CN103937761B - Biscarbonyl reductase, and coding gene and application thereof - Google Patents
Biscarbonyl reductase, and coding gene and application thereof Download PDFInfo
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- CN103937761B CN103937761B CN201410189187.4A CN201410189187A CN103937761B CN 103937761 B CN103937761 B CN 103937761B CN 201410189187 A CN201410189187 A CN 201410189187A CN 103937761 B CN103937761 B CN 103937761B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- 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
Technical field
The present invention relates to living things catalysis synthesis technical field, in particular to a kind of di-carbonyl reduction enzyme, its coding base
Cause and application.
Background technology
Chiral alcohol is the important midbody compound of a class, is widely used in chiral drug and other chiral fine chemicals
Synthesis.3R, 5S- dihydroxy -6- benzyloxies-hecanoic acid t-butyl ester is the suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase
The crucial chiral intermediate of agent statins antilipemic drugs, the method that can pass through chemical synthesis and living things catalysis synthesis are obtained.
However, by chemical synthesis 3R, there is following point in the double hydroxy products of 5S-：Chiral metal catalyst, production cost need to typically be used
It is high；The optical purity of product is relatively inaccessible to require；Organic solvent is used in a large number, causes environmental pollution serious.
It is a kind of effective method to synthesize this kind of compound using living things catalysis, will the substrate containing carbonyl by specific
Enzymatic be reduced to corresponding chiral alcohol.Wolberg etc. utilizes bacterium Lactobacllus brevis by a kind of β, δ-bis- carbonyls
Into δ-hydroxyl, ee is 98.1% to δ-carbonyl reduction in substrate；Deoxyribose -5- phosphate aldolases (DERA) can be with acetaldehyde
It is substrate with chloroacetaldehyde, two chiral centres is introduced simultaneously by two step aldehyde contracting reaction and obtains 3R, the double hydroxy products of 5S-, ee>
99.9%, de=96.6%, but deoxyribose -5- phosphate aldolase catalytic reaction production costs are higher.Guo etc. is utilized
Acinetobacter species SC13874 reduction prepares 3R, 5S- dihydroxy -6- benzyloxies-capronate, but de=
63.3%.Also have been reported that itrile group hydrolase by obtaining monohydroxy intermediate R-4 cyano group -3- to 3-HGN desymmetrization
Hydroxybutyric acid, then 3R, 5S- dihydroxy -6- benzyloxies-capronate is obtained through multistep reaction.It can be seen that, by Biocatalysis method
The prior art of the double hydroxy products of synthesis 3R, 5S- also still suffers from some technical problems：The longer difficulty of nitrilase reaction scheme compared with
Greatly, it is relatively costly；Wolberg etc. is another with additive method introducing using needing in bacterium Lactobacllus brevis synthetic methods
One chiral centre；Larger with enzyme amount using needing in deoxyribose -5- phosphate aldolases (DERA) synthetic method, substrate suppresses
Effect is very strong and initial reaction raw material is inflammable and explosive reagent, and industrialized production difficulty is higher.
The content of the invention
The present invention is intended to provide a kind of di-carbonyl reduction enzyme, its encoding gene and application, to simplify 3R, 5S- dihydroxies
The synthesis step of compound, reduces production pollution and production cost.
To achieve these goals, according to an aspect of the invention, there is provided a kind of di-carbonyl reduction enzyme.This pair of carbonyl
Reductase has one of following amino acid sequence：1) amino acid sequence of SEQ NO.1；2) by the amino acid sequence of SEQ NO.1
Arrange replacement, and/or disappearance, and/or addition through one or several amino acid and have willIt is three-dimensional
Optionally it is reduced toThe amino acid sequence by derived from SEQ NO.1 of function, and SEQ NO.1 spread out
Raw amino acid sequence is with SEQ NO.1 with more than 80% homology, wherein, R1Selected from aromatic radical, alkyl, cycloalkyl, alkane
Aromatic radical, the aromatic radical of halogen substiuted, aralkyl heterocyclic radical, cycloheteroalkyl or the miscellaneous alkanisation alkyl of ring-type that base replaces, R2It is selected from
Alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl.
According to another aspect of the present invention, there is provided a kind of encoding gene of above-mentioned di-carbonyl reduction enzyme.
Further, above-mentioned encoding gene, the deoxynucleotide sequence with one of the following：1) the deoxidation core of SEQ NO.2
Nucleotide sequence；2) there is 80% homology with the deoxynucleotide sequence of SEQ NO.2 and the protein that encodes have willStereoselective reduction isThe deoxynucleotide sequence of function.
According to a further aspect of the invention, there is provided a kind of restructuring table of the encoding gene containing above-mentioned di-carbonyl reduction enzyme
Up to carrier.
According to a further aspect of the invention, there is provided a kind of transgenosis of the encoding gene containing above-mentioned di-carbonyl reduction enzyme
Clone.
According to a further aspect of the invention, there is provided a kind of transgenosis of the encoding gene containing above-mentioned di-carbonyl reduction enzyme
Recombinant bacterium.
According to a further aspect of the invention, there is provided a kind of above-mentioned di-carbonyl reduction enzyme is being incited somebody to action
Stereoselective reduction isIn application.
Further, di-carbonyl reduction enzyme is preparing 3R, the application in 5S- dihydroxy -6- benzyloxies-hecanoic acid t-butyl ester.
Further,For
It is biocatalyst using the di-carbonyl reduction enzyme (Diketoreductase, DKR) of the present invention, can be with a step also
Former diketone substrate, prepares the 3R of single optical purity, and 5S- dihydroxy compounds applies it to synthesis statins
Intermediate, obtains the 3R that ee values are that 99%, de values are 93% or so, and 5S- dihydroxy compounds simplifies synthesis step, reduces
Production pollution and production cost, are suitable for large-scale industrial production.
Specific embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combine.The present invention is described in detail below in conjunction with embodiment.
The carbonyl reductase gene source of the present invention is in Mycobacterium fortuitum subsp.fortuitum
The DSM46621 carbonyl reductases have the function of di-carbonyl reduction enzyme.
According to a kind of typical embodiment of the present invention, there is provided a kind of di-carbonyl reduction enzyme.The di-carbonyl reduction enzyme has
One of following amino acid sequence：1) (SEQ NO.1 are the amino acid sequence of SEQ NO.1：
MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLEAAKARFEKLAETYANEVPDARDGKAREALGRLTLT
SDLKAAVADADLVIEAIPEILDLKRETYQKLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNT
AEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNSLLVPFLNAAAELAAGGYADPSAVDDTWRIATGAPV
GPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKLGLASGEGFYKYN)；2) by the amino acid of SEQ NO.1
Sequence through one or several amino acid replacement, and/or disappearance, and/or addition and have willIt is vertical
Body selective reduction isThe amino acid sequence by derived from SEQ NO.1 of function, wherein, R1Selected from virtue
Perfume base, R2 are selected from alkyl.It is biocatalyst using the di-carbonyl reduction enzyme (Diketoreductase, DKR) of the present invention, can
Diketone substrate is reduced with a step, the 3R of single optical purity is prepared, 5S- dihydroxy compounds is applied it to and synthesizes him
Spit of fland class pharmaceutical intermediate, simplifies synthesis step, reduces production pollution and production cost, is suitable for large-scale industry metaplasia
Produce.
According to a kind of typical embodiment of the present invention, there is provided a kind of encoding gene of above-mentioned di-carbonyl reduction enzyme, the volume
Code gene has the deoxynucleotide sequence of one of the following：1) (SEQ NO.2 are the deoxynucleotide sequence of SEQ NO.2：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)；2) there is 80% homology with the deoxynucleotide sequence of SEQ NO.2 and the protein that encodes have willStereoselective reduction isThe deoxynucleotide sequence of function.
According to a kind of typical embodiment of the present invention, there is provided a kind of encoding gene containing above-mentioned di-carbonyl reduction enzyme
Recombinant expression carrier.The 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 overexpression in Escherichia coli,
The molecular weight presented on SDS-PAGE through the di-carbonyl reduction enzyme of amount expression is about 30KD, at 30 DEG C, under the conditions of pH6.0,
The higher 3R of optical purity, 5S- dihydroxyl compounds can be obtained with step reduction.
Certainly, transgenic cell line including above-mentioned encoding gene, transgenosis recombinant bacterium also protection scope of the present invention it
It is interior.According to a kind of typical embodiment of the present invention, there is provided a kind of above-mentioned di-carbonyl reduction enzyme is being incited somebody to action
Stereoselective reduction isIn application.The enzyme can reduce diketone substrate with a step, prepare list
The 3R of one optical purity, 5S- dihydroxy compounds, may apply to synthesize statins drug midbody.Preferably, double carbonyls are also
Protoenzyme is preparing 3R, applies in 5S- dihydroxy -6- benzyloxies-hecanoic acid t-butyl ester, wherein,Can be
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Embodiment 1
(1) from the di-carbonyl reduction of Mycobacterium fortuitum subsp.fortuitum DSM46621
The cloning and expression of enzyme
For the ease of the expression and identification of di-carbonyl reduction enzyme gene (DKR), the 5 ' and 3 ' of oligonucleotide primer
The compatible restriction enzyme site of tip designs.Its primer pair is as follows：Upstream primer SEQ ID NO.3：5’-
GGAATTCCATATGACTAACTCTGTAAAAACGGTGACTGTTC-3’；Downstream primer SEQ ID NO.4：5’-
CCGCTCGAGGTTATATTTGTAGAAACCTTCACCAGAAGCC-3’.Expand to obtain gene order (SEQ ID NO.2：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)。
Its amino acid sequence is (SEQ ID NO.1：MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLE
AAKARFEKLAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQKLGDLAPAKTIFA
TNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNS
LLVPFLNAAAELAAGGYADPSAVDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKL
GLASGEGFYKYN)。
After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, with Nde I and Xho I respectively by genes of interest
With pET22b (+) (pET-22b (+) can also be 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, pUC-19) plasmid simultaneously carries out digestion, and T4DNA ligases are attached reaction, by connection product turn
Change in E. coli competent DH5 α bacterial strains, coat the solid LB media containing ampicillin containing 50 μ g/ml
In (Luria-Bertani culture mediums), 37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in containing 50 μ g/
In LB fluid nutrient mediums of the ml containing ampicillin, overnight, through plasmid extraction, PCR is identified and double digestion 37 DEG C of shaken cultivations
After identification, correct cloning vector pET-22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated containing 50 μ
In LB culture mediums of the g/ml containing ampicillin, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml
In containing LB fluid nutrient mediums of the 50 μ g/ml containing ampicillin, correct table is identified through bacterium colony PCR (PCR)
Abduction delivering is carried out up to carrier, above-mentioned bacterium solution is transferred in 5ml is containing LB fluid nutrient mediums of the 50 μ g/ml containing ampicillin,
37 DEG C of shaken cultivations are to OD600When=0.6, IPTG (isopropylthiogalactoside) is added 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 DEG C, and set up and be not added with
The negative control of IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min collects thallines is taken out.Thalline is used super
The broken instrument smudge cells of sound, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, supernatant vertical electrophoresis apparatus
Carry out SDS-PAGE detections.As a result find the amount of the abduction delivering di-carbonyl reduction enzyme in final concentration of 0.02mM IPTG concentration
At most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid (DE3) in IPTG final concentrations
For 0.02mM, 8h is induced respectively at 37 DEG C, is expressed, and be not added with while setting up under the conditions of 8h and 25 DEG C of induction 16h of 30 DEG C of inductions
The negative control of IPTG derivants.After abduction delivering terminates, respectively the induction thalline of three temperature is centrifuged in 8000rpm/min
10min collects thallines.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and
Precipitation, supernatant carry out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to express di-carbonyl reduction when 8h is induced for 30 DEG C
The amount of enzyme is most.
(2) from the di-carbonyl reduction of Mycobacterium fortuitum subsp.fortuitum DSM46621
The activity identification of enzyme
From commercially business-like raw material or the easily ketone compounds of preparation
For initial feed, wherein R1 is selected from aromatic radical；R2Selected from alkyl.Described double hydroxy products are expressed by following chemical general formula：Wherein R1Selected from aromatic radical；R2Selected from alkyl.
To in 500ml reaction bulbs, 10g main materials are added40ml polyethylene glycol PEG-
400 (PEG400s), after dissolution of raw material, add 360ml phosphate buffers (100mM, pH=6.0), and substrate is dispersed
In buffer solution；Plus ketoreductase：Add 0.6g NAD+ (NADH), 118g D-Glucoses, 0.6g
Coenzyme GDH and 1g di-carbonyl reduction enzymes, system pH=6.0, and 40h is incubated in 30 ± 3 DEG C；With 400ml acetic acid second
Ester stops reaction, is filtered with 250g diatomite, and 400ml ethyl acetate is extracted twice, and stands a point liquid, and organic phase drying is filtered,
Crude product is concentrated to give, then Jing column chromatographies purifying obtains the higher product of 4g purityPurity
98.0%, yield 50%, ee values 99%, de values 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.
To in 500ml reaction bulbs, 10g main materials are added40ml polyethylene glycol PEG-
400, after dissolution of raw material, 360ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；Plus
Ketoreductase：Add 0.3g NAD+, 41.2g ammonium formates, 0.5g coenzyme hydrogenlyase and 1g di-carbonyl reduction enzymes, system pH
=6.0, and 17h is incubated in 30 ± 3 DEG C；Stop reacting with 400ml ethyl acetate, filtered with 250g diatomite, 400ml acetic acid second
Ester is extracted twice, and stands a point liquid, and organic phase drying is filtered, is concentrated to give crude product, dihydroxy
Product 65.0%, yield 90%, ee values are more than 90-99%, de values 80-95%.
Products obtained therefrom mass spectrometric data is：MW=282 ± 1.
To in 500ml reaction bulbs, 10g main materials are added40ml polyethylene glycol PEG-400,
After dissolution of raw material, 360ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；Plus ketone is also
Protoenzyme：Add 0.3g NAD+, 41.2g ammonium formates, 0.5g coenzyme hydrogenlyase and 1g di-carbonyl reduction enzymes, system pH=
6.0, and 17h is incubated in 30 ± 3 DEG C；Stop reacting with 400ml ethyl acetate, filtered with 250g diatomite, 400ml ethyl acetate
It is extracted twice, stands a point liquid, organic phase drying is filtered, is concentrated to give crude product, dihydroxyProduce
Product 50%, purity 98.0%, yield 90%, ee values 90-99%, de values 80-95%.
Products obtained therefrom mass spectrometric data is as follows：MW=268 ± 1.
To in 500ml reaction bulbs, 10g main materials are added40ml polyethylene glycol PEG-
400, after dissolution of raw material, 360ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；Plus ketone
Reductase：Add 0.3g NAD+, 41.2g ammonium formates, 0.5g coenzyme hydrogenlyase and 3g di-carbonyl reduction enzymes, system pH=
6.0, and 17h is incubated in 30 ± 3 DEG C；Stop reacting with 400ml ethyl acetate, filtered with 250g diatomite, 400ml ethyl acetate extraction
Take twice, stand a point liquid, organic phase drying is filtered, is concentrated to give crude product, dihydroxyProduce
Product 60%, purity 98.0%, yield 90%, ee values 90-99%, de values 80-95%.
Products obtained therefrom mass spectrometric data is as follows：MW=324 ± 1.
Embodiment 2
With di-carbonyl reduction enzyme (SEQ ID NO.5) of the sequence homology in embodiment 1 more than 90%, the gene is SEQ
The mutant of NO.1.
For the ease of the expression and identification of di-carbonyl reduction enzyme gene, set in 5 ' and 3 ' ends of oligonucleotide primer
The restriction enzyme site of compatibility is counted.Its primer pair is as follows：Upstream primer SEQ ID NO.6：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.7：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ ID NO.5：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCATGTGTGGCAGTTCAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)。
Its amino acid sequence is (SEQ ID NO.8：MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVLE
AAKARFEKLAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQKLGDLAPAKTIFA
TNSSTLLPSDLKDSTGRPDKFLALHFANHVWQFNTAEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLNS
LLVPFLNAAAELAAGGYADPSAVDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGKL
GLASGEGFYKYN)。
After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, with Nde I and Xho I respectively by genes of interest
With pET-22b (+) (pET-22b (+) can also be 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, pUC-19) plasmid simultaneously carries out digestion, and T4DNA ligases are attached reaction, by connection product turn
Change in the competence of e.colistraindh5α, in coating containing solid LB culture dishes of the 50 μ g/ml containing ampicillin,
37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in be trained containing LB liquid of the 50 μ g/ml containing ampicillin
In foster base, overnight, through plasmid extraction, PCR is identified after identifying with double digestion 37 DEG C of shaken cultivations, by correct cloning vector
PET22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated and is cultivated containing LB of the 50 μ g/ml containing ampicillin
In base, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml and contains ampicillin containing 50 μ g/ml
In LB fluid nutrient mediums, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, above-mentioned bacterium solution is transferred in 5ml
Contain in the LB fluid nutrient mediums of ampicillin containing 50 μ g/ml, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added to end
Concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is induced at 18 DEG C
Expression, and set up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution is taken out, 8000rpm/min centrifugation 10min are received
Collection thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, on
Clear liquid carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the abduction delivering in final concentration of 0.02mM IPTG concentration
The amount of di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid
(DE3) in the final concentration of 0.02mM of IPTG, 8h, 8h and 25 DEG C of induction 16h condition following table of 30 DEG C of inductions is induced respectively at 37 DEG C
Reach, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction thalline of three temperature
10min collects thallines are centrifuged in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugations
20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to induce at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme during 16h is most.From commercially business-like raw material or the easily ketone of preparation
CompoundFor initial feed, wherein R1Selected from aromatic radical；R2Selected from alkyl.Described double hydroxyls are produced
Thing is expressed by following chemical general formula：Wherein R1Selected from aromatic radical；R2Selected from alkyl.
To in 10ml reaction bulbs, 0.01g main materials are added0.04ml polyethylene glycol PEG-
400, after dissolution of raw material, 0.86ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；
Add 1mgNAD+, 4.12mg ammonium formates, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzymes, system pH=6.0, and in
30 ± 3 DEG C of insulation 15-24h；Stop reacting with 1ml ethyl acetate, filtered with 0.25g diatomite, 1ml ethyl acetate extraction two
It is secondary, a point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, product67.35%, yield
90%, ee value 92.1%, de values 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
With di-carbonyl reduction enzyme (SEQ ID NO.9) of the sequence homology in embodiment 1 more than 90%；The gene is SEQ
The mutant of NO.1.
For the ease of the expression and identification of di-carbonyl reduction enzyme gene, set in 5 ' and 3 ' ends of oligonucleotide primer
The restriction enzyme site of compatibility is counted.Its primer pair is as follows：Upstream primer SEQ ID NO.10：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.11：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ ID NO.9：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGAACAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)。
Its amino acid sequence is (SEQ ID NO.12：MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVL
EAAKARFEKLAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQKLGDLAPAKTIF
ATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQNNTAEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLN
SLLVPFLNAAAELAAGGYADPSAVDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGK
LGLASGEGFYKYN)。
After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, with Nde I and Xho I respectively by genes of interest
With pET-22b (+) (pET-22b (+) can also be 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, pUC-19) plasmid simultaneously carries out digestion, and T4DNA ligases are attached reaction, by connection product turn
Change in the competence of e.colistraindh5α, in coating containing solid LB medias of the 50 μ g/ml containing ampicillin,
37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in be trained containing LB liquid of the 50 μ g/ml containing ampicillin
In foster base, overnight, through plasmid extraction, PCR is identified after identifying with double digestion 37 DEG C of shaken cultivations, by correct cloning vector
PET22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated and is cultivated containing LB of the 50 μ g/ml containing ampicillin
In base, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml and contains ampicillin containing 50 μ g/ml
In LB fluid nutrient mediums, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, above-mentioned bacterium solution is transferred in 5ml
Contain in the LB fluid nutrient mediums of ampicillin containing 50 μ g/ml, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added to end
Concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is induced at 18 DEG C
Expression, and set up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution is taken out, 8000rpm/min centrifugation 10min are received
Collection thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, on
Clear liquid carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the abduction delivering in final concentration of 0.02mM IPTG concentration
The amount of di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid
(DE3) in the final concentration of 0.02mM of IPTG, 8h, 8h and 25 DEG C of induction 16h condition following table of 30 DEG C of inductions is induced respectively at 37 DEG C
Reach, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction thalline of three temperature
10min collects thallines are centrifuged in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugations
20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to induce at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme during 16h is most.
From commercially business-like raw material or the easily ketone compounds of preparation
For initial feed, wherein R1Selected from aromatic radical；R2Selected from alkyl.Described double hydroxy products are expressed by following chemical general formula：Wherein R1Selected from aromatic radical；R2Selected from alkyl.
To in 10ml reaction bulbs, 0.01g main materials are added0.04ml polyethylene glycol PEG-
400, after dissolution of raw material, 0.86ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；
Add 1mgNAD+, 4.12mg ammonium formates, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzymes, system pH=6.0, and in
30 ± 3 DEG C of insulation 15-24h；Stop reacting with 1ml ethyl acetate, filtered with 0.25g diatomite, 1ml ethyl acetate extraction two
It is secondary, a point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, product59.46%, yield
90%, ee value 93.9%, de values 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：
With di-carbonyl reduction enzyme (SEQ ID NO.13) of the sequence homology in embodiment 1 more than 90%；The gene is SEQ
The mutant of NO.1.
For the ease of the expression and identification of di-carbonyl reduction enzyme gene, set in 5 ' and 3 ' ends of oligonucleotide primer
The restriction enzyme site of compatibility is counted.Its primer pair is as follows：Upstream primer SEQ ID NO.14：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.15：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ ID NO.13：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGATCCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACAAGACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)。
Its amino acid sequence is (SEQ ID NO.16：MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVL
EAAKARFEKLAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAIPEILDLKRETYQKLGDLAPAKTIF
ATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLN
SLLVPFLNAAAELAAGGYADPSAVDKTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGK
LGLASGEGFYKYN)。
After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, with Nde I and Xho I respectively by genes of interest
With pET-22b (+) (pET-22b (+) can also be 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, pUC-19) plasmid simultaneously carries out digestion, and T4DNA ligases are attached reaction, by connection product turn
Change in the competence of e.colistraindh5α, in coating containing solid LB medias of the 50 μ g/ml containing ampicillin,
37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in be trained containing LB liquid of the 50 μ g/ml containing ampicillin
In foster base, overnight, through plasmid extraction, PCR is identified after identifying with double digestion 37 DEG C of shaken cultivations, by correct cloning vector
PET22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated and is cultivated containing LB of the 50 μ g/ml containing ampicillin
In base, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml and contains ampicillin containing 50 μ g/ml
In LB fluid nutrient mediums, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, above-mentioned bacterium solution is transferred in 5ml
Contain in the LB fluid nutrient mediums of ampicillin containing 50 μ g/ml, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added to end
Concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is induced at 18 DEG C
Expression, and set up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution is taken out, 8000rpm/min centrifugation 10min are received
Collection thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, on
Clear liquid carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the abduction delivering in final concentration of 0.02mM IPTG concentration
The amount of di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid
(DE3) in the final concentration of 0.02mM of IPTG, 8h, 8h and 25 DEG C of induction 16h condition following table of 30 DEG C of inductions is induced respectively at 37 DEG C
Reach, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction thalline of three temperature
10min collects thallines are centrifuged in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugations
20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to induce at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme during 16h is most.
From commercially business-like raw material or the easily ketone compounds of preparation
For initial feed, wherein R1Selected from aromatic radical；R2Selected from alkyl.Described double hydroxy products are expressed by following chemical general formula：Wherein R1Selected from aromatic radical；R2Selected from alkyl.
To in 10ml reaction bulbs, 0.01g main materials are added0.04ml polyethylene glycol PEG-
400, after dissolution of raw material, 0.86ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；
Add 1mgNAD+, 4.12mg ammonium formates, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzymes, system pH=6.0, and in
30 ± 3 DEG C of insulation 15-24h；Stop reacting with 1ml ethyl acetate, filtered with 0.25g diatomite, 1ml ethyl acetate extraction two
It is secondary, a point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, product50.75%, yield
90%, ee value 91.74%, de values 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：
With di-carbonyl reduction enzyme (SEQ ID NO.17) of the sequence homology in embodiment 1 more than 90%；The gene is SEQ
The mutant of NO.1.
For the ease of the expression and identification of di-carbonyl reduction enzyme gene, set in 5 ' and 3 ' ends of oligonucleotide primer
The restriction enzyme site of compatibility is counted.Its primer pair is as follows：Upstream primer SEQ ID NO.18：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.19：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ ID NO.17：
ATGACGAACTCCGTGAAGACGGTGACCGTTCTCGGGACCGGAGTGCTCGGTTCCCAGATCGCGTTCCAATCCGCGTT
CAAGGGTTTCACCGTCACCGCGTACGACATCAATGATGAGGTTCTCGAAGCGGCCAAAGCGCGGTTCGAGAAGCTGG
CCGAGACCTACGCCAACGAGGTACCCGACGCGCGTGACGGCAAGGCCCGGGAAGCGTTGGGCCGGCTCACGCTGACC
TCCGACCTGAAGGCAGCGGTGGCCGACGCCGATCTGGTGATCGAGGCGGTTCCGGAGATCCTCGATCTCAAGCGGGA
GACCTACCAGAAGCTCGGGGACCTCGCGCCGGCCAAGACCATCTTCGCCACCAACTCCTCGACGCTGCTGCCCAGCG
ACCTCAAGGATTCCACCGGCAGGCCCGACAAGTTCCTGGCACTGCACTTCGCAAACCGGGTGTGGCAGTTCAACACT
GCCGAGATCATGGGGACCGCGGACACCGACCCGAGCGTTTTCGCCGCGGTGGTCGAGTTCGCCAAGGCCATCGGGAT
GGTGCCGATCGAGCTGCACAAGGAGAAGGCCGGCTACGTCCTCAACTCACTTCTGGTCCCGTTCCTCAATGCCGCCG
CCGAACTCGCGGCCGGTGGCTACGCCGACCCCAGCGCCGTGGACGACACCTGGCGGATCGCCACCGGCGCACCGGTG
GGCCCGTTCCAGATCTACGACATCATCGGCCTGACCACGCCGTACAACATCATGGCCAATGGGGACGCCGAAGCTCA
GAAGCTCGCCGCGTGGCTGAAAGAGAACTACATCGACAAAGGCAAGTTGGGCCTCGCCAGCGGCGAAGGCTTCTACA
AGTACAACTGA)。
Its amino acid sequence is (SEQ ID NO.20：MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINDEVL
EAAKARFEKLAETYANEVPDARDGKAREALGRLTLTSDLKAAVADADLVIEAVPEILDLKRETYQKLGDLAPAKTIF
ATNSSTLLPSDLKDSTGRPDKFLALHFANRVWQFNTAEIMGTADTDPSVFAAVVEFAKAIGMVPIELHKEKAGYVLN
SLLVPFLNAAAELAAGGYADPSAVDDTWRIATGAPVGPFQIYDIIGLTTPYNIMANGDAEAQKLAAWLKENYIDKGK
LGLASGEGFYKYN)。
After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, with Nde I and Xho I respectively by genes of interest
With pET-22b (+) (pET-22b (+) can also be 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, pUC-19) plasmid simultaneously carries out digestion, and T4DNA ligases are attached reaction, by connection product turn
Change in the competence of e.colistraindh5α, in coating containing solid LB medias of the 50 μ g/ml containing ampicillin,
37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in be trained containing LB liquid of the 50 μ g/ml containing ampicillin
In foster base, overnight, through plasmid extraction, PCR is identified after identifying with double digestion 37 DEG C of shaken cultivations, by correct cloning vector
PET22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated and is cultivated containing LB of the 50 μ g/ml containing ampicillin
In base, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml and contains ampicillin containing 50 μ g/ml
In LB fluid nutrient mediums, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, above-mentioned bacterium solution is transferred in 5ml
Contain in the LB fluid nutrient mediums of ampicillin containing 50 μ g/ml, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added to end
Concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is induced at 18 DEG C
Expression, and set up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution is taken out, 8000rpm/min centrifugation 10min are received
Collection thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, on
Clear liquid carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the abduction delivering in final concentration of 0.02mM IPTG concentration
The amount of di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid
(DE3) in the final concentration of 0.02mM of IPTG, 8h, 8h and 25 DEG C of induction 16h condition following table of 30 DEG C of inductions is induced respectively at 37 DEG C
Reach, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction thalline of three temperature
10min collects thallines are centrifuged in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugations
20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to induce at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme during 16h is most.
From commercially business-like raw material or the easily ketone compounds of preparation
For initial feed, wherein R1Selected from aromatic radical；R2Selected from alkyl.Described double hydroxy products are expressed by following chemical general formula：Wherein R1Selected from aromatic radical；R2Selected from alkyl.
To in 10ml reaction bulbs, 0.01g main materials are added0.04ml polyethylene glycol PEG-
400, after dissolution of raw material, 0.86ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；
Add 1mgNAD+, 4.12mg ammonium formates, 2mg coenzyme hydrogenlyase and 4mg di-carbonyl reduction enzymes, system pH=6.0, and in
30 ± 3 DEG C of insulation 15-24h；Stop reacting with 1ml ethyl acetate, filtered with 0.25g diatomite, 1ml ethyl acetate extraction two
It is secondary, a point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, product73.19%, yield
90%, ee value 93.8%, de values 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.
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：Using this
Bright di-carbonyl reduction enzyme (Diketoreductase, DKR) is biocatalyst, can reduce diketone substrate with a step, be prepared into
To the 3R of single optical purity, 5S- dihydroxy compounds, synthesis statins drug midbody is applied it to, simplify synthesis
Step, reduces production pollution and production cost, is suitable for large-scale industrial production.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is all within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (4)
1. di-carbonyl reduction enzyme is being incited somebody to actionStereoselective reduction isIn
Using；Wherein, R1Selected from aromatic radical, alkyl, cycloalkyl, alkyl-substituted aromatic radical, the aromatic radical of halogen substiuted, aralkyl heterocycle
The miscellaneous alkanisation alkyl of base, cycloheteroalkyl or ring-type, R2Selected from alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl,
The amino acid sequence of the di-carbonyl reduction enzyme such as one of following amino acid sequences：SEQ ID NO.1、SEQ ID NO.8、
Amino acid sequence shown in SEQ ID NO.12, SEQ ID NO.16 or SEQ ID NO.20.
2. application according to claim 1, it is characterised in that the deoxynucleotide sequence of the coding di-carbonyl reduction enzyme
Such as one of following deoxynucleotide sequence：SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13 or
Deoxynucleotide sequence shown in SEQ ID NO.17.
3. application according to claim 1, it is characterised in that the di-carbonyl reduction enzyme prepare 3R, 5S- dihydroxy-
Application in 6- benzyloxies-hecanoic acid t-butyl ester.
4. application according to claim 1, it is characterised in that describedFor
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| CN103966176B (en) | 2014-05-09 | 2017-12-22 | 凯莱英医药集团（天津）股份有限公司 | Diketoreductase mutant and its application |
| CN104560907B (en) * | 2014-12-19 | 2017-12-22 | 凯莱英医药集团（天津）股份有限公司 | Diketoreductase mutant and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018914A2 (en) * | 1998-09-25 | 2000-04-06 | Amgen Inc. | Novel dkr polypeptides |
| CN101429514A (en) * | 2007-11-08 | 2009-05-13 | 陈依军 | Di-carbonyl reduction enzyme, its gene and uses thereof |
| CN102277338A (en) * | 2011-08-02 | 2011-12-14 | 中国药科大学 | Diketoreductase mutant and application thereof |
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2014
- 2014-05-06 CN CN201410189187.4A patent/CN103937761B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018914A2 (en) * | 1998-09-25 | 2000-04-06 | Amgen Inc. | Novel dkr polypeptides |
| CN101429514A (en) * | 2007-11-08 | 2009-05-13 | 陈依军 | Di-carbonyl reduction enzyme, its gene and uses thereof |
| CN102277338A (en) * | 2011-08-02 | 2011-12-14 | 中国药科大学 | Diketoreductase mutant and application thereof |
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| Title |
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| Accession:WP_003883220;NCBI;《Genbank》;20130801;序列 * |
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