CN104342412B

CN104342412B - For producing the Ketoreductase mutant of (S) -4- chloro-3-hydroxyl ethyl butyrate

Info

Publication number: CN104342412B
Application number: CN201310345823.3A
Authority: CN
Inventors: 丁雪峰
Original assignee: NANJING LANG'EN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Current assignee: NANJING LANG'EN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Priority date: 2013-08-09
Filing date: 2013-08-09
Publication date: 2018-12-21
Anticipated expiration: 2033-08-09
Also published as: CN104342412A

Abstract

The present invention relates to a kind of Ketoreductase mutants, its wild type ketoreductase for being derived from Kefir grains lactobacillus (Lactobacillus kefir), 4- chloroacetyl acetacetic ester can be converted into (S) -4- chloro-3-hydroxyl ethyl butyrate, with A94S, F147V, one or more mutation in L199P, A202V.Ketoreductase mutant of the invention has apparent high specific enzyme activity, improves 2-20 times than wild type ketoreductase, using the enzyme can biocatalysis 4- chloroacetyl acetacetic ester is converted into (S) -4- chloro-3-hydroxyl ethyl butyrate；Reaction condition is mild, low for equipment requirements, and production process is not necessarily to high temperature or cooling, low energy consumption, since enzymatic has efficiently, single-minded selectivity, therefore production statins key intermediate (S) -4- chloro-3-hydroxyl ethyl butyrate no coupling product generates in this way, purifying is convenient；Furthermore reacting most solvents is water, and three waste discharge is low, environmentally protective.

Description

For producing the Ketoreductase mutant of (S) -4- chloro-3-hydroxyl ethyl butyrate

Technical field

The present invention relates to Ketoreductase mutant and its statins drug midbody production in application, in particular to one Kind is derived from the Ketoreductase mutant of Kefir grains lactobacillus (Lactobacillus kefir) and its in the chloro- 3- of chirality (S) -4- Application in hydroxy-butyric acid ethyl ester production.

Background technique

Atorvastatin calcium (trade name LIPITOR is sold by Pfizer), rosuvastatin calcium (trade name CRESTOR Sold by Astrazeneca AB) and Pitavastatin (trade (brand) name Lipalo is by Nissan Chemical and Kowa company in Japan Sale), it is important norcholesterol statins.And chirality (S) -4- chloro-3-hydroxyl-ethyl butyrate is these important they The crucial chiral intermediate of spit of fland class drug.The yield of the domestic intermediate in 2009 is 400 tons, it is contemplated that in the coming years, state Interior yield is up to 1000 tons or more, and direct economic benefit is in several hundred million members.The many of these chiral intermediates are prepared known In approach, including chemical method and enzyme process, all tool have disadvantages that.Since chemical method synthesis process condition is harsh, side reaction is more, produces Product isolate and purify that difficulty is big, and yield is low, at high cost, it is made to be difficult to become the ideal method for commercial scale synthesis.Using enzyme Method preparation and chirality (S) -4- chloro-3-hydroxyl-ethyl butyrate need to use the ketoreductase with stereoselectivity, but existing Wild type ketoreductase catalytic activity is very lower, and the ketone substrate of 1g/L can be only converted within 20 hours using the thick enzyme powder of 10g/L, is also made It is difficult to become the ideal method for commercial scale synthesis.

Summary of the invention

The object of the present invention is to provide a kind of ketoreductases and preparation method thereof that enzymatic activity is high.

Realizing the technical solution of the object of the invention is: naturally depositing in Kefir grains lactobacillus (Lactobacillus kefir) Wild type ketoreductase, 4- chloroacetyl acetacetic ester (COBE) can be converted to (S) -4- chloro-3-hydroxyl ethyl butyrate (S- CHBE).The inventor of present disclosure has found ketoreductase and Kefir grains lactobacillus including being mutated in certain position The wild type ketoreductase (SEQ ID NO:2) that (Lactobacillus kefir) is generated is lived compared to increased catalysis is shown Property." wild type ketoreductase ", " wild type KRED enzyme " and " wild type KRED ketoreductase " refers to by being originated from Kefir grains lactobacillus The wild type ketoreductase genes of SEQ ID NO:1 coding of (Lactobacillus kefir), and there is SEQ ID NO:2 Amino acid sequence ketoreductase.4- chloroacetyl acetacetic ester asymmetric reduction can be generated (S) -4- chloro-3-hydroxyl by the enzyme Ethyl butyrate." wild type " refers to the form of material or substance as found in nature.Such as the protein of wild type Or nucleic acid sequence be can from nature separation and without artificial modification, the original series present in organism Form." increased catalytic activity " refers to measured compared with wild type ketoreductase in test in vitro or in vivo, shows Make substrate (such as 4- chloroacetyl acetacetic ester) to product (such as S-4- chloro-3-hydroxyl ethyl butyrate) the increased ketone of conversion ratio Reductase.

Provided by the present invention for the Ketoreductase mutant of production (S) -4- chloro-3-hydroxyl ethyl butyrate, feature exists In being derived from the wild type ketoreductase of Kefir grains lactobacillus (Lactobacillus kefir), wherein mutation is in parent It is inserted into the amino acid sequence of this ketoreductase, replaces or lack one or more amino acid, or in parent's ketoreductase One or two end of amino acid sequence adds or deletes one or more amino acid, and compared with using parent's ketoreductase, Its 2-20 times of ketoreductase increased activity.

Further, the mutant has the amino acid substitution on following one or more amino acid positions, respectively takes Alternative triplet indicates: alphabet-numeric playing-letter, wherein the position of digital representation mutating acid, and the letter before number is corresponding prominent Becoming the amino acid of design, the letter after number indicates the amino acid for replacing amino acid before number: A94S, F147V, L199P, A202V.

Ketoreductase mutant provided by the invention, derived from Kefir grains lactobacillus (Lactobacillus kefir) Wild type ketoreductase, performance send as an envoy to substrate (such as 4- chloroacetyl acetacetic ester) to product (such as S-4- chloro-3-hydroxyl fourth Acetoacetic ester) the increased ketoreductase of conversion ratio.The Ketoreductase mutant, the wild type ketoreductase with SEQ ID NO.2 Compared to showing stronger catalytic activity.Ability can be used in Ketoreductase mutant and the polynucleotides for encoding this mutant The preparation of field technique personnel's commonly used approach.Mutant can by make to encode the vitro recombination of the enzyme, polynucleotides mutagenesis, DNA reorganization, fallibility PCR and directed evolution method etc. obtain.

Further, the mutant includes the amino acid sequence of SEQ ID NO.4.

The ketoreductase of overall length mutation is for keeping the catalytic activity of enzyme to be not required in that.Correspondingly, it is considered as ketone also The truncated analog of protoenzyme mutant and the segment for having catalytic activity.For example, in some embodiments, the number of C-terminal or N-terminal A amino acid can be left out.Any specific truncated analog or segment can use corresponding test and live to assess catalysis Property.Likewise, additional amino acid residue can be added to one or two end without influencing catalytic activity.Additional sequences It can be functional or non-functional.For example, Additional amino acid sequences can be used to assist in purifying, as label, or Execute some other functions.Therefore, the Ketoreductase mutant of present disclosure can be the form of fusion protein, wherein ketone Enzyme mutant (or its segment) is restored such as by hydrotropy label (such as SUMO albumen), purification tag (as combined metal His label) and bacterium positioning signal (such as secretion signal) example rather than limitation mode be fused to other oroteins.

Further, the present invention provides the encoding gene of above-mentioned mutant.

Further, encoding gene provided by the present invention, sequence include DNA molecular described in SEQ ID NO.3.Its Sequence optimisation is had been subjected to be suitable in expression in escherichia coli.In some embodiments, polynucleotides include being optimized for The codon expressed in certain types of host cell.The use of codon for various types of microorganism and partially Good property is known, because it is the codon for the optimization of the specific amino acid of expression in these microorganisms.

The present invention provides a kind of recombinant plasmid, and sequence preferably is selected from SEQ ID NO.5, than pET series and the expression of pQE series Carrier compared to it there is more rigorous expression to control.In some embodiments, control sequence include promoter, leader sequence, Polyadenylation sequences, propeptide sequence, signal peptide sequence and transcription terminator etc..For bacterial host cell, code sequence is instructed The suitable promoter of the transcription of column includes but is not limited to from Phage T5, phage t7, bacteriophage lambda, Escherichia coli LacUV5 operon, Escherichia coli trp operon, Escherichia coli tac operon etc..

The present invention provides a kind of host cell, preferably is selected from Escherichia coli W3110, one of DH1 and JM109.Express ketone The expression vector of reduction enzyme mutant may include permission, and vector integration is into host cell gene group or carrier is in bacterium The element independently replicated independently of genome.To be integrated into host cell gene group, carrier can make to carry by recombined engineering Body is integrated into genome.

The present invention provides a kind of method for preparing Ketoreductase mutant, it is characterised in that the following steps are included: (a) is constructed The genetic engineering bacterium of Ketoreductase mutant is expressed, the genetic engineering bacterium includes host cell, expression vector and ketone reduction Enzyme mutant gene；(b) it cultivates and induces the genetic engineering bacterium；(c) it is crushed the genetic engineering bacterium, collects crude enzyme liquid simultaneously Carry out Enzyme activity assay.

The step (a) be will from Kefir grains lactobacillus (Lactobacillus kefir) encoding wild type ketone also After the polynucleotides (SEQ ID NO:1) of protoenzyme carry out sequence optimisation according to bacterial strain DH1 codon preference, pass through full genome The mode of synthesis obtains.The polynucleotides of coding ketoreductase after optimization are cloned into expression vector (SEQ ID NO. 5) Under the control of promoter, the plasmid that can express wild type ketoreductase is obtained.Gained plasmid is transformed by standard method In Escherichia coli DH1.Cloning process used is the mode of homologous recombination, used amplimer are as follows:

F:5'ATTAAAGAGGAGAAATTAACATATGACTGATCGTTTAAAAGGCAAAG 3'；

R:5'AACAGGAGTCCAAGCTCAGCTTATTATTGAGCAGTGTATCCACCATCG 3'.

The polynucleotides for encoding Ketoreductase mutant (SEQ ID NO:3) are cloned into expression vector with same method Under the control of the promoter of (SEQ ID NO. 5), the plasmid that can express Ketoreductase mutant is obtained.Gained plasmid is led to Standard method is crossed to be transformed into Escherichia coli DH1.

The step (b) contains for picking has the Escherichia coli single colonie of purpose expression vector to be inoculated in 10ml high pressure sterilization In the first culture medium afterwards, 30 DEG C, 250rpm is incubated overnight；Next day takes 1L triangular flask, is linked by the inoculative proportion of 1:100 In the second culture medium after 100ml high pressure sterilization, triangular flask is placed in 25 DEG C at once and shaken by culture to thallus OD 5-6 in 30 DEG C In bed, 250rpm cultivates 1h, and IPTG to final concentration 0.1mM is added, and in 25 DEG C, 250rpm continues to cultivate 15h.

First culture medium are as follows: 10 g/L of tryptone, 5 g/L of yeast extract, 3.55 g/L of disodium hydrogen phosphate, 3.4 g/L of potassium dihydrogen phosphate, 2.68 g/L of ammonium chloride, 0.71 g/L of sodium sulphate, 0.493 g/L of epsom salt, six water chlorinations Iron 0.027 g/L, glycerol 5g/L, glucose 0.8g/L add ampicillin to 100mg/L after sterilizing.

Second culture medium are as follows: 10 g/L of tryptone, 5 g/L of yeast extract, 3.55 g/L of disodium hydrogen phosphate, 3.4 g/L of potassium dihydrogen phosphate, 2.68 g/L of ammonium chloride, 0.71 g/L of sodium sulphate, 0.493 g/L of epsom salt, six water chlorinations Iron 0.027 g/L, glycerol 5g/L, glucose 0.3g/L.

The present invention has the effect of positive: (1) Ketoreductase mutant of the invention has apparent high specific enzyme activity, than open country Raw type ketoreductase improves 2-20 times, and 4- chloroacetyl acetacetic ester can be converted into (S) -4- chloro-3-hydroxyl using the enzyme Ethyl butyrate；(2) reaction condition of the present invention is mild, low for equipment requirements, and production process is not necessarily to high temperature or cooling, and low energy consumption, Since enzymatic has efficiently, single-minded selectivity, therefore the chloro- 3- hydroxyl of statins key intermediate (S) -4- is produced in this way Base ethyl butyrate no coupling product generates, and purifying is convenient；(3) reacting most solvents is water, and three waste discharge is low, environmentally protective.

Specific embodiment

Embodiment 1 constructs wild type and Ketoreductase mutant engineering bacteria

It will be from the polynucleotides of the encoding wild type ketoreductase of Kefir grains lactobacillus (Lactobacillus kefir) (SEQ ID NO:1) sequence optimisation is carried out according to bacterial strain DH1 codon preference after (see Puigb ò P, Guzm á n E, Romeu A, Garcia-Vallvé S. OPTIMIZER: a web server for optimizing the codon Usage of DNA sequences. Nucleic Acids Res. 2007), pass through the full genome synthesis mode of based on PCR (PCR-based gene synthesis method) carries out gene chemical synthesis.By the multicore glycosides of the coding ketoreductase after optimization Acid is cloned under the control of the promoter of expression vector (SEQ ID NO. 5), obtains the matter that can express wild type ketoreductase Grain.Gained plasmid is transformed by standard method in Escherichia coli DH1.Cloning process used is the mode of homologous recombination, institute The amplimer used are as follows:

F:5'ATTAAAGAGGAGAAATTAACATATGACTGATCGTTTAAAAGGCAAAG 3'；

R:5'AACAGGAGTCCAAGCTCAGCTTATTATTGAGCAGTGTATCCACCATCG 3'.

Similar, the polynucleotides for encoding Ketoreductase mutant (SEQ ID NO:3) are cloned into expression vector (SEQ ID NO. 5) promoter control under, obtain the plasmid that can express Ketoreductase mutant.Gained plasmid is passed through into standard Method is transformed into Escherichia coli DH1.

The preparation of 2 ketoreductase of embodiment

Picking, which contains, has the Escherichia coli DH1 single colonie of purpose expression vector to be inoculated in the culture medium after 10ml high pressure sterilization In: 10 g/L of tryptone, 5 g/L of yeast extract, 3.55 g/L of disodium hydrogen phosphate, 3.4 g/L of potassium dihydrogen phosphate, chlorination 2.68 g/L of ammonium, 0.71 g/L of sodium sulphate, 0.493 g/L of epsom salt, Iron trichloride hexahydrate 0.027 g/L, glycerol 5g/L, Portugal Grape sugar 0.8g/L adds ampicillin to 100mg/L after sterilizing.30 DEG C, 250rpm is incubated overnight.Next day takes 1L triangular flask, In culture medium after being linked into 100ml high pressure sterilization by the inoculative proportion of 1:100: 10 g/L of tryptone, yeast extract 5 G/L, 3.55 g/L of disodium hydrogen phosphate, 3.4 g/L of potassium dihydrogen phosphate, 2.68 g/L of ammonium chloride, 0.71 g/L of sodium sulphate, seven water sulphur Sour 0.493 g/L of magnesium, Iron trichloride hexahydrate 0.027 g/L, glycerol 5g/L, glucose 0.3g/L.Kanamycins is added extremely after sterilizing 50mg/L.Triangular flask is placed in 25 DEG C of shaking tables, 250rpm cultivates 1h by culture at once to thallus OD 5-6 in 30 DEG C.It is added IPTG to final concentration 0.1mM, and in 25 DEG C, 250rpm continues to cultivate 15h.

After culture, 20min is centrifuged by culture solution in 4 DEG C, under 6000g and finally obtains wet thallus 3.6g.Then it will sink Twice, collect thallus wash with distilled water in shallow lake.It is resuspended again with distilled water, clarification is crushed under Ultrasonic Cell Disruptor.After broken It is centrifuged 30min under 4 DEG C, 12000g, collects supernatant, prepares freeze-dried powder with freeze drier after being cooled to -70 DEG C in advance.It finally obtains Thick 0. 39g of enzyme freeze-dried powder.

The active measurement of 3 ketoreductase of embodiment

Since NADPH has an absorption peak at 340nm, and NADP at 340nm without absorption peak, therefore can be reacted by detection The variation of NADPH light absorption value in journey, and calculate the activity of ketoreductase.Ketoreductase vitality test system are as follows: take 100ul appropriate The enzyme solution of concentration is added to final concentration of 100mM pH7.0 Triethanolamine buffer, 1g/L NADP, 50% isopropanol, 1mM sulphur It is reacted in sour magnesium reaction system.Before enzyme solution is added, need first to mix well reaction system to be placed in 25 DEG C of water-baths.It will Implement to take in 100ul addition reaction system after the ketoreductase dry powder for preparing dilute in appropriate proportion in 2, after mixing in Absorbance change value per minute is detected at 340nm.The enzyme activity of ketoreductase is calculated referring to NADPH standard curve.Unit enzyme activity (U) it is defined as generating enzyme amount required for 1 μm of ol NADP per minute.With the enzyme activity of same method detection wild type ketoreductase. For the albumen of phase homogenous quantities, improved Ketoreductase mutant improves 18 times than wild type ketoreductase enzyme activity.

Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects It describes in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention Within the scope of shield.

SEQUENCE LISTING

<110>Nanjing Lang En Biotechnology Co., Ltd

<120>for producing the Ketoreductase mutant of (S) -4- chloro-3-hydroxyl ethyl butyrate

<130> 2013

<160> 5

<170> PatentIn version 3.3

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<213>Kefir grains lactobacillus (Lactobacillus kefir)

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tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300

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ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420

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gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600

ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660

aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720

gcagaattcg ttgtcgatgg tggatacact gctcaataa 759

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<213>Kefir grains lactobacillus (Lactobacillus kefir)

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catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 1200

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tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga 1320

gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 1380

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 1440

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 1500

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 1560

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 1620

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1680

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1740

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1800

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1860

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1920

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1980

tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 2040

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 2100

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 2160

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 2220

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 2280

tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 2340

agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 2400

gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 2460

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 2520

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 2580

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 2640

acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 2700

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 2760

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 2820

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 2880

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 2940

atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 3000

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 3060

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 3120

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 3180

ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 3240

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3300

cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 3360

aggcgtatca cgaggccctt tcgtcttcac 3390

Claims

1. the Ketoreductase mutant for producing (S) -4- chloro-3-hydroxyl ethyl butyrate, which is characterized in that the mutant Amino acid sequence be SEQ ID NO.4.

2. described in claim 1 for producing the coding base of the Ketoreductase mutant of (S) -4- chloro-3-hydroxyl ethyl butyrate Cause.

3. encoding gene according to claim 2, which is characterized in that the sequence of the gene is SEQ ID NO.3.

4. containing the recombinant plasmid of encoding gene as claimed in claim 2.

5. the host cell containing recombinant plasmid described in claim 4.

6. the Ketoreductase mutant described in claim 1 for producing (S) -4- chloro-3-hydroxyl ethyl butyrate is producing (S) application in -4- chloro-3-hydroxyl-ethyl butyrate.

7. a kind of production (S) -4- chloro-3-hydroxyl-ethyl butyrate method, which is characterized in that the method is with 4- chloracetyl Ethyl acetate is substrate, under the catalytic action of the mutant described in claim 1, obtains (S) -4- chloro-3-hydroxyl-butyric acid second Ester.