CN103937761A - Biscarbonyl reductase, and coding gene and application thereof - Google Patents

Biscarbonyl reductase, and coding gene and application thereof Download PDF

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CN103937761A
CN103937761A CN201410189187.4A CN201410189187A CN103937761A CN 103937761 A CN103937761 A CN 103937761A CN 201410189187 A CN201410189187 A CN 201410189187A CN 103937761 A CN103937761 A CN 103937761A
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pet
seq
carbonyl reduction
reduction enzyme
alkyl
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CN103937761B (en
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洪浩
詹姆斯·盖吉
陈朝勇
周炎
高峰
吕彤
郭丽娜
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ASYCHEM PHARMACEUTICALS (TIANJIN) Co.,Ltd.
Shanghai kailaiying Biotechnology Co., Ltd
Asymchem Laboratories Fuxin Co Ltd
Asymchem Laboratories Tianjin Co Ltd
Asymchem Laboratories Jilin Co Ltd
Asymchem Life Science Tianjin Co Ltd
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Asymchem Laboratories Fuxin Co Ltd
Asymchem Laboratories Tianjin Co Ltd
Asymchem Laboratories Jilin Co Ltd
Asymchem Life Science Tianjin Co Ltd
Tianjin Asymchem Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)

Abstract

The invention discloses a biscarbonyl reductase, and a coding gene and application thereof. The biscarbonyl reductase has one of the following amino acid sequences: 1) amino acid sequence disclosed as SEQ NO.1; or 2) SEQ NO.1-derived amino acid sequence subjected to substitution, and/or deletion and/or addition of one or more amino acids with the function of stereoselectively reducing the formula disclosed in the specification into the formula disclosed in the specification, wherein the SEQ NO.1-derived amino acid sequence has more than 80% of homology with SEQ NO.1.R1 is selected from aryl group, alkyl group, cycloalkyl group, alkyl-substituted aryl group, halogen-substituted aryl group, aryl alkyl heterocyclic group, and cycloheteroalkyl or cyclohetero alkylated alkyl group; and R2 is selected from alkyl group, cycloalkyl group, and haloalkyl group or halocycloalkyl group. The biscarbonyl reductase can reduce a diketone substrate in one step to obtain 3R,5S-dihydroxy compounds with single optical purity.

Description

Di-carbonyl reduction enzyme, its encoding gene and application
Technical field
The present invention relates to biocatalysis synthesis technical field, in particular to a kind of di-carbonyl reduction enzyme, its encoding gene and application.
Background technology
Chiral alcohol is the important midbody compound of a class, is widely used in the synthetic of chiral drug and other chiral fine chemicals.3R, 5S-dihydroxyl-6-benzyloxy-hecanoic acid t-butyl ester is the crucial chiral intermediate of the inhibitor statins antilipemic drugs of 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme, can make by chemosynthesis and the synthetic method of biocatalysis.Yet by chemosynthesis 3R, there is following point in the two hydroxyl products of 5S-: general need chiral metal catalyst, production cost is high; The optical purity of product is more difficult reaches requirement; In a large number with an organic solvent, cause environmental pollution serious.
Adopting synthetic this compounds of biocatalysis is a kind of effective means, and the substrate that is about to contain carbonyl is reduced to corresponding chiral alcohol by specific enzyme catalysis.Wolberg etc. utilize bacterium Lactobacllus brevis by a kind of β, and the δ-carbonyl reduction in δ-bis-carbonyl substrates becomes δ-hydroxyl, and ee is 98.1%; It is substrate that deoxyribose-5-phosphoric acid zymohexase (DERA) can be take acetaldehyde and monochloroacetaldehyde, by two step aldehyde contracting reactions, introduce two chiral centres simultaneously and obtain 3R, the two hydroxyl products of 5S-, ee>99.9%, de=96.6%, but deoxyribose-5-phosphoric acid zymohexase catalyzed reaction production cost is higher.Guo etc. utilize Acinetobacter species SC13874 reduction to prepare 3R, 5S-dihydroxyl-6-benzyloxy-capronate, but de=63.3%.Also there is report itrile group lytic enzyme by 3-HGN desymmetrization being obtained to monohydroxy intermediate R-4 cyano-3-hydroxy butyric acid, then obtain 3R, 5S-dihydroxyl-6-benzyloxy-capronate through polystep reaction.Visible, by Biocatalysis method, synthesize 3R, also still there are some technical problems in the prior art of the two hydroxyl products of 5S-: the longer difficulty of nitrilase reaction scheme is larger, and cost is higher; Wolberg etc. utilize in bacterium Lactobacllus brevis synthetic method and need to introduce another chiral centre with additive method; Utilizing needs by enzyme amount greatlyr in deoxyribose-5-phosphoric acid zymohexase (DERA) synthetic method, the very strong and initial reaction raw material of substrate inhibition is inflammable and explosive reagent, and suitability for industrialized production difficulty is higher.
Summary of the invention
The present invention aims to provide a kind of di-carbonyl reduction enzyme, its encoding gene and application, and to simplify 3R, the synthesis step of 5S-dihydroxy compound, reduces to produce and pollute and production cost.
To achieve these goals, according to an aspect of the present invention, provide a kind of di-carbonyl reduction enzyme.This di-carbonyl reduction enzyme has one of following aminoacid sequence: the 1) aminoacid sequence of SEQ NO.1; 2) by the aminoacid sequence of SEQ NO.1 through one or several amino acid whose replacement and/or disappearance and/or add and have by stereoselective is reduced to the aminoacid sequence being derived by SEQ NO.1 of function, and derivative aminoacid sequence and the SEQ NO.1 of SEQ NO.1 have more than 80% homology, wherein, and R 1be selected from aromatic base, alkyl, cycloalkyl, the aromatic base that alkyl replaces, the assorted alkyl of aromatic base, aralkyl heterocyclic radical, ring-type or the assorted alkanisation alkyl of ring-type that halogen replaces, R 2be selected from alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl.
A kind of encoding gene of above-mentioned di-carbonyl reduction enzyme is provided according to another aspect of the present invention.
Further, above-mentioned encoding gene, has the deoxynucleoside acid sequence of one of the following: 1) the deoxynucleoside acid sequence of SEQ NO.2; 2) protein that there is 80% homology and coding with the deoxynucleoside acid sequence of SEQ NO.2 have by stereoselective reduction is the deoxynucleoside acid sequence of function.
A kind of recombinant expression vector of the encoding gene that contains above-mentioned di-carbonyl reduction enzyme is provided according to a further aspect of the invention.
A kind of transgenic cell line of the encoding gene that contains above-mentioned di-carbonyl reduction enzyme is provided according to a further aspect of the invention.
A kind of transgenosis recombinant bacterium of the encoding gene that contains above-mentioned di-carbonyl reduction enzyme is provided according to a further aspect of the invention.
According to a further aspect of the invention, provide a kind of above-mentioned di-carbonyl reduction enzyme inciting somebody to action stereoselective reduction is in application.
Further, di-carbonyl reduction enzyme is in preparation 3R, the application in 5S-dihydroxyl-6-benzyloxy-hecanoic acid t-butyl ester.
Further, for
Adopt di-carbonyl reduction enzyme (Diketoreductase of the present invention, DKR) be biological catalyst, can one step reduction diketone substrate, prepare the 3R of single optical purity, 5S-dihydroxy compound, apply it to synthetic statins drug midbody, obtaining ee value is that 99%, de value is the 3R of 93% left and right, 5S-dihydroxy compound, simplify synthesis step, reduced to produce and polluted and production cost, be suitable for large-scale industrial production.
Embodiment
It should be noted that, in the situation that not conflicting, embodiment and the feature in embodiment in the application can combine mutually.Below in conjunction with embodiment, describe the present invention in detail.
Carbonyl reductase gene source of the present invention has the function of di-carbonyl reduction enzyme in this carbonyl reductase of Mycobacterium fortuitum subsp.fortuitum DSM46621.
According to the present invention, a kind of typical embodiment, provides a kind of di-carbonyl reduction enzyme.This di-carbonyl reduction enzyme has one of following aminoacid sequence: 1) (SEQ NO.1 is the aminoacid sequence of SEQ NO.1: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEKLAETYA NEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQKLGDLA PAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTADTDPSVF AAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSAVDDTWR IATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLGLASGEG FYKYN); 2) by the aminoacid sequence of SEQ NO.1 through one or several amino acid whose replacement and/or disappearance and/or add and have by stereoselective reduction is the aminoacid sequence being derived by SEQ NO.1 of function, wherein, R 1be selected from aromatic base, R2 is selected from alkyl.Adopt di-carbonyl reduction enzyme (Diketoreductase of the present invention, DKR) be biological catalyst, can one step reduction diketone substrate, prepare the 3R of single optical purity, 5S-dihydroxy compound, applies it to synthetic statins drug midbody, has simplified synthesis step, reduced to produce and polluted and production cost, be suitable for large-scale industrial production.
According to the present invention, an exemplary embodiment; provides a carbonyl reductase of the double-encoding gene, the genes encoding one of the following deoxyribonucleotide sequence: deoxynucleotide sequence 1) SEQ NO.2 (the SEQ NO.2 as:ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) ；2) protein that there is 80% homology and coding with the deoxyribonucleoside acid sequence of SEQ NO.2 have by Stereoselective reduction is The deoxyribonucleoside acid sequence of function.
A kind of typical embodiment according to the present invention, provides a kind of recombinant expression vector of the encoding gene that contains above-mentioned di-carbonyl reduction enzyme.This carrier can be pET-22b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.Expression vector is overexpression in intestinal bacteria, and the molecular weight presenting on SDS-PAGE through the di-carbonyl reduction enzyme of overexpression is about 30KD, at 30 ℃, under pH6.0 condition, can a step reduction obtain the 3R that optical purity is higher, 5S-dihydroxyl compound.
Certainly, the transgenic cell line, transgenosis recombinant bacterium that comprise above-mentioned encoding gene are also within protection scope of the present invention.A kind of typical embodiment according to the present invention, provides a kind of above-mentioned di-carbonyl reduction enzyme inciting somebody to action stereoselective reduction is in application.This enzyme can one step reduction diketone substrate, prepare the 3R of single optical purity, 5S-dihydroxy compound, can be applied to synthetic statins drug midbody.Preferably, di-carbonyl reduction enzyme, at preparation 3R, is applied in 5S-dihydroxyl-6-benzyloxy-hecanoic acid t-butyl ester, wherein, can be
Below in conjunction with embodiment, further illustrate beneficial effect of the present invention.
Embodiment 1
(1) derive from the cloning and expression of the di-carbonyl reduction enzyme of Mycobacterium fortuitum subsp.fortuitum DSM46621
For the ease of expression and the evaluation of di-carbonyl reduction enzyme gene (DKR), in 5 ' and 3 ' tip designs of oligonucleotide primer compatible restriction enzyme site.Its primer pair is as follows: upstream primer SEQ ID NO.3:5 '-GGAATTC cATATGaCTAACTCTGTAAAAACGGTGACTGTTC-3 '; Downstream primer SEQ ID NO.4:5 '-CCG cTCGAGgTTATATTTGTAGAAACCTTCACCAGAAGCC-3 '.Was amplified gene sequence (SEQ ID NO.2:ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) 。
Its aminoacid sequence is (SEQ ID NO.1:MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEKL AETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQK LGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTADT DPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSAV DDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLGL ASGEGFYKYN).
After di-carbonyl reduction enzyme total gene synthesis, be connected on pUC57 carrier, by Nde I and Xho I, by goal gene and pET22b (+), (pET-22b (+) can be also pET-3a (+) respectively, pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid carries out enzyme simultaneously and cuts, T4DNA ligase enzyme carries out ligation, connection product is transformed in intestinal bacteria competence DH5 α bacterial strain, coat and contain 50 μ g/ml containing in the solid LB substratum (Luria-Bertani substratum) of penbritin, 37 ℃ of overnight incubation.Single colony inoculation on the above-mentioned substratum of picking is in containing 50 μ g/ml containing in the LB liquid nutrient medium of penbritin, 37 ℃ of shaking culture are spent the night, through plasmid extraction, after PCR evaluation and double digestion are identified, correct cloning vector pET-22b (+)-DKR is transformed in e. coli bl21 (DE3), coat and contain 50 μ g/ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation in the above-mentioned substratum of picking contains in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, through bacterium colony PCR (polymerase chain reaction), identify that correct expression vector carries out abduction delivering, above-mentioned bacterium liquid is transferred and contained in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, and 37 ℃ of shaking culture are to OD 600=0.6 o'clock, add IPTG (isopropylthiogalactoside) to be respectively 0.02mM to final concentration, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM carry out abduction delivering at 18 ℃, and set up the negative control that does not add IPTG inductor.After induction 16h, take out bacterium liquid, the centrifugal 10min of 8000rpm/min collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.The amount that found that abduction delivering di-carbonyl reduction enzyme when final concentration is 0.02mM IPTG concentration is maximum.For improving the expression amount of di-carbonyl reduction enzyme, by the e. coli bl21 that contains recombinant plasmid (DE3), at IPTG final concentration, be 0.02mM, respectively at 37 ℃ of induction 8h, under 30 ℃ of induction 8h and 25 ℃ of induction 16h conditions, express, and set up the negative control that does not add IPTG inductor simultaneously.After abduction delivering finishes, respectively the induction thalline of three temperature is collected to thalline in the centrifugal 10min of 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.Found that the amount of expressing di-carbonyl reduction enzyme during 8h 30 ℃ of inductions is maximum.
(2) derive from the activity identification of the di-carbonyl reduction enzyme of Mycobacterium fortuitum subsp.fortuitum DSM46621
Select the ketone compounds of business-like raw material or easy preparation on market for initial feed, wherein R1 is selected from aromatic base; R 2be selected from alkyl.Described two hydroxyl products are expressed by following chemical general formula: r wherein 1be selected from aromatic base; R 2be selected from alkyl.
In 500ml reaction flask, add 10g main raw material 40ml polyoxyethylene glycol PEG-400 (poly(oxyethylene glycol) 400), after material dissolution, adds 360ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add ketoreductase: add 0.6g NAD+ (Reduced nicotinamide-adenine dinucleotide), 118g D-Glucose, 0.6g coenzyme Hexose phosphate dehydrogenase and 1g di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 40h; With 400ml ethyl acetate stopped reaction, use 250g diatomite filtration, 400ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, and concentrates and obtains crude product, then obtain through column chromatography purification the product that 4g purity is higher purity 98.0%, yield 50%, ee value 99%, de value 93%.
The nuclear magnetic data of products obtained therefrom is as follows: 400Hz, CDCl3: δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45～3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
Products obtained therefrom mass-spectrometric data is as follows: MW=310 ± 1.
In 500ml reaction flask, add 10g main raw material 40ml polyoxyethylene glycol PEG-400, after material dissolution, adds 360ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add ketoreductase: add 0.3g NAD+, 41.2g ammonium formiate, 0.5g coenzyme hydrogenlyase and 1g di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 17h; With 400ml ethyl acetate stopped reaction, use 250g diatomite filtration, 400ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, dihydroxyl of obtaining product 65.0%, yield 90%, ee value is greater than 90-99%, de value 80-95%.
Products obtained therefrom mass-spectrometric data is: MW=282 ± 1.
In 500ml reaction flask, add 10g main raw material 40ml polyoxyethylene glycol PEG-400, after material dissolution, adds 360ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add ketoreductase: add 0.3g NAD+, 41.2g ammonium formiate, 0.5g coenzyme hydrogenlyase and 1g di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ insulation 17h; With 400ml ethyl acetate stopped reaction, use 250g diatomite filtration, 400ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, dihydroxyl of obtaining product 50%, purity 98.0%, yield 90%, ee value 90-99%, de value 80-95%.
Products obtained therefrom mass-spectrometric data is as follows: MW=268 ± 1.
In 500ml reaction flask, add 10g main raw material 40ml polyoxyethylene glycol PEG-400, after material dissolution, adds 360ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add ketoreductase: add 0.3g NAD+, 41.2g ammonium formiate, 0.5g coenzyme hydrogenlyase and 3g di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ insulation 17h; With 400ml ethyl acetate stopped reaction, use 250g diatomite filtration, 400ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, dihydroxyl of obtaining product 60%, purity 98.0%, yield 90%, ee value 90-99%, de value 80-95%.
Products obtained therefrom mass-spectrometric data is as follows: MW=324 ± 1.
Embodiment 2
With the di-carbonyl reduction enzyme (SEQ ID NO.5) that sequence homology in embodiment 1 is greater than 90%, this gene is the mutant of SEQ NO.1.
For the ease of expression and the evaluation of di-carbonyl reduction enzyme gene, in 5 ' and 3 ' tip designs of oligonucleotide primer compatible restriction enzyme site.Its primer pair is as follows: upstream primer SEQ ID NO.6:5 '-GGAATTC cATATGaCCGAACTGAAACAAATCACC-3 '; Downstream primer SEQ ID NO.7:5 '-CCG cTCGAGaCCTTTGTAGTTGTAAAAGCCGTCAC-3 '.Was amplified gene sequence (SEQ ID NO.2:ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCATGTGTGGCAGTTCAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) 。
Its aminoacid sequence is (SEQ ID NO.8:MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEKL AETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQK LGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANHVWQFNTAEIMGTADT DPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSAV DDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLGL ASGEGFYKYN).
After di-carbonyl reduction enzyme total gene synthesis, be connected on pUC57 carrier, by Nde I and Xho I, by goal gene and pET-22b (+), (pET-22b (+) can be also pET-3a (+) respectively, pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid carries out enzyme simultaneously and cuts, T4DNA ligase enzyme carries out ligation, by connecting product, be transformed in the competence of e.colistraindh5α, coat and contain 50 μ g/ml containing in the solid LB culture dish of penbritin, 37 ℃ of overnight incubation.Single colony inoculation on the above-mentioned substratum of picking is in containing 50 μ g/ml containing in the LB liquid nutrient medium of penbritin, 37 ℃ of shaking culture are spent the night, through plasmid extraction, after PCR evaluation and double digestion are identified, correct cloning vector pET22b (+)-DKR is transformed in e. coli bl21 (DE3), coat and contain 50 μ g/ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation in the above-mentioned substratum of picking contains in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, through bacterium colony PCR, identify that correct expression vector carries out abduction delivering, above-mentioned bacterium liquid is transferred and contained in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, and 37 ℃ of shaking culture are to OD 600=0.6 o'clock, add IPTG to be respectively 0.02mM to final concentration, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM carry out abduction delivering at 18 ℃, and set up the negative control that does not add IPTG inductor.After induction 16h, take out bacterium liquid, the centrifugal 10min of 8000rpm/min collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.The amount that found that abduction delivering di-carbonyl reduction enzyme when final concentration is 0.02mM IPTG concentration is maximum.For improving the expression amount of di-carbonyl reduction enzyme, by the e. coli bl21 that contains recombinant plasmid (DE3), at IPTG final concentration, be 0.02mM, respectively at 37 ℃ of induction 8h, under 30 ℃ of induction 8h and 25 ℃ of induction 16h conditions, express, and set up the negative control that does not add IPTG inductor simultaneously.After abduction delivering finishes, respectively the induction thalline of three temperature is collected to thalline in the centrifugal 10min of 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.Found that the amount of expressing di-carbonyl reduction enzyme during 16h 25 ℃ of inductions is maximum.Select the ketone compounds of business-like raw material or easy preparation on market for initial feed, R wherein 1be selected from aromatic base; R 2be selected from alkyl.Described two hydroxyl products are expressed by following chemical general formula: r wherein 1be selected from aromatic base; R 2be selected from alkyl.
In 10ml reaction flask, add 0.01g main raw material 0.04ml polyoxyethylene glycol PEG-400, after material dissolution, adds 0.86ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add 1mgNAD +, 4.12mg ammonium formiate, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 15-24h; With 1ml ethyl acetate stopped reaction, use 0.25g diatomite filtration, 1ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, product of obtaining 67.35%, yield 90%, ee value 92.1%, de value 78.9%.
The nuclear magnetic data of products obtained therefrom is as follows: 400Hz, CDCl3: δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45～3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
Products obtained therefrom mass-spectrometric data is as follows: MW=310 ± 1.
Embodiment 3
The di-carbonyl reduction enzyme (SEQ ID NO.9) that is greater than 90% with sequence homology in embodiment 1; This gene is the mutant of SEQ NO.1.
For the ease of expression and the evaluation of di-carbonyl reduction enzyme gene, in 5 ' and 3 ' tip designs of oligonucleotide primer compatible restriction enzyme site.Its primer pair is as follows: upstream primer SEQ ID NO.10:5 '-GGAATTC cATATGaCCGAACTGAAACAAATCACC-3 '; Downstream primer SEQ ID NO.11:5 '-CCG cTCGAGaCCTTTGTAGTTGTAAAAGCCGTCAC-3 '.Was amplified gene sequence (SEQ ID NO.9:ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGAACAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) 。
Its aminoacid sequence is (SEQ ID NO.12:MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEK LAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQ KLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQNNTAEIMGTAD TDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSA VDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLG LASGEGFYKYN).
After di-carbonyl reduction enzyme total gene synthesis, be connected on pUC57 carrier, by Nde I and Xho I, by goal gene and pET-22b (+), (pET-22b (+) can be also pET-3a (+) respectively, pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid carries out enzyme simultaneously and cuts, T4DNA ligase enzyme carries out ligation, by connecting product, be transformed in the competence of e.colistraindh5α, coat and contain 50 μ g/ml containing in the solid LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation on the above-mentioned substratum of picking is in containing 50 μ g/ml containing in the LB liquid nutrient medium of penbritin, 37 ℃ of shaking culture are spent the night, through plasmid extraction, after PCR evaluation and double digestion are identified, correct cloning vector pET22b (+)-DKR is transformed in e. coli bl21 (DE3), coat and contain 50 μ g/ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation in the above-mentioned substratum of picking contains in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, through bacterium colony PCR, identify that correct expression vector carries out abduction delivering, above-mentioned bacterium liquid is transferred and contained in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, and 37 ℃ of shaking culture are to OD 600=0.6 o'clock, add IPTG to be respectively 0.02mM to final concentration, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM carry out abduction delivering at 18 ℃, and set up the negative control that does not add IPTG inductor.After induction 16h, take out bacterium liquid, the centrifugal 10min of 8000rpm/min collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.The amount that found that abduction delivering di-carbonyl reduction enzyme when final concentration is 0.02mM IPTG concentration is maximum.For improving the expression amount of di-carbonyl reduction enzyme, by the e. coli bl21 that contains recombinant plasmid (DE3), at IPTG final concentration, be 0.02mM, respectively at 37 ℃ of induction 8h, under 30 ℃ of induction 8h and 25 ℃ of induction 16h conditions, express, and set up the negative control that does not add IPTG inductor simultaneously.After abduction delivering finishes, respectively the induction thalline of three temperature is collected to thalline in the centrifugal 10min of 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.Found that the amount of expressing di-carbonyl reduction enzyme during 16h 25 ℃ of inductions is maximum.
Select the ketone compounds of business-like raw material or easy preparation on market for initial feed, R wherein 1be selected from aromatic base; R 2be selected from alkyl.Described two hydroxyl products are expressed by following chemical general formula: r wherein 1be selected from aromatic base; R 2be selected from alkyl.
In 10ml reaction flask, add 0.01g main raw material 0.04ml polyoxyethylene glycol PEG-400, after material dissolution, adds 0.86ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add 1mgNAD +, 4.12mg ammonium formiate, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 15-24h; With 1ml ethyl acetate stopped reaction, use 0.25g diatomite filtration, 1ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, product of obtaining 59.46%, yield 90%, ee value 93.9%, de value 90.7%.
The nuclear magnetic data of products obtained therefrom is as follows: 400Hz, CDCl3: δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45～3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
Products obtained therefrom mass-spectrometric data is as follows: MW=310 ± 1.
Embodiment 4:
The di-carbonyl reduction enzyme (SEQ ID NO.13) that is greater than 90% with sequence homology in embodiment 1; This gene is the mutant of SEQ NO.1.
For the ease of expression and the evaluation of di-carbonyl reduction enzyme gene, in 5 ' and 3 ' tip designs of oligonucleotide primer compatible restriction enzyme site.Its primer pair is as follows: upstream primer SEQ ID NO.14:5 '-GGAATTC cATATGaCCGAACTGAAACAAATCACC-3 '; Downstream primer SEQ ID NO.15:5 '-CCG cTCGAGaCCTTTGTAGTTGTAAAAGCCGTCAC-3 '.Was amplified gene sequence (SEQ ID NO.13 :ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACAAGACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) 。
Its aminoacid sequence is (SEQ ID NO.16:MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEK LAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQ KLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTAD TDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSA VDKTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLG LASGEGFYKYN).
After di-carbonyl reduction enzyme total gene synthesis, be connected on pUC57 carrier, by Nde I and Xho I, by goal gene and pET-22b (+), (pET-22b (+) can be also pET-3a (+) respectively, pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid carries out enzyme simultaneously and cuts, T4DNA ligase enzyme carries out ligation, by connecting product, be transformed in the competence of e.colistraindh5α, coat and contain 50 μ g/ml containing in the solid LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation on the above-mentioned substratum of picking is in containing 50 μ g/ml containing in the LB liquid nutrient medium of penbritin, 37 ℃ of shaking culture are spent the night, through plasmid extraction, after PCR evaluation and double digestion are identified, correct cloning vector pET22b (+)-DKR is transformed in e. coli bl21 (DE3), coat and contain 50 μ g/ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation in the above-mentioned substratum of picking contains in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, through bacterium colony PCR, identify that correct expression vector carries out abduction delivering, above-mentioned bacterium liquid is transferred and contained in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, and 37 ℃ of shaking culture are to OD 600=0.6 o'clock, add IPTG to be respectively 0.02mM to final concentration, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM carry out abduction delivering at 18 ℃, and set up the negative control that does not add IPTG inductor.After induction 16h, take out bacterium liquid, the centrifugal 10min of 8000rpm/min collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.The amount that found that abduction delivering di-carbonyl reduction enzyme when final concentration is 0.02mM IPTG concentration is maximum.For improving the expression amount of di-carbonyl reduction enzyme, by the e. coli bl21 that contains recombinant plasmid (DE3), at IPTG final concentration, be 0.02mM, respectively at 37 ℃ of induction 8h, under 30 ℃ of induction 8h and 25 ℃ of induction 16h conditions, express, and set up the negative control that does not add IPTG inductor simultaneously.After abduction delivering finishes, respectively the induction thalline of three temperature is collected to thalline in the centrifugal 10min of 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.Found that the amount of expressing di-carbonyl reduction enzyme during 16h 25 ℃ of inductions is maximum.
Select the ketone compounds of business-like raw material or easy preparation on market for initial feed, R wherein 1be selected from aromatic base; R 2be selected from alkyl.Described two hydroxyl products are expressed by following chemical general formula: r wherein 1be selected from aromatic base; R 2be selected from alkyl.
In 10ml reaction flask, add 0.01g main raw material 0.04ml polyoxyethylene glycol PEG-400, after material dissolution, adds 0.86ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add 1mgNAD +, 4.12mg ammonium formiate, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 15-24h; With 1ml ethyl acetate stopped reaction, use 0.25g diatomite filtration, 1ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, product of obtaining 50.75%, yield 90%, ee value 91.74%, de value 91.6%.
The nuclear magnetic data of products obtained therefrom is as follows: 400Hz, CDCl3: δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45～3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
Products obtained therefrom mass-spectrometric data is as follows: MW=310 ± 1.
Embodiment 5:
The di-carbonyl reduction enzyme (SEQ ID NO.17) that is greater than 90% with sequence homology in embodiment 1; This gene is the mutant of SEQ NO.1.
For the ease of expression and the evaluation of di-carbonyl reduction enzyme gene, in 5 ' and 3 ' tip designs of oligonucleotide primer compatible restriction enzyme site.Its primer pair is as follows: upstream primer SEQ ID NO.18:5 '-GGAATTC cATATGaCCGAACTGAAACAAATCACC-3 '; Downstream primer SEQ ID NO.19:5 '-CCG cTCGAGaCCTTTGTAGTTGTAAAAGCCGTCAC-3 '.Was amplified gene sequence (SEQ ID NO.13 :ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTTCAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGGCCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACCTCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGGTTCCGGAGATCCTCGATCTCAAGCGGGAGACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCGACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACTGCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGATGGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCGCCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTGGGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCAGAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACAAGTACAACTGA ) 。
Its aminoacid sequence is (SEQ ID NO.20:MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEK LAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAVPEILDLKRETYQ KLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTAD TDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSA VDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLG LASGEGFYKYN).
After di-carbonyl reduction enzyme total gene synthesis, be connected on pUC57 carrier, by Nde I and Xho I, by goal gene and pET-22b (+), (pET-22b (+) can be also pET-3a (+) respectively, pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid carries out enzyme simultaneously and cuts, T4DNA ligase enzyme carries out ligation, by connecting product, be transformed in the competence of e.colistraindh5α, coat and contain 50 μ g/ml containing in the solid LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation on the above-mentioned substratum of picking is in containing 50 μ g/ml containing in the LB liquid nutrient medium of penbritin, 37 ℃ of shaking culture are spent the night, through plasmid extraction, after PCR evaluation and double digestion are identified, correct cloning vector pET22b (+)-DKR is transformed in e. coli bl21 (DE3), coat and contain 50 μ g/ml containing in the LB substratum of penbritin, 37 ℃ of overnight incubation.Single colony inoculation in the above-mentioned substratum of picking contains in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, through bacterium colony PCR, identify that correct expression vector carries out abduction delivering, above-mentioned bacterium liquid is transferred and contained in the LB liquid nutrient medium of penbritin containing 50 μ g/ml in 5ml, and 37 ℃ of shaking culture are to OD 600=0.6 o'clock, add IPTG to be respectively 0.02mM to final concentration, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM carry out abduction delivering at 18 ℃, and set up the negative control that does not add IPTG inductor.After induction 16h, take out bacterium liquid, the centrifugal 10min of 8000rpm/min collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.The amount that found that abduction delivering di-carbonyl reduction enzyme when final concentration is 0.02mM IPTG concentration is maximum.For improving the expression amount of di-carbonyl reduction enzyme, by the e. coli bl21 that contains recombinant plasmid (DE3), at IPTG final concentration, be 0.02mM, respectively at 37 ℃ of induction 8h, under 30 ℃ of induction 8h and 25 ℃ of induction 16h conditions, express, and set up the negative control that does not add IPTG inductor simultaneously.After abduction delivering finishes, respectively the induction thalline of three temperature is collected to thalline in the centrifugal 10min of 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 ℃, the centrifugal 20min of 12000rpm/min obtains supernatant liquor and precipitation, and supernatant liquor carries out SDS-PAGE detection with vertical electrophoresis apparatus.Found that the amount of expressing di-carbonyl reduction enzyme during 16h 25 ℃ of inductions is maximum.
Select the ketone compounds of business-like raw material or easy preparation on market for initial feed, R wherein 1be selected from aromatic base; R 2be selected from alkyl.Described two hydroxyl products are expressed by following chemical general formula: r wherein 1be selected from aromatic base; R 2be selected from alkyl.
In 10ml reaction flask, add 0.01g main raw material 0.04ml polyoxyethylene glycol PEG-400, after material dissolution, adds 0.86ml phosphate buffered saline buffer (100mM, pH=6.0), and substrate is dispersed in damping fluid; Add 1mgNAD +, 4.12mg ammonium formiate, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzyme, system pH=6.0, and in 30 ± 3 ℃ of insulation 15-24h; With 1ml ethyl acetate stopped reaction, use 0.25g diatomite filtration, 1ml ethyl acetate extracting twice, standing separatory, organic phase drying, filters, the concentrated crude product, product of obtaining 73.19%, yield 90%, ee value 93.8%, de value 85.9%.
The nuclear magnetic data of products obtained therefrom is as follows: 400Hz, CDCl3: δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45～3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
Products obtained therefrom mass-spectrometric data is as follows: MW=310 ± 1.
From above description, can find out, the above embodiments of the present invention have realized following technique effect: adopting di-carbonyl reduction enzyme of the present invention (Diketoreductase, DKR) is biological catalyst, can one step reduction diketone substrate, prepare the 3R of single optical purity, 5S-dihydroxy compound, applies it to synthetic statins drug midbody, has simplified synthesis step, reduced to produce and polluted and production cost, be suitable for large-scale industrial production.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (9)

1. a di-carbonyl reduction enzyme, is characterized in that, has one of following aminoacid sequence:
1) aminoacid sequence of SEQ NO.1;
2) by the aminoacid sequence of SEQ NO.1 through one or several amino acid whose replacement and/or disappearance and/or add and have by stereoselective is reduced to the aminoacid sequence being derived by SEQ NO.1 of function, and derivative aminoacid sequence and the described SEQ NO.1 of described SEQ NO.1 have more than 80% homology, wherein, and R 1be selected from aromatic base, alkyl, cycloalkyl, the aromatic base that alkyl replaces, the assorted alkyl of aromatic base, aralkyl heterocyclic radical, ring-type or the assorted alkanisation alkyl of ring-type that halogen replaces, R 2be selected from alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl.
2. the encoding gene of di-carbonyl reduction enzyme claimed in claim 1.
3. encoding gene according to claim 2, is characterized in that, has the deoxynucleoside acid sequence of one of the following:
1) the deoxynucleoside acid sequence of SEQ NO.2;
2) protein that there is 80% homology and coding with the deoxynucleoside acid sequence of SEQ NO.2 have by stereoselective reduction is the deoxynucleoside acid sequence of function.
4. the recombinant expression vector that contains the encoding gene of di-carbonyl reduction enzyme claimed in claim 1.
5. the transgenic cell line that contains the encoding gene of di-carbonyl reduction enzyme claimed in claim 1.
6. the transgenosis recombinant bacterium that contains the encoding gene of di-carbonyl reduction enzyme claimed in claim 1.
7. di-carbonyl reduction enzyme as claimed in claim 1 is being incited somebody to action stereoselective reduction is in application.
8. application according to claim 7, is characterized in that, described di-carbonyl reduction enzyme is in preparation 3R, the application in 5S-dihydroxyl-6-benzyloxy-hecanoic acid t-butyl ester.
9. application according to claim 8, is characterized in that, described in for
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WO2015168866A1 (en) * 2014-05-06 2015-11-12 凯莱英医药集团（天津）股份有限公司 Double-carbonyl reductase, coding gene of same, and application thereof
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