CN103937759B - Biscarbonyl reductase, and coding gene and application thereof - Google Patents
Biscarbonyl reductase, and coding gene and application thereof Download PDFInfo
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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
The replacement, and/or disappearance, and/or addition through one or several amino acid is arranged, and there is generalIt is vertical
Body is optionally reduced toThe amino acid sequence by derived from SEQ NO.1 of function, and SEQ NO.1
Derivative amino acid sequence is with SEQ NO.1 with more than 80% homology, wherein, R1Selected from aromatic radical, alkyl, cycloalkyl,
The miscellaneous alkanisation alkyl of alkyl-substituted aromatic radical, the aromatic radical of halogen substiuted, aralkyl heterocyclic radical, cycloheteroalkyl or ring-type, R2Choosing
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 has the deoxynucleotide sequence of 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 Or
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 90% 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 reduction enzyme gene of the present invention, from the Rhodococcus erythropolis SK121 carbonyl reductions
Enzyme has 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) amino acid sequence of SEQ NO.1；SEQ NO.1 are：
MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSLAAAYKAENVEGAKEGKADEALQRITYS
YDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNT
AEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPF
GPFQIMDVVGLTTVYNISSQGGEKQREFADYIKKNYIDEGKLGVAVGDGFYNYKG；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, 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.Using the di-carbonyl reduction enzyme (Diketoreductase, DKR) of the present invention it is
Biocatalyst, can reduce diketone substrate with a step, prepare the 3R of single optical purity, and 5S- dihydroxy compounds, by which
Synthesis statins drug midbody is applied to, synthesis step is simplified, production pollution and production cost is reduced, is suitable for big rule
Mould industrialized production.
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) deoxynucleotide sequence of SEQ NO.2；SEQ NO.2 are：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA；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 are also in protection scope of the present invention
Within.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 actionStereoselective reduction isIn application.The enzyme can reduce two with a step
Ketone substrate, prepares the 3R of single optical purity, and 5S- dihydroxy compounds may apply to synthesize in the middle of statins
Body.Preferably, di-carbonyl reduction enzyme is preparing 3R, applies in 5S- dihydroxy -6- benzyloxies-hecanoic acid t-butyl ester, wherein,Can be
Or
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Embodiment 1
(1) from the cloning and expression of the di-carbonyl reduction enzyme of Rhodococcus erythropolis SK121 bacterial strains
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.3：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.4：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ ID NO.2：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)。
Amino acid sequence is (SEQ ID NO.1：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAK
ARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVFATN
SSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLL
VPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISSQGGEKQREFADYIKKNYIDEGKLGV
AVGDGFYNYKG)。
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 E. coli competent DH5 α bacterial strains, coat the LB solid mediums 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 solid LB medias of the g/ml containing ampicillin, 37 DEG C of overnight incubations.Single bacterium colony inoculation in the above-mentioned culture medium of picking
In 5ml is containing LB fluid nutrient mediums of the 50 μ g/ml containing ampicillin, just identify through bacterium colony PCR (PCR)
True expression vector carries out abduction delivering, and above-mentioned bacterium solution is transferred in 5ml containing LB Liquid Cultures of the 50 μ g/ml containing ampicillin
In base, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG (isopropylthiogalactoside) is added to be respectively to final concentration
0.02mM, 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
The vertical negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min collects thallines is taken out.Bacterium
Body Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation, and supernatant is with vertically
Electrophoresis apparatus carries out SDS-PAGE detections.As a result find the abduction delivering di-carbonyl reduction enzyme in final concentration of 0.02mMIPTG concentration
Amount it is most.For improving the expression of di-carbonyl reduction enzyme, by the e. coli bl21 containing recombinant plasmid (DE3) at IPTG ends
Concentration is 0.02mM, induces 8h respectively at 37 DEG C, expresses, and while set up under the conditions of 8h and 25 DEG C of induction 16h of 30 DEG C of inductions
It is not added with the negative control of IPTG derivants.After abduction delivering terminates, respectively by the induction thalline of three temperature in 8000rpm/min
Centrifugation 10min collects thallines.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant
Liquid and precipitation, supernatant carry out SDS-PAGE detections with vertical electrophoresis apparatus.As a result the double carbonyls of expression when 16h is induced for 25 DEG C are found
The amount of reductase is most.
(2) from the activity identification of the di-carbonyl reduction enzyme of Rhodococcus erythropolis SK121 bacterial strains
From commercially business-like raw material or the easily ketone compounds of preparation
For initial feed, wherein R1Selected from aromatic radical, alkyl, cycloalkyl, alkyl-substituted aromatic radical, the aromatic radical of halogen substiuted, virtue
The miscellaneous alkanisation alkyl of alkane heterocyclic radical, cycloheteroalkyl or ring-type；R2Selected from alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl.Described
Double hydroxy products are expressed by following chemical general formula：Wherein R1Selected from aromatic radical, alkyl, cycloalkyl,
The miscellaneous alkanisation alkyl of alkyl-substituted aromatic radical, the aromatic radical of halogen substiuted, aralkyl heterocyclic radical, cycloheteroalkyl or ring-type；R2Choosing
From alkyl, cycloalkyl, alkylhalide group or halogen cycloalkyl.
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, dihydroxyProduce
Product 92.0%, yield 90%, ee values are more than 99.5%, de values 90.4%.
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：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 85.0%, 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；Add
0.3g NAD+, 41.2g ammonium formate, 0.5g coenzyme hydrogenlyase and 1g di-carbonyl reduction enzymes, system pH=6.0, and in 30
± 3 DEG C of insulation 17h；Stop reacting with 400ml ethyl acetate, filtered with 250g diatomite, 400ml ethyl acetate is extracted twice,
A point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, dihydroxyProduct 80% is pure
Degree 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
Enter 0.3g NAD+, 41.2g ammonium formate, 0.5g coenzyme hydrogenlyase and 3g di-carbonyl reduction enzymes, system pH=6.0, and in
30 ± 3 DEG C of insulation 17h；Stop reacting with 400ml ethyl acetate, filtered with 250g diatomite, 400ml ethyl acetate extraction two
It is secondary, a point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, dihydroxyProduct
75%, 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 sequence homology in embodiment 1 more than 80% di-carbonyl reduction enzyme (SEQ ID NO.5), the gene source in
Rhodococcus erythropolis PR4。
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：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCGCAGATCGCCTATCAGACCGCCTATCA
CGGATTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAGAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAAAACGTCGAGGGCGCCAAGGAAGGCAAGGCCGACGAAGCGCTACAACGTATTACGTACTCG
TACGATCTCGCCGAAGCCGTGACCAAGGCCGATCTTGTCATCGAGGCAATTCCCGAGGACTTCGCCATCAAGCGCGA
CACCTACGAGAAGCTCGCGGCAGTAGCTCCTGAGCACACGGTGTTCGCAACCAACTCCTCGACGCTTCTGCCGAGCG
ACCTCAAGGAGTTCACCGGCCGCCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAATTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAATGCAGCAT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGATGTCGTCGGGTTGACCACCGTCTTCAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGCGTTCGCCGACTACATCAAGAAGAACTACATCGACGAAGGCAAGCTCGGCGTCGCTGTCGGCGAGGGCTTCTACA
ACTACAAAGGCTGA)
Amino acid sequence is (SEQ ID NO.8：MTELKQITVLGTGVLGSQIAYQTAYHGFDVVAYDINAEVIEKAK
ARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLAEAVTKADLVIEAIPEDFAIKRDTYEKLAAVAPEHTVFATN
SSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLL
VPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVFNISSQGGEKQRAFADYIKKNYIDEGKLGV
AVGEGFYNYKG)。
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, 0.05g main materials are added0.5ml polyethylene glycol PEG-
400, after dissolution of raw material, 4.5ml phosphate buffers (100mM, pH=6.0) are added, substrate is dispersed in buffer solution；Plus
Enter 1.5mgNAD+, 20.6mg ammonium formates, 10mg coenzyme hydrogenlyase and 20mg di-carbonyl reduction enzymes, system pH=6.0, and
17h is incubated in 30 ± 3 DEG C；Stop reacting with 5ml ethyl acetate, filtered with 1.25g diatomite, 5ml ethyl acetate is extracted twice,
A point liquid is stood, organic phase drying is filtered, is concentrated to give crude product, product83.0%, yield
90%, ee value 98%, de values 91%.
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 80%；, 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：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAGTTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)。
Its amino acid sequence is (SEQ ID NO.12：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAVPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISSQGGEKQREFADYIKKNYIDEGK
LGVAVGDGFYNYKG)。
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), coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml moulds of benzyl containing ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of element, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution transfer in
5ml contains containing 50 μ g/ml in the LB fluid nutrient mediums of ampicillin, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added extremely
Final concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is lured at 18 DEG C
Expression is led, and sets up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min is taken out
Collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation,
Supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the induction table in final concentration of 0.02mM IPTG concentration
Amount up to di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the Escherichia coli containing recombinant plasmid
BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h condition of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Lower expression, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction of three temperature
Thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min
Centrifugation 20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to lure at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme when leading 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 R1 is selected from aromatic radical；R2 is selected 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, product80.06%, yield
90%, ee value 99.5%, de values 90.2%.
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 80%；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 IDNO.13：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCGCGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)
Its amino acid sequence is (SEQ ID NO.16：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANRVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISSQGGEKQREFADYIKKNYIDEGK
LGVAVGDGFYNYKG)。
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 dish 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), coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml moulds of benzyl containing ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of element, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution transfer in
5ml contains containing 50 μ g/ml in the LB fluid nutrient mediums of ampicillin, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added extremely
Final concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is lured at 18 DEG C
Expression is led, and sets up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min is taken out
Collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation,
Supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the induction table in final concentration of 0.02mM IPTG concentration
Amount up to di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the Escherichia coli containing recombinant plasmid
BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h condition of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Lower expression, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction of three temperature
Thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min
Centrifugation 20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to lure at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme when leading 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, product70%, yield
90%, ee value 100%, de values 87.95%.
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 80%；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 IDNO.17：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACATTGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)
Its amino acid sequence is (SEQ ID NO.20：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDIVGLTTVYNISSQGGEKQREFADYIKKNYIDEGK
LGVAVGDGFYNYKG)。
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 dish 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), coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml moulds of benzyl containing ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of element, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution transfer in
5ml contains containing 50 μ g/ml in the LB fluid nutrient mediums of ampicillin, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added extremely
Final concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is lured at 18 DEG C
Expression is led, and sets up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min is taken out
Collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation,
Supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the induction table in final concentration of 0.02mM IPTG concentration
Amount up to di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the Escherichia coli containing recombinant plasmid
BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h condition of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Lower expression, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction of three temperature
Thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min
Centrifugation 20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to lure at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme when leading 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, product73.24%, yield
90%, ee value 100%, de values 88.98%.
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 6：
With di-carbonyl reduction enzyme (SEQ ID NO.21) of the sequence homology in embodiment 1 more than 80%；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.22：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.23：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ IDNO.21：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGAA
AGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)。
Its amino acid sequence is (SEQ ID NO.24：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISSQGGEKQKEFADYIKKNYIDEGK
LGVAVGDGFYNYKG)。
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), coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml moulds of benzyl containing ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of element, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution transfer in
5ml contains containing 50 μ g/ml in the LB fluid nutrient mediums of ampicillin, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added extremely
Final concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is lured at 18 DEG C
Expression is led, and sets up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min is taken out
Collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation,
Supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the induction table in final concentration of 0.02mM IPTG concentration
Amount up to di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the Escherichia coli containing recombinant plasmid
BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h condition of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Lower expression, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction of three temperature
Thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min
Centrifugation 20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to lure at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme when leading 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, product72.31%, receive
Rate 90%, ee values 100%, de values 89.16%.
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 7
With di-carbonyl reduction enzyme (SEQ ID NO.25) of the sequence homology in embodiment 1 more than 80%；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.26：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.27：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ IDNO.25：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGCTGTCGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)。
Its amino acid sequence is (SEQ ID NO.28：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDAVGLTTVYNISSQGGEKQREFADYIKKNYIDEGK
LGVAVGDGFYNYKG).After di-carbonyl reduction enzyme total gene synthesis, it is connected on pUC57 carriers, respectively will with Nde I and Xho I
(pET-22b (+) can also be pET-3a (+), pET-3d (+), pET-11a (+), pET-12a for genes of interest and pET-22b (+)
(+), 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, will company
Thing of practicing midwifery is transformed in the competence of e.colistraindh5α, coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony on the above-mentioned culture medium of picking is inoculated in and contains ampicillin containing 50 μ g/ml
In LB fluid nutrient mediums, overnight, through plasmid extraction, PCR is identified after identifying with double digestion 37 DEG C of shaken cultivations, by correct gram
Grand carrier pET22b (+)-DKR is transformed in e. coli bl21 (DE3), is coated and is contained ampicillin containing 50 μ g/ml
In solid LB media, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml and contains ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of parasiticin, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution
Transfer in the LB fluid nutrient mediums that 5ml contains ampicillin containing 50 μ g/ml, 37 DEG C of shaken cultivations to OD600When=0.6, add
IPTG is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM to final concentration, at 18 DEG C
Abduction delivering is carried out, and sets up the negative control for being not added with IPTG derivants.Induction 16h after, take out bacterium solution, 8000rpm/min from
Heart 10min collects thallines.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant
And precipitation, supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find in final concentration of 0.02mM IPTG concentration
The amount of abduction delivering di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the large intestine containing recombinant plasmid
Bacillus BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Under the conditions of express, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by three temperature
Induction thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/
Min centrifugation 20min obtain supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find 25
DEG C induction 16h when express di-carbonyl reduction enzyme amount it 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, product56.96%, receive
Rate 90%, ee values 100%, de values 92.36%.
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 8
With di-carbonyl reduction enzyme (SEQ ID NO.29) of the sequence homology in embodiment 1 more than 80%；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.30：5’-
GGAATTCCATATGACCGAACTGAAACAAATCACC-3’；Downstream primer SEQ ID NO.31：5’-
CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3’.Expand to obtain gene order (SEQ IDNO.29：
ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCA
CGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCG
CGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCG
TACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGA
CACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCG
ATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACT
GCCGAGGTCATGGGCACCGAGTCCACCGACCCCGCCGTGTACCGCGAGGTCGTCGAGTTCGCGAAGAACATCGGCAT
GGTGCCGATCGAACTCAAGAAGGAGAAGGCGGGCTACGTACTCAACTCGCTCCTGGTCCCGCTCCTCAACGCGGCCT
CCGACCTGCTGATCGACGGCATCGCCGATCCCGACATGGTCGACAAGACGTGGCGTATCGGCACCGGAGCCCCGTTC
GGCCCCTTCCAGATCATGGACGTCGCTGGGTTGACCACCGTCTACAACATCTCCTCCCAGGGCGGCGAGAAGCAGCG
CGAATTCGCCGACTACATCAAGAAGAACTACATCGACGAGGGCAAGCTCGGCGTTGCTGTCGGCGACGGCTTCTACA
ACTACAAGGGCTGA)。
Its amino acid sequence is (SEQ ID NO.32：MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIE
KAKARFDSLAAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAIKRDTYEKLATVAPEHTVF
ATNSSTLLPSDLKEFTGRPEKFLALHFANHVWVNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLN
SLLVPLLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVAGLTTVYNISSQGGEKQREFADYIKKNYIDEGK
LGVAVGDGFYNYKG)。
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), coats the solid LB containing ampicillin containing 50 μ g/ml
In culture medium, 37 DEG C of overnight incubations.Single bacterium colony in the above-mentioned culture medium of picking is inoculated in 5ml moulds of benzyl containing ammonia containing 50 μ g/ml
In the LB fluid nutrient mediums of element, identify that correct expression vector carries out abduction delivering through bacterium colony PCR, by above-mentioned bacterium solution transfer in
5ml contains containing 50 μ g/ml in the LB fluid nutrient mediums of ampicillin, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG is added extremely
Final concentration is respectively 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM, is lured at 18 DEG C
Expression is led, and sets up the negative control for being not added with IPTG derivants.After induction 16h, bacterium solution, 8000rpm/min centrifugation 10min is taken out
Collects thalline.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min centrifugation 20min obtain supernatant and precipitation,
Supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find the induction table in final concentration of 0.02mM IPTG concentration
Amount up to di-carbonyl reduction enzyme is most.For improving the expression of di-carbonyl reduction enzyme, by the Escherichia coli containing recombinant plasmid
BL21 (DE3) induces 8h, 8h and 25 DEG C of induction 16h condition of 30 DEG C of inductions in the final concentration of 0.02mM of IPTG respectively at 37 DEG C
Lower expression, and while set up the negative control for being not added with IPTG derivants.After abduction delivering terminates, respectively by the induction of three temperature
Thalline is centrifuged 10min collects thallines in 8000rpm/min.Thalline Ultrasonic Cell Disruptor smudge cells, 4 DEG C, 12000rpm/min
Centrifugation 20min obtains supernatant and precipitation, and supernatant carries out SDS-PAGE detections with vertical electrophoresis apparatus.As a result find to lure at 25 DEG C
The amount for expressing di-carbonyl reduction enzyme when leading 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, product71.94%, yield
90%, ee value 100%, de values 88.29%.
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 should
With, 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, described pair of carbonyl is also
The amino acid sequence of protoenzyme such as one of following amino acid sequences：SEQ ID NO.1、SEQ ID NO.8、SEQ ID NO.12、SEQ
Amino acid sequence shown in ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28 or SEQ ID NO.32
Row.
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、SEQ
Deoxynucleotide sequence shown in ID NO.17, SEQ ID NO.21, SEQ ID NO.25 or SEQ ID NO.29.
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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| CN107603961B (en) * | 2014-05-09 | 2020-04-24 | 凯莱英医药集团（天津）股份有限公司 | Bischarbonylreductase mutant and application thereof |
| 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 |
| WO2016095223A1 (en) * | 2014-12-19 | 2016-06-23 | 凯莱英医药集团（天津）股份有限公司 | Double-carbonyl reductase mutant and application thereof |
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| US6344541B1 (en) * | 1998-09-25 | 2002-02-05 | Amgen Inc. | DKR polypeptides |
| CN101429514B (en) * | 2007-11-08 | 2011-06-15 | 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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