Bis- (trifluoromethyl) benzyl carbinols of R-3,5- are the key intermediates for synthesizing Aprepitant.Aprepitant is write from memory by the U.S. A kind of neurokinine-1 (NK-1) acceptor inhibitor of gram company research and development, the medicine 2003 through food and medicine Surveillance Authority (FDA) approval listing, was approved in Discussion on Chinese Listed in 2014, was mainly used for preventing and treating caused acute and delayed after chemotherapy Property bad habit vomiting etc. symptoms.

Bis- (trifluoromethyl) benzyl carbinols of R-3,5- can perhaps split acquisition by chemistry but chemical method or Split Method office Limit is that yield is relatively low, Split Method theoretically highest yield 50%.

Biologically, chiral hydroxyl group compound can be by Enzyme catalyzed synthesis, and reaction condition is mild, can make product pair 99% should selectively be reached.Currently, more and more drugs or intermediate are synthesized by way of biocatalysis.It is raw Object catalysis method be by bis- (trifluoromethyl) the acetophenone asymmetric reductions of precursor 3,5- at bis- (trifluoromethyl) the benzene second of R-3,5- Alcohol, synthetic method are as follows:

Patent CN102382780 provides a kind of side for aoxidizing microbacterium and its preparing chiral bis trifluoromethyl benzyl carbinol Method, patent CN104212841 are provided in one kind medium containing ionic liquid cosolvent and are prepared (R) -3,5- dual-trifluoromethyl benzene second The method of alcohol all synthesizes bis- (trifluoromethyl) benzyl carbinols of (R) -3,5- in the two patents, still with carbonyl reductase Concentration of substrate is all relatively low.Patent CN106399398 provides the biology system of bis- (trifluoromethyl) benzyl carbinols of one kind (R) -3,5- Preparation Method prepares bis- (trifluoromethyl) benzyl carbinols of (R) -3,5- with the ketoreductase of QnR, but organic examination is utilized in the enzyme Agent isopropanol carries out coenzyme circulation, and organic reagent, which has enzyme activity, largely to be damaged, and have apparent inhibiting effect, It is become apparent when amplification production.

Summary of the invention

For reported asymmetric reduction reaction prepare yield in (R) -3,5- bis- (trifluoromethyl) benzyl carbinols reaction it is low, The problem that reaction condition is unfriendly and cost is relatively high, high, mapping choosing that the purpose of the present invention is to provide a kind of catalytic activity The ketoreductase that selecting property is strong, yield is relatively high additionally provides the nucleic acid for encoding the ketoreductase, the recombination containing the nucleic acid sequence Expression plasmid, recombinant strains comprising the recombinant expression plasmid and preparation method thereof and the ketoreductase be not right Claim the application in synthesis bis- (trifluoromethyl) benzyl carbinols of R-3,5-.

The technical solution adopted by the present invention is that:

A kind of ketoreductase is following (a), (b) or protein (c):

(a) polypeptide that the amino acid sequence shown in SEQ ID NO:2 forms；

(b) on the basis of (a) described polypeptide by mutation, lack or add one or several amino acid have ketone also The polypeptide of protoenzyme property；The mutation is that the mutation of 1-10 amino acid is carried out on the basis of the polypeptide, i.e., by mutation Polypeptide afterwards still has the property of ketoreductase, several amino acid referred at 1-30, for example, increase His Tag label or Add the signal peptide of one section of secreting, expressing；

(c) at least 90% identity and there is the active polypeptide of ketoreductase with (a) described polypeptide.

A kind of nucleic acid encoding ketoreductase of the present invention, nucleic acid DNA sequence dna group as shown in SEQ ID NO:1 At.

A kind of recombinant expression plasmid pET21a-MT-KRED01 comprising nucleic acid of the present invention.

A kind of recombinant strains comprising recombinant expression plasmid of the present invention.

The preparation method of recombinant strains of the present invention, comprising the following steps:

(a) recombinant expression plasmid is transformed into host cell, be coated on the LB plate containing 50 μ g/mL ammonia benzyls, 37 DEG C After being incubated overnight, therefrom selects positive bacterium colony and be inoculated into 50mL LB liquid medium and cultivated；After cultivating 4h, 10mL is drawn Seed liquor is transferred to the fresh LB liquid medium of 1L；

(b) when the concentration of culture reaches OD₆₀₀When 0.6-1.0, the isopropyl-of final concentration of 0.1~0.2mmol/L is added β-D- Thiogalactopyranoside (IPTG) is induced, and inducing temperature is 16-30 DEG C, Fiber differentiation 10-16h, induction training By being centrifuged or being concentrated to get the wet thallus of higher concentration after supporting, after generally going through ultrasonic disruption or high-pressure homogeneous crusher machine Supernatant, the as thick enzymatic lysis liquid of recombinant strains is collected by centrifugation.

The preparation method of recombinant strains of the present invention, wherein host cell described in step (a) is Escherichia coli E.coli BL21(DE3)。

The preparation method of recombinant strains of the present invention, wherein final concentration of 0.15mmol/L is added in step (b) Isopropyl-beta D-thio galactopyranoside (IPTG) induced, inducing temperature be 28 DEG C, Fiber differentiation 12h.

Ketoreductase of the present invention prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-, In the buffer of pH 6.0~8.0, in the presence of glucose, glucose dehydrogenase (GDH) and coenzyme, the ketoreductase is to preceding Body carbonyls carries out asymmetric reduction reaction, forms R-3, bis- (trifluoromethyl) benzyl carbinols of 5-, the precursor carbonyl chemical combination Object is bis- (trifluoromethyl) acetophenones of 3,5-, and the coenzyme is NADP coenzyme.

Ketoreductase of the present invention prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-, In, comprising the following steps:

(1) phosphate buffer of 500mL 50mM is added in flask, the phosphate buffer pH is 6.0-8.0, Stirring；

(2) the thick enzymatic lysis liquid of ketoreductase, glucose dehydrogenase and glucose are added, add NADP coenzyme and Precursor carbonyl compound reacts at a temperature of 28-32 DEG C, controls pH with the sodium carbonate of 2mol/L, uses TLC contact plate after reacting 16h Detect fully reacting；

(3) reaction system is warming up to 70-80 DEG C of heating 1-2h, diatomite is added and filters removing protein, with isometric acetic acid Ethyl ester aqueous phase extracted and washing filter cake；

(4) merge organic phase after being extracted twice, obtain grease crude product with pressure rotation steam is subtracted after anhydrous sodium sulfate drying to get R- Bis- (trifluoromethyl) benzyl carbinols of 3,5-.

Ketoreductase of the present invention prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-, In, the NADP coenzyme is NADP⁺Coenzyme, the dosage of the NADP coenzyme are 0.05~0.1g/L；Bis- (the fluoroforms of 3,5- Base) concentration of the acetophenone in reaction solution be 100-200g/L；The dosage of the ketoreductase is 500-1000U/L；The Portugal The dosage of grape sugar is 120-300g/L, and the dosage 200-2000U/L of the glucose dehydrogenase, the concentration of the glucose is institute The 1.2-1.5 equivalent of precursor carbonyl compound is stated, above-mentioned each dosage reacts bad very little, and dosage is too many, increases cost.

The invention has the advantages that:

Ketoreductase of the present invention can convert R-3, the bis- (trifluoros of 5- for substrate 3, bis- (trifluoromethyl) acetophenones of 5- Methyl) benzyl carbinol, amino acid sequence still belongs in synthesis R-3, bis- (trifluoromethyl) benzyl carbinols of 5- to be reported for the first time.

Ketoreductase of the present invention prepares R-3 in asymmetric reduction, and the application in bis- (trifluoromethyl) benzyl carbinols of 5- is preceding Body carbonyls may be up to 200g/L, and the reaction time is less than for 24 hours；NADP⁺As coenzyme, added in liquid enzyme reaction, and The expensive coenzyme NAD P of addition is not needed in full cell⁺；Bis- (trifluoromethyl) the benzyl carbinol yield of product R-3,5- are 96.8%, Its e.e. value > 99%, realizes NADP using glucose dehydrogenase⁺Circulation between/NADPH is avoided using isopropanol etc. Injury of the organic reagent to environment and ketoreductase.Relative to existing other preparation methods, restored using ketone of stating of the invention Not only concentration and optical purity are high for bis- (trifluoromethyl) benzyl carbinols of R-3 prepared by enzyme, 5-, and reaction condition is mild, to environment Close friend, it is easy to operate, it is easy to industrial amplification, there is good prospects for commercial application.

Detailed description of the invention

Fig. 1 is PCR amplification electropherogram in embodiment 2, and M is DNA molecular amount standard, and swimming lane 1 is that the ketone of PCR amplification restores Enzyme gene；

Fig. 2 is SDS-PAGE electrophoresis in embodiment 3, and M is molecular weight standard, and swimming lane 1 is the albumen supernatant of ketoreductase Liquid.

Below in conjunction with specific embodiments and the drawings, the invention will be further described.

Specific embodiment

Embodiment 1

The acquisition of ketoreductase MT-KRED01 gene

(1) soil sample is acquired from China University of Science and Technology, Hefei, Anhui Province and extract DNA (gene extracting method ginseng Examine Chroma Spin TE-1000, Clontech Laboratories, Inc., USA), by the DNA sample Sau3AI of extraction After digestion, the DNA fragmentation of 0.5~4kb of gel extraction after electrophoresis is connected on pUC19 plasmid by the site BamHI, thus To the plasmid library of genome；

(2) plasmid library described in step (1) is transformed into E. coli Top10 (purchased from the full Shi Jinsheng in Beijing Object Technology Co., Ltd.), and be applied on the LB plate of corresponding ammonia benzyl antibiotic, it selects positive colony and is inoculated into added with 500 μ Final concentration of 0.2mmol/L isopropyl-β-is added after 37 DEG C of culture 4h in the 96 deep hole orifice plates of L LB (contain corresponding ammonia benzyl antibiotic) D- Thiogalactopyranoside (IPTG) induction, 28 DEG C are continued to cultivate overnight induction；Deep hole culture is collected by centrifugation and is added The sodium phosphate buffer of the 10mmol/LpH7.5 of 50ul, -80 DEG C of multigelations make bacteria lysis afterwards three times；

(3) it is substrate, 20mmol/L grape that bis- (trifluoromethyl) acetophenones of 1mmol/L precursor carbonyl compound 3,5-, which are added, Sugar, 1U glucose dehydrogenase (GDH), 0.001% phenol red, 30 DEG C of culture 4h, hole that picking color reddens obvious institute is right The deep hole culture answered extracts plasmid and send sequencing；With the ORF Finder of National Center for Biotechnology Information (NCBI) The opening code-reading frame (ORF) of analytical sequence, by filtering out ORF nucleic acid sequence SEQ after a large amount of data mining and comparing ID NO:1, and the amino acid sequence SEQ ID NO:2 of its coding is further obtained, obtain the reduction of ketone shown in SEQ.ID.N02 Zymoprotein sequence.

Embodiment 2

The clone of ketoreductase MT-KRED01 gene

According to SEQ ID NO:1 synthetic primer, to F1 (nucleotides sequence is classified as SEQ ID NO:3) and F2, (nucleotides sequence is classified as SEQ ID NO:4)；PCR 20ul system is as follows: 2 μ L, 10 × PCR buffer of 2mmol/L dNTP, 2 μ L, PCR high fidelity enzyme 0.5 μ L, DNA profiling 1 the μ L, ddH that embodiment 1 obtains₂Each 1 μ L (5mmol/L) of 12 μ L, F1 and F2 of O.PCR amplification step are as follows: 1. 95 DEG C, initial denaturation 5min；2. 98 DEG C, being denaturalized 15s；3. 56 DEG C of annealing 30s；4. 72 DEG C of extension 45s；Step 2.~4. repeat 30 It is secondary；5. 72 DEG C are continued to extend 10min, it is cooled to 16 DEG C.PCR product is purified through agarose electrophoresis, and PCR amplification electrophoresis result is as schemed Shown in 1, there is target stripe in 1000bp or so, recycles the target stripe with Ago-Gel QIAquick Gel Extraction Kit, obtain one The complete sequence of item, through DNA sequencing, overall length 840bp obtains the PCR product of ketoreductase gene shown in SEQ.ID.N01.

Embodiment 3

The expression of ketoreductase MT-KRED01 gene

It is solidifying through agarose after PCR product described in embodiment 2 and pET21a carrier are used NdeI/XhoI double digestion simultaneously Gel electrophoresis purifying recycles target fragment using Ago-Gel QIAquick Gel Extraction Kit；It, will be described under the action of T4DNA ligase Target fragment connect to obtain recombinant expression plasmid pET21a-MT-KRED01 with the pET21a carrier after double digestion.

Above-mentioned recombinant expression plasmid pET21a-MT-KRED01 is transformed into E.coli BL21 (DE3) (purchased from the full formula in Beijing Golden Bioisystech Co., Ltd) competent cell, it is coated on the LB plate containing 50 μ g/mL ammonia benzyls, after 37 DEG C are incubated overnight, It therefrom selects positive bacterium colony and is inoculated into 50mL LB liquid medium and cultivated, LB liquid medium ingredient are as follows: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.2；After cultivating 4h, draws 10mL seed liquor and be transferred to the fresh LB liquid of 1L OD is arrived in culture medium, culture₆₀₀Up to after 0.6~1.0,28 DEG C are cooled to, and the IPTG that final concentration of 0.15mmol/L is added is lured Culture 12h is led, thalline were collected by centrifugation by 5000rpm (obtaining the wet thallus of recombinant strains), with the phosphorus of 50mmol/LpH7.0 Sour sodium buffer is washed once, and every gram of thallus is resuspended in the above-mentioned phosphate buffer of 10mL, the ultrasonication in ice bath, and centrifugation is received Collect supernatant, the as thick enzymatic lysis liquid of recombinant strains.Part supernatant is taken to carry out SDS-PAGE protein electrophoresis check table Up to level, SDS-PAGE electrophoresis result is as shown in Fig. 2, as shown in Figure 2, target protein size and expection are consistent.

Embodiment 4

A kind of ketoreductase prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-, including following Step:

(1) phosphate buffer of 500mL 50mmol/L, the phosphate buffer pH are added in 2L three-necked flask It is 7.0, stirring；

(2) thick enzymatic lysis liquid 200mL described in embodiment 3 is added, glucose dehydrogenase (GDH) 2000U (is purchased from Sigama) and glucose 120g, NADP coenzyme 0.05g and precursor carbonyl compound 3, bis- (trifluoromethyl) the benzene second of 5- are added Ketone 100g, then it is settled to 1L with phosphate buffer, it is reacted at a temperature of 30 DEG C, controls pH with the sodium hydroxide of 2mol/L, instead TLC contact plate is used to detect fully reacting after answering 16h, the NADP coenzyme is NADP⁺Coenzyme；

(3) reaction system is warming up to 70 DEG C of heating 1h, 10g diatomite is added and filters removing protein, with isometric acetic acid second Ester (EA) aqueous phase extracted and washing filter cake 2 times；

Measure the purity 98.0% of the present embodiment crude product, molar yield 97%, e.e. value > 99%.

The HPLC of product e.e. value makes a concrete analysis of condition are as follows: Daicel IC-3 (250*4.6mm, 3 μm)；Flow velocity 1mL/ min；Mobile phase: n-hexane: isopropanol=95:5；Ultraviolet detection wavelength 254nm；25 DEG C of column temperature；Sample is dissolved in methanol, concentration 5mg/mL；Sampling volume 2ul.

Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.

Sequence table

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Claims

1. a kind of ketoreductase, it is characterised in that: be following (a), (b) or protein (c):

(a) polypeptide that the amino acid sequence shown in SEQ ID NO:2 forms；

(b) on the basis of (a) described polypeptide by mutation, lack or add one or several amino acid have ketoreductase The polypeptide of property；The mutation is that the mutation of 1-10 amino acid is carried out on the basis of the polypeptide, several to refer in 1-30 A amino acid；

2. a kind of nucleic acid of ketoreductase described in coding claim 1, it is characterised in that: the nucleic acid is by SEQ ID NO:1 institute The DNA sequence dna composition shown.

3. a kind of recombinant expression plasmid pET21a-MT-KRED01 comprising nucleic acid described in claim 2.

4. a kind of recombinant strains comprising recombinant expression plasmid described in claim 3.

5. the preparation method of recombinant strains described in claim 4, it is characterised in that: the following steps are included:

(a) recombinant expression plasmid described in claim 3 is transformed into host cell, it is flat is coated on the LB containing 50 μ g/mL ammonia benzyls On plate, after 37 DEG C are incubated overnight, therefrom select positive bacterium colony and be inoculated into 50mL LB liquid medium and cultivated；Cultivate 4h Afterwards, it draws 10ml seed liquor and is transferred to the fresh LB liquid medium of 1L；

(b) when the concentration of culture reaches OD₆₀₀When 0.6-1.0, the isopropyl-β-D- of final concentration of 0.1~0.2mmol/L is added Thiogalactopyranoside is induced, and inducing temperature is 16-30 DEG C, Fiber differentiation 10-16h, after Fiber differentiation by from The heart or the wet thallus for being concentrated to get higher concentration are collected by centrifugation after generally going through ultrasonic disruption or high-pressure homogeneous crusher machine Clear liquid, the as thick enzymatic lysis liquid of recombinant strains.

6. the preparation method of recombinant strains according to claim 5, it is characterised in that: host described in step (a) is thin Born of the same parents are E. coli BL21.

7. according to the preparation method of the recombinant strains of claim 5 or 6, it is characterised in that: be added in step (b) dense eventually Degree is that the isopropyl-beta D-thio galactopyranoside of 0.15mmol/L is induced, and inducing temperature is 28 DEG C, and Fiber differentiation is 12h。

8. ketoreductase described in claim 1 prepares R-3 in asymmetric reduction, the application in bis- (trifluoromethyl) benzyl carbinols of 5-, It is characterized by: in the presence of glucose, glucose dehydrogenase and coenzyme, the ketone is also in the buffer of pH 6.0~8.0 Protoenzyme carries out asymmetric reduction reaction to precursor carbonyl compound, forms R-3, bis- (trifluoromethyl) benzyl carbinols of 5-, the precursor Carbonyls is bis- (trifluoromethyl) acetophenones of 3,5-, and the coenzyme is NADP coenzyme.

9. ketoreductase is prepared in (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidine in asymmetric reduction according to claim 8 Application, it is characterised in that: the following steps are included:

(2) the thick enzymatic lysis liquid of ketoreductase, glucose dehydrogenase and glucose are added, NADP coenzyme and precursor are added Carbonyls reacts at a temperature of 28-32 DEG C, controls pH with the sodium carbonate of 2mol/L, is detected after reacting 16h with TLC contact plate Fully reacting；

(3) reaction system is warming up to 70-80 DEG C of heating 1-2h, diatomite is added and filters removing protein, with isometric ethyl acetate Aqueous phase extracted and washing filter cake are twice；

(4) merge organic phase after being extracted twice, obtain grease crude product with pressure rotation steam is subtracted after anhydrous sodium sulfate drying to get R-3,5- Bis- (trifluoromethyl) benzyl carbinols.

10. ketoreductase is prepared in bis- (trifluoromethyl) benzyl carbinols of R-3,5- in asymmetric reduction according to claim 9 Using, it is characterised in that: the NADP coenzyme is NADP⁺Coenzyme, the dosage of the NADP coenzyme are 0.05~0.1g/L；It is described Concentration of bis- (trifluoromethyl) acetophenones of 3,5- in reaction solution is 100~200g/L；The dosage of the ketoreductase be 500~ 1000U/L；The dosage of the glucose is 120~300g/L, and the dosage of the glucose dehydrogenase is 200~2000U/L, institute The concentration for stating glucose is the 1.2-1.5 equivalent of the precursor carbonyl compound.