CN104830921A - Enzymatic method for preparing statins intermediates - Google Patents
Enzymatic method for preparing statins intermediates Download PDFInfo
- Publication number
- CN104830921A CN104830921A CN201510209354.1A CN201510209354A CN104830921A CN 104830921 A CN104830921 A CN 104830921A CN 201510209354 A CN201510209354 A CN 201510209354A CN 104830921 A CN104830921 A CN 104830921A
- Authority
- CN
- China
- Prior art keywords
- compound
- formula
- alcoholdehydrogenase
- cmadh3
- polynucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 0 C*C(CC1*C(C)(C)O[C@](CC#N)C1)=O Chemical compound C*C(CC1*C(C)(C)O[C@](CC#N)C1)=O 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing statins intermediates through biosynthesis, and specifically discloses an enzymatic method for preparing statins intermediates. The enzymatic method comprises the following steps: (a) carrying out catalytic reduction reactions on a compound (III) in the presence of alcohol dehydrogenase CmADH3, co-enzyme factors, and a regeneration system of an optional co-enzyme factor to produce a compound (II); (b) reacting the compound (II) with 2,2-dimethoxy propane to generate a compound (I). The provided method can reduce the production cost and environmental pollution, and thus has a great application value.
Description
Technical field
The present invention relates to a kind of biological technical field, particularly, relate to a kind of method that enzyme process prepares statin compound intermediate.
Background technology
Atorvastatin is Statins blood lipid regulation medicine, belongs to HMG-CoA reductase inhibitor.Hydrolysate after oral absorption suppresses the rate-limiting enzyme HMG-CoA reductase in cholesterol biosynthesis process in vivo competitively, thus reduce the synthesis of cholesterol, increase the synthesis of low density lipoprotein receptor, thus for preventing and treating hypercholesterolemia, combined hyperlipidemia familial, coronary heart disease and cerebral apoplexy.
Usually adopt the intermediate of chemical process synthesis atorvastatin at present, because the method has high, the heavy-polluted shortcoming of production cost, thus start gradually recently to replace this chemical process with enzyme-chemical method.But the general catalytic activity of enzyme adopted at present is lower, causes catalyzed reaction enzyme amount very large, affect extract yield, and production cost is still higher.
Therefore, this area can reduce production cost and environment amenable biosynthetic means in the urgent need to developing one, particularly, needs to develop the method that enzyme that a kind of utilization has a high-efficiency catalytic activity prepares statin compound intermediate.
Summary of the invention
The object of this invention is to provide and a kind ofly there is the enzyme of high-efficiency catalytic activity and prepare the method for statin compound intermediate with this enzyme.
First aspect present invention provides a kind of method of preparation formula II compound, and described method comprises step:
A (), under alcoholdehydrogenase CmADH3 exists, carries out catalytic reduction reaction to formula III compound, thus production II compound;
Wherein, R is selected from C1-C6 alkyl or C1-C6 haloalkyl.
In another preference, R is C1-C4 alkyl, is preferably the tertiary butyl, ethyl or methyl.
In another preference, described alcoholdehydrogenase CmADH3 derives from Candida magnoliae.
In another preference, described alcoholdehydrogenase CmADH3 is the alcoholdehydrogenase of wild-type/saltant type.
In another preference, the alcoholdehydrogenase CmADH3 of described saltant type has the aminoacid sequence shown in SEQ ID NO.:1.
In another preference, described sequence comprises the aminoacid sequence with SEQ ID NO.:1 with 90% (preferably, 95%) homology.
In another preference, described method is carried out under the existence of cofactors.
In another preference, described method is carried out under the existence of the regeneration system rapidly of cofactors and described cofactors.
In another preference, described cofactors is NADPH and/or NADH.
In another preference, described cofactors is NADPH.
In another preference, the regeneration system rapidly of described cofactors is selected from lower group: Hexose phosphate dehydrogenase GDH and glucose, hydrogenlyase and formic acid, Virahol and dehydrogenating para-alcohol enzyme or its combination.
In another preference, the regeneration system rapidly of described cofactors is Hexose phosphate dehydrogenase GDH and the glucose of wild-type/saltant type.
In another preference, the Hexose phosphate dehydrogenase GDH of described saltant type sports leucine at the 252nd, and sports arginine at the 170th.
In another preference, described Hexose phosphate dehydrogenase GDH has the encoding sequence shown in SEQ ID NO.:2.
In another preference, described sequence comprises the encoding sequence with SEQ ID NO.:2 with 90% (preferably, 95%) homology.
In another preference, the consumption (g/L) of described cofactors is 0.005-0.2, preferably, and 0.01-0.1, more preferably, 0.03-0.08.
In another preference, described Hexose phosphate dehydrogenase GDH derives from prokaryotic organism.
In another preference, described Hexose phosphate dehydrogenase GDH derives from subtilis, bacillus megaterium and mutant thereof.
In another preference, described step (a) has following one or more feature:
(1) temperature of reaction is 20-40 DEG C, preferably, and 25-35 DEG C, more preferably 28-32 DEG C;
(2) reaction times is 0.5-24 hour, preferably, is 1-10 hour, more preferably, is 2-7 hour;
(3) pH is 5-8, preferably, and 6.5-7.5.
In another preference, described formula III compound and described alcoholdehydrogenase CmADH3 weight ratio are 10000:1-10:1, preferably, and 1000:1-20:1, more preferably, 500:1-50:1.
In another preference, described alcoholdehydrogenase CmADH3 adds with the form of bacterium mud.
In another preference, the weight ratio of described formula III compound and described bacterium mud is 100:1-1:1, preferably, and 80:1-8:1, more preferably, 50:1-10:1.
Second aspect present invention provides the method for the ring-4-alkyl acetate compound of (4R-Cis)-6-cyano group-2,2-dimethyl-1, the 3-dioxy shown in a kind of preparation formula I, comprises step:
A (), under alcoholdehydrogenase CmADH3 exists, carries out generation catalytic reduction reaction to formula III compound, thus production II compound;
B formula II compound and 2,2-dimethoxypropane, in inert solvent, react, form formula I by ():
Wherein, above-mentioned various in, R is selected from C1-C6 alkyl or C1-C6 haloalkyl.
In another preference, in step (b), react under methylsulfonic acid exists.
In another preference, described method also comprises step between step (a) and (b): the formula II compound generated in step (a) is carried out separation and purification, and the formula II compound of separation and purification is used for step (b).
In another preference, described separation and purification comprises: filter and extraction.
In another preference, the temperature in step (b) is 0-50 DEG C, preferably 4-35 DEG C; Time is 0.1-24 hour, is preferably 0.5-12 hour.
Third aspect present invention provides a kind of method of synthesizing statin compound, and described method comprises step:
A () formula III compound is by catalytic reduction reaction production II compound, wherein, described step (a) is carried out under alcoholdehydrogenase CmADH3 existent condition;
B () formula II compound and 2,2-dimethoxypropane react, form formula I; With
C described formula I as intermediate, is prepared statin compound by ().
In another preference, described statin compound is selected from lower group: atorvastatin, superstatin, pitavastatin or its combination.
Fourth aspect present invention provides a kind of reaction mixture, and described mixture comprises:
(i) alcoholdehydrogenase CmADH3;
(ii) formula III compound and/or formula II compound; And
(iii) described cofactors.
In another preference, described mixture also comprises the regeneration system rapidly of described cofactors.
In another preference, described cofactors is NADPH and/or NADH.
In another preference, described cofactors is NADPH.
In another preference, the regeneration system rapidly of described cofactors is selected from lower group: Hexose phosphate dehydrogenase GDH and glucose, hydrogenlyase and formic acid, Virahol and dehydrogenating para-alcohol enzyme or its combination.
In another preference, the regeneration system rapidly of described cofactors is Hexose phosphate dehydrogenase GDH and glucose.
In another preference, described alcoholdehydrogenase CmADH3 joins in reaction formula with zymin or fermented liquid form.
In another preference, the consumption (g/L) of described cofactors is 0.005-0.2, preferably, and 0.01-0.1, more preferably, 0.03-0.08.
In another preference, described formula III compound and described alcoholdehydrogenase CmADH3 weight ratio are 10000:1-10:1, preferably, and 1000:1-20:1, more preferably, 500:1-50:1.
In another preference, described alcoholdehydrogenase CmADH3 adds with the form of bacterium mud.
In another preference, the weight ratio of described formula III compound and described bacterium mud is 100:1-1:1, preferably, and 80:1-8:1, more preferably, 50:1-10:1.
Fifth aspect present invention provides the purposes of the engineering bacteria of a kind of alcoholdehydrogenase or expression recombinase, and the engineering bacteria of described alcoholdehydrogenase or expression recombinase is used for catalytic reduction formula III compound, thus production II compound.
In another preference, described alcoholdehydrogenase derives from candida magnoliae (Candida magnoliae).
In another preference, described reaction is also carried out under the regeneration system rapidly of cofactors and optional cofactors.
In another preference, described cofactors is NADPH and/or NADH.
In another preference, described cofactors is NADPH.
In another preference, the regeneration system rapidly of described cofactors is selected from lower group: Hexose phosphate dehydrogenase GDH and glucose, hydrogenlyase and formic acid, Virahol and dehydrogenating para-alcohol enzyme or its combination.
In another preference, the regeneration system rapidly of described cofactors is Hexose phosphate dehydrogenase GDH and glucose.
In another preference, described engineering bacteria is selected from lower group: intestinal bacteria, pichia spp, yeast saccharomyces cerevisiae, genus bacillus or its combination.
Sixth aspect present invention provides a kind of polynucleotide, and described Nucleotide is selected from lower group:
The polynucleotide of (a) nucleotide sequence as shown in SEQ ID NO.:3;
Homology >=90% (preferably >=95%) of sequence shown in (b) nucleotide sequence and SEQ ID NO.:3, and there are the polynucleotide of formula III compound for catalysis reducing activity;
(c) polynucleotide sudden change 1-60 as shown in SEQ ID NO.:3 (preferably 1-30, more preferably 1-6) Nucleotide, and there are the polynucleotide of formula III compound for catalysis reducing activity;
D () is for the aminoacid sequence shown in SEQ ID NO.:1 of encoding.
In another preference, described polynucleotide derive from candida magnoliae (Candida magnoliae).
Seventh aspect present invention provides a kind of expression vector, and described carrier contains the polynucleotide described in sixth aspect present invention.
Eighth aspect present invention provides a kind of host cell, and described host cell contains the polynucleotide described in sixth aspect present invention that expression vector described in seventh aspect present invention or its chromosomal integration have external source.
In another preference, described host cell is selected from lower group: intestinal bacteria, pichia spp, yeast saccharomyces cerevisiae, genus bacillus or its combination.
In another preference, described host cell is intestinal bacteria.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the solubility expression situation of kred316 enzyme and CmADH3 enzyme.
Fig. 2 shows the catalytic activity comparative result of CmADH3 enzyme and Kred316 enzyme.
Fig. 3 shows the purity check situation of formula I.
Fig. 4 shows the chiral analysis situation of formula I.
Embodiment
The present inventor is through extensive and deep research, by screening in a large number desaturase, a kind of alcoholdehydrogenase CmADH3 of unexpected discovery, it has very high catalytic activity, can under cofactors (as NADPH) and Hexose phosphate dehydrogenase GDH existent condition, the intermediate formula III compound of catalytic reduction atorvastatin, production II compound.Further, catalyze and synthesize atorvastatin intermediate with alcoholdehydrogenase CmADH3, can not only production cost be reduced, can also environmental pollution be reduced, there is great using value.
Alcoholdehydrogenase CmADH3 and uses thereof
As used herein, described " alcoholdehydrogenase CmADH3 " refers to that one derives from the alcoholdehydrogenase of candida magnoliae (Candidamagnoliae), it is suitable for more at expression in escherichia coli, and the alcoholdehydrogenase CmADH3 that expression obtains has very high catalytic activity.
In the present invention, alcoholdehydrogenase CmADH3 is used to catalytic reduction formula III compound, generates the intermediate formula II compound of statin compound.
As compared to kred316 enzyme (Chinese Patent Application No.: 200780036841.6, SEQ ID NO..315), alcoholdehydrogenase CmADH3 of the present invention has higher catalytic activity.
The regeneration system rapidly of cofactors and cofactors
As used herein, described " cofactors " refers to and participates in the non-protein compound that alcoholdehydrogenase CmADH3 carries out catalytic reduction reaction.
In the present invention, described " cofactors " includes but not limited to: NADPH (NADP
+reduction form) and NADP
+, NADH (NAD
+reduction form) and NAD
+.
As used herein, described " regeneration system rapidly of cofactors " refers to that the oxidized form form participating in the dihydrocoenzyme factor is (as NADP
+to NADPH) a group reaction thing of reaction.Usually, the regeneration system rapidly of cofactors includes but not limited to: Hexose phosphate dehydrogenase GDH and glucose, hydrogenlyase and formic acid, Virahol and dehydrogenating para-alcohol enzyme, phosphorous acid and phosphorous acid desaturase.
In the present invention, with Hexose phosphate dehydrogenase GDH and glucose as " regeneration system rapidly of cofactors ".Particularly, alcoholdehydrogenase CmADH3 enzyme catalysis reduction formula III compound prepares formula II compound, and this reduction reaction relies on coenzyme NADP 11 and provides reducing power, and after reaction, NADPH is converted into NADP
+, with Hexose phosphate dehydrogenase GDH and glucose regeneration coenzyme NADP 11, glucose is converted into gluconic acid.Under the effect of described " regeneration system rapidly of cofactors ", cofactors is able to continuous recycling.
As used herein, described " Hexose phosphate dehydrogenase GDH " comprises the Hexose phosphate dehydrogenase GDH of wild-type and the Hexose phosphate dehydrogenase GDH of saltant type.Wherein, the Hexose phosphate dehydrogenase GDH of saltant type can produce by known method (as mutagenesis, orthogenesis) and other similar approach.In a preference of the present invention, described " Hexose phosphate dehydrogenase GDH " derives from subtilis (Bacillus subtilis subsp.subtilis str.168), there is the encoding sequence shown in SEQ ID NO.:2, described sequence comprises and has 90% (preferably with SEQ ID NO.:2,95%) encoding sequence of homology, and the Hexose phosphate dehydrogenase GDH of described saltant type sports leucine at the 252nd, and sport arginine at the 170th.The enzyme measuring the Hexose phosphate dehydrogenase GDH of saltant type by the method for the embodiment of the present invention 6 is lived, and result shows its enzyme work up to 4190U/ml.
The preparation of formula I and statin compound
As used herein, the structural formula of formula I is
be the most important intermediate of statin compound, usually adopt chemical process to synthesize.
The invention provides a kind of method of biosynthesizing formula I, particularly, described method comprises the steps: that (a) is under the existence of the regeneration system rapidly (as Hexose phosphate dehydrogenase GDH and glucose) of cofactors (as NADPH) and cofactors, with alcoholdehydrogenase CmADH3, catalytic reduction reaction is carried out to formula III compound, production II compound; B formula II compound and 2,2-dimethoxypropane react by (), form formula I.The formula I obtained by the method has very high purity, and the content of diastereomer is only 0.0106%, and the content of enantiomer is only 0.01%.Wherein, the R of formula I, formula II, formula III compound as defined hereinabove.
Present invention also offers the method for a kind of biosynthesizing synthesis statin compound, comprise step:
A () formula III compound is by catalytic reduction reaction production II compound, wherein, described step (a) is carried out under alcoholdehydrogenase CmADH3 existent condition;
B () formula II compound and 2,2-dimethoxypropane react, form formula I; With
C described formula I as intermediate, is prepared statin compound by ().
With aforesaid method preparation I compound and statin compound, can not only reduce costs, can also environmental pollution be reduced, there is great using value.
Major advantage of the present invention comprises:
(1) first alcoholdehydrogenase CmADH3 is used in the catalytic reduction reaction of atorvastatin intermediate (formula II compound).
(2) compared with Kred316, alcoholdehydrogenase CmADH3 has higher catalytic activity, in 5.5 hours, just can complete catalytic reduction reaction.TLC shows, and after 5.5 hours, alcoholdehydrogenase CmADH3 is complete by substrate reactions, and Kred316 still has a large amount of substrate unreacted complete.
(3) the Hexose phosphate dehydrogenase GDH participating in catalytic reduction reaction has very high enzymic activity, can reach 4190U/ml.
(4) the atorvastatin intermediate (formula I) that catalytic reduction reaction obtains has high purity, and the content of diastereomer is only 0.0106%, and the content of enantiomer is only 0.01%.
Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted detailed conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Universal method
Strain fermentation:
(1) substratum preparation:
LB substratum: 10g/L Tryptones, 5g/L yeast extract, 10g/L sodium-chlor, pH7.2,121 DEG C of autoclave sterilization 20min;
TB substratum: 24g/L yeast extract, 12g/L Tryptones, 16.43g/L K
2hPO
4.3H
2o, 2.31g/L KH
2pO
4, 5g/L glycerine, pH 7.0-7.5,121 DEG C of autoclave sterilization 20min;
Slant medium: 10g/L Tryptones, 5g/L yeast extract, 10g/L sodium-chlor, 20g/L agar powder, eggplant bottle is dispensed into by 30-40mL liquid amount after mixing, setting is positioned over 121 DEG C of autoclave sterilization 20min, 100 μ g/mL sulphuric acid kanamycins are added after cooling, put into inclined-plane, one-tenth solid to be condensed.
(2) fermentation culture
Seed activation: get seed glycerol stocks pipe, gets 100 μ l Seed Deposit liquid, with transfering loop uniform application in eggplant bottle inclined-plane, is then placed in 37 DEG C of incubator overnight incubation (18h);
Seed culture: get 100ml sterilized water importing eggplant bottle and make bacteria suspension, get the 250ml shaking flask that 50ml TB substratum is equipped with in bacteria suspension 50 μ l access, 30 DEG C, 220rpm cultivates 16h;
Fermentation: the 5L shaking flask access of first order seed nutrient solution being equipped with 1l TB substratum, 37 DEG C, 220rpm adds 0.3mM IPTG and is cooled to 28 DEG C, 220rpm inducing culture 12h after cultivating 4-6h.
(3) microorganism collection: collect fermented liquid, be placed in the centrifugal 30min of whizzer 4000rpm, collect bacterium mud after abandoning supernatant, be positioned over-20 DEG C of freezing collections.
(4) preparation of enzyme liquid: take each 5g of GDH, CmAdH3, Kred316 bacterium mud respectively, then after respectively adding the deionized water resuspension of 20ml, carry out cytoclasis by ultrasonic wave, broken liquid gets supernatant after the centrifugal 10min of 12000rpm, be enzyme liquid, be positioned in ice bath stand-by;
Embodiment 1 CmADH3 express bacterial classification structure
Candida magnoliae (Candida magnoliae) to be originated the protein sequence (NCBI accession number: ABB91667) of alcoholdehydrogenase CmADH3, carry out being suitable for the codon optimized of escherichia coli expression, and full genome synthesizes this gene order, at two ends design restriction enzyme site NdeI and BamHI, and be subcloned into the upper corresponding site of carrier pET24a (purchased from Novagen), thus obtain recombinant plasmid pET24a-CmADH3.
By recombinant plasmid pET24a-CmADH3 Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built, obtain BL21 (DE3)/pET24a-CmADH3.
Codon optimized gene order (SEQ ID NO.:3):
Protein sequence (SEQ ID NO.1):
Embodiment 2 KRED316 expresses the structure of bacterial classification
According to the gene order (Chinese Patent Application No.: 200780036841.6 of the Saccharomyces cerevisiae source carbonyl reduction enzyme mutant reported, SEQ ID NO.315), carry out full genome and synthesize this sequence, at two ends design restriction enzyme site NdeI and BamHI, and be subcloned into the upper corresponding site of carrier pET24a (purchased from Novagen), thus obtain recombinant plasmid pET24a-KRED316.
By recombinant plasmid pET24a-KRED316 Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built, obtain BL21 (DE3)/pET24a-KRED316.
Gene order is as follows:
Embodiment 3 GDH expresses the structure of bacterial classification
Subtilis (Bacillus subtilis subsp.subtilis str.168) is inoculated in LB liquid nutrient medium, 30 DEG C, 220rpm cultivates 24 hours.Reference gene group extracts (biological purchased from raw work) that test kit specification sheets extracts STb gene.
According to the gene order (NCBI accession number: AL009126.3) of the Hexose phosphate dehydrogenase (GDH) that the subtilis reported (Bacillus subtilis subsp.subtilis str.168) is originated, design primer pair: GDH-F:5 '-CGGGATCCATGTATCCGGATTTAAAAG-3 ' (BamHI), GDH-R:5 '-CCCAAGCTTTTAACCGCGGCCTGCCTGG-3 ' (HindIII).
PCR reaction system comprises: each 50pmol of GDH-F and GDH-R, STb gene 100ng, 1X KODplus buffer, 0.2mM dNTP, 25mM MgSO4, KOD plus 2U, and moisturizing is to total system 50 μ L.Pcr amplification condition is: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 1min, repeats 30 circulations, 68 DEG C of 10min.
After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of an about 800bp detected, for required.Glue reclaims test kit and reclaims pcr amplification product in a small amount, with BamHI and HindIII in 37 DEG C of double digestion 3-6 hour, crosses column purification and reclaims.Reclaim expression vector pET24a that product and same enzyme cut process to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, Transformed E .coli DH5 α competent cell, picking transformant carries out sequence verification, acquisition recombinant plasmid.
Rite-directed mutagenesis is done to 252 and 170 two sites of GDH aminoacid sequence: E170R, Q252L.According to amino acid to be suddenlyd change and mutational site design primer, MEGA WHOP method (Arnold andGeorgiou 2003) is adopted to suddenly change.
Design primer pair: GDHE170R-F:AAGCTGATGACACGAACATTAGCGTT, GDHQ252L-R:AATGAAGGATATAGTGTCATACCGC.
With this, sequence containing mutational site Q252L/E170R is gone out to primer amplification.PCR reaction system comprises: each 50pmol of GDHE170R-F and GDHQ252L-R, plasmid template pET24a-GDH 50ng, 1X KODplus buffer, 0.2mM dNTP, 25mM MgSO4, KOD plus 2U, and moisturizing is to total system 50 μ L.Pcr amplification condition is: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 30s, repeats 30 circulations, 68 DEG C of 10min.After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of an about 250bp detected, for required.Glue reclaims test kit and reclaims pcr amplification product in a small amount.Using this PCR primer as large primer, take pET24a-GDH as template, high-fidelity DNA polymerase KOD plus is adopted to do full plasmid linear amplification, PCR reaction system comprises: large primer segments 50-100pmol, plasmid template pET24a-GDH 50ng, 1X KOD plus buffer, 0.2mM dNTP, 25mM MgSO4, KODplus 2U, moisturizing is to total system 50 μ L.Pcr amplification condition is: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 6min, repeats 25 circulations, 68 DEG C of 10min.After having increased, in system, add DpnI remove plasmid template, then by direct for digestion product Transformed E .coli BL21 (DE3) competent cell in 37 DEG C of digestion.Picked clones carries out sequence verification, expression bacterial classification called after BL21 (the DE3)/pET24a-GDH checking order correct.
Gene order (SEQ ID NO.:2):
Embodiment 4 strain fermentation
By universal method, bacterial strain is fermented, collect bacterium mud afterwards, be positioned over-20 DEG C of freezing collections, and prepare enzyme liquid.
Embodiment 5 protein electrophoresis detects expression product
Take that appropriate thalline deionized water is resuspended makes the bacteria suspension that concentration is 20g/l, ultrasonic cell disintegration.Get 1ml and break the centrifugal rear sucking-off supernatant liquor of cytosol, then add the resuspended precipitation of equal-volume water, obtained precipitated liquid.Get 15 μ l supernatant liquors and precipitated liquid and 5 μ l 4XSDS albumen sample-loading buffer (Takara Code:D621) respectively to mix and boil 5min, get 10 μ l after centrifugal and carry out SDS-PAGE analysis.Adopt 5% concentrated glue and 12% separation gel, electrophoresis terminates rear gel coomassie brilliant blue R250 and dyes.
As shown in Figure 1, result shows, the solubility expression of kred316 and CmADH3 is all relatively good.
The enzyme activity determination of embodiment 6 Hexose phosphate dehydrogenase GDH
Take each 5g of GDH, CmADH3, Kred316 bacterium mud respectively, after then respectively adding the deionized water resuspension of 20ml, carry out cytoclasis by ultrasonic wave, broken liquid gets supernatant after the centrifugal 10min of 12000rpm, is enzyme liquid, is positioned in ice bath stand-by.
With document (Fujita Y, Ramaley R, Freese E.Location and properties ofglucose dehydrogenase in sporulating cells and spores of Bacillussubtilis.J.Bacteriol, 1977, method 132:282-293.) measures, and during mensuration, coenzyme adopts NADP.
Result shows, GDH enzyme liquid has higher enzyme and lives, up to 4190U/ml.
The catalytic activity comparative result of embodiment 7 CmADH3 enzyme and Kred316 enzyme
Experimental technique:
Get the beaker of 2 250ml, add 0.1g trolamine, 5ml water, 4.5g glucose, 5g substrate (6-cyano group-(5R)-hydroxyl-3-carbonyl tert-butyl acrylate) respectively, be warming up to 30 DEG C, pH7.0 is regulated with 20% sulfuric acid, add 1.2ml GDH enzyme liquid, then add kred316 and the CmADH3 enzyme liquid of 3ml respectively, be settled to 50ml with water and after being warming up to 30 DEG C, the NaNADP adding 0.05g/L starts reaction, with the Na of 1M
2cO
3solution control pH7.0.
The Na of table 1 Different periods
2cO
3the additional amount of solution
Enzyme is detected to the Expenditure Levels of substrate with TLC.Result shows, as shown in Figure 2, after 5.5h, CmADH3 is complete by substrate reactions, and kred316 still has a large amount of substrate unreacted complete.Therefore, the catalytic efficiency of CmADH3 is apparently higher than kred316.
Embodiment 8 synthetic compound of formula i
(1)
6-cyano group-(5R)-hydroxyl-3-carbonyl tert-butyl acrylate prepares 6-cyano group-(3R, 5R)-dihydroxyl tert-butyl acrylate
Water 100ml, 2g trolamine is added in 1L flask, sulfuric acid with 20% is by pH regulator to 7.0, add glucose 80g, stir, adding 50g 6-cyano group-(5R)-hydroxyl-3-carbonyl tert-butyl acrylate again, is 7.0 by 20% dilute sulphuric acid adjust ph, then adds GDH enzyme liquid 2ml, CmADH3 enzyme liquid 20ml of 4190U/ml, reaction is started, with the Na of 1M after adding the NaNADP of 0.05g/L after being warming up to 30 DEG C
2cO
3water-soluble is 7.0 by pH control, reacts pH after 5 hours and no longer changes, detect transform completely through TLC.
Add ethyl acetate 500ml and diatomite 50g, be warming up to 50 ~ 60 DEG C, layering after filtering, water layer 300ml extraction into ethyl acetate twice, merges organic layer, the concentrated crude product 6-cyano group-(3R obtaining 48g, 5R)-dihydroxyl tert-butyl acrylate is oily matter.
(2)
6-cyano group-(3R, 5R)-dihydroxyl is tert-butyl acrylate preparation (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate
In flask, add 48g 6-cyano group-(3R, 5R)-dihydroxyl tert-butyl acrylate, 2,2-dimethoxypropane 50g, toluene 100ml and methylsulfonic acid 0.4g that step (1) obtains, be warming up to 25 DEG C, insulation reaction 3 hours.After reaction terminates, add 200ml toluene, add saturated sodium bicarbonate aqueous solution and be neutralized to pH7.5, branch vibration layer, concentrated dried organic layer.Add 150ml hexane, rising temperature for dissolving, be cooled to 0 DEG C, crystallization, filter, obtain 45.5g (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate crude product.Crude product uses hexane (100ml) and ethanol (5ml) mixed solvent recrystallization again, filters, dries to obtain 26.5g (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate.
The purity of embodiment 9 analysis mode I (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate
Measuring method:
Gas chromatographic column: SPBTM-50 30m × 0.32mm × 0.25um;
Column temperature: 200 DEG C, detector: 300 DEG C
Vaporizing chamber: 280 DEG C of carrier gas: N2, column head pressure: 0.05MPa
Sample size: 0.2 μ l, air: 300ml/min
Splitting ratio: 50:1 hydrogen: 30ml/min
Diastereomer retention time is 5.85min, and (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate is 6.38min
Sample analysis: take above-mentioned sample and be about 0.2g in 25mm × 25mm weighing bottle, add analytically pure DMF1ml to dissolve, after instrument stabilizer, with microsyringe extracting sample solution 0.2 μ l inject gas chromatograph, record color atlas is to two times of main composition peak retention time, delete DMF and solvent peak, by area normalization method calculation result.
As shown in Figure 3, result shows, the content of diastereomer is only 0.0106%.Therefore, formula I (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate there is very high purity.
The chiral analysis of embodiment 10 formula I (4R-Cis)-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate
Measuring method:
Liquid phase flow phase: normal hexane: the mellow 98:2 of isopropyl
Wavelength X=215nm, flow velocity=0.6ml/min, solvent: moving phase;
Chromatographic column: Chiralcel OD-H 250 × 4.6mm 5um;
Sample size: 20 μ l, column temperature: 30 DEG C, working time: 28min.
(4R-Cis) retention time of-6-cyano group-2,2-dimethyl-1,3-dioxy ring-4-tert.-butyl acetate is about 19.8min; 4R, 6S-isomer about retention time 14.1min; 4S, 6S-isomer about retention time 15.5min; 4S, 6R-isomer about retention time 16.9min.
Sample analysis: take above-mentioned sample and be about 200mg and put in the volumetric flask of 10ml, with dissolution with solvents, constant volume, shake up, as sample solution.Extracting sample solution 20 μ l, injection liquid chromatography, detects.
As shown in Figure 4, result shows, under liquid phase chiral analysis condition, the content of enantiomer is only 0.01%, and other isomer almost do not detect.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a method for preparation formula II compound, is characterized in that, described method comprises step:
A (), under alcoholdehydrogenase CmADH3 exists, carries out catalytic reduction reaction to formula III compound, thus production II compound;
Wherein, R is selected from C1-C6 alkyl or C1-C6 haloalkyl.
2. the method for claim 1, is characterized in that, described method is carried out under the existence of cofactors.
3. the method for the ring-4-alkyl acetate compound of (4R-Cis)-6-cyano group-2,2-dimethyl-1, the 3-dioxy shown in a preparation formula I, is characterized in that, comprise step:
A (), under alcoholdehydrogenase CmADH3 exists, carries out generation catalytic reduction reaction to formula III compound, thus production II compound;
B formula II compound and 2,2-dimethoxypropane, in inert solvent, react, form formula I by ():
Wherein, above-mentioned various in, R is selected from C1-C6 alkyl or C1-C6 haloalkyl.
4. synthesize a method for statin compound, it is characterized in that, described method comprises step:
A () formula III compound is by catalytic reduction reaction production II compound, wherein, described step (a) is carried out under alcoholdehydrogenase CmADH3 existent condition;
B () formula II compound and 2,2-dimethoxypropane react, form formula I; With
C described formula I as intermediate, is prepared statin compound by ().
5. a reaction mixture, is characterized in that, described mixture comprises:
(i) alcoholdehydrogenase CmADH3;
(ii) formula III compound and/or formula II compound; And
(iii) described cofactors.
6. reaction mixture as claimed in claim 5, it is characterized in that, described mixture also comprises the regeneration system rapidly of described cofactors.
7. a purposes for the engineering bacteria of alcoholdehydrogenase or expression recombinase, is characterized in that, the engineering bacteria of described alcoholdehydrogenase or expression recombinase is used for catalytic reduction formula III compound, thus production II compound.
8. polynucleotide, is characterized in that, described Nucleotide is selected from lower group:
The polynucleotide of (a) nucleotide sequence as shown in SEQ ID NO.:3;
Homology >=90% (preferably >=95%) of sequence shown in (b) nucleotide sequence and SEQ ID NO.:3, and there are the polynucleotide of formula III compound for catalysis reducing activity;
(c) polynucleotide sudden change 1-60 as shown in SEQ ID NO.:3 (preferably 1-30, more preferably 1-6) Nucleotide, and there are the polynucleotide of formula III compound for catalysis reducing activity;
D () is for the aminoacid sequence shown in SEQ ID NO.:1 of encoding.
9. an expression vector, is characterized in that, described carrier contains polynucleotide according to claim 8.
10. a host cell, is characterized in that, described host cell contains the polynucleotide according to claim 8 that expression vector according to claim 9 or its chromosomal integration have external source.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510209354.1A CN104830921B (en) | 2015-04-27 | 2015-04-27 | A kind of method that enzyme process prepares statin compound intermediate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510209354.1A CN104830921B (en) | 2015-04-27 | 2015-04-27 | A kind of method that enzyme process prepares statin compound intermediate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104830921A true CN104830921A (en) | 2015-08-12 |
| CN104830921B CN104830921B (en) | 2019-07-02 |
Family
ID=53809118
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510209354.1A Active CN104830921B (en) | 2015-04-27 | 2015-04-27 | A kind of method that enzyme process prepares statin compound intermediate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104830921B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636248A (en) * | 2016-12-21 | 2017-05-10 | 浙江海洋大学 | Method for preparing crizotinib intermediate by using carbonyl reductase |
| CN107779459A (en) * | 2016-08-31 | 2018-03-09 | 安琪酵母股份有限公司 | Glucose dehydrogenase DNA molecular, carrier and bacterial strain and application |
| CN109897872A (en) * | 2017-12-11 | 2019-06-18 | 湖州颐辉生物科技有限公司 | Enzyme process prepares (2S, 3S)-N- tertbutyloxycarbonyl -3- amino -1- chlorine-2-hydroxyl -4- phenyl butane |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1152054A1 (en) * | 1999-12-03 | 2001-11-07 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
| US20080220484A1 (en) * | 2005-09-19 | 2008-09-11 | Basf Aktiengesellschaft | Novel Dehydrogenases, the Derivatives thereof, and Method for the Production of Optically Active Alkanols |
| CN101528917A (en) * | 2006-10-02 | 2009-09-09 | 科德克希思公司 | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
| CN102676596A (en) * | 2011-03-16 | 2012-09-19 | 苏州国镝医药科技有限公司 | Bio-enzyme chiral synthesis Lipitor midbody ATS-7 |
| CN103014080A (en) * | 2012-12-19 | 2013-04-03 | 苏州汉酶生物技术有限公司 | Biological preparation method of 6-cyano-(3R, 5R)-dihydroxyhexanoate |
| CN103642765A (en) * | 2013-12-25 | 2014-03-19 | 南京工业大学 | Alcohol dehydrogenase mutant and application thereof |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
| CN104195191A (en) * | 2014-09-12 | 2014-12-10 | 尚科生物医药（上海）有限公司 | Method for preparing (3R,5R)-6-cyano-3,5-dihydroxy hexanoic acid tert-butyl ester by enzyme process |
| CN104388488A (en) * | 2014-10-28 | 2015-03-04 | 尚科生物医药（上海）有限公司 | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis |
-
2015
- 2015-04-27 CN CN201510209354.1A patent/CN104830921B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1152054A1 (en) * | 1999-12-03 | 2001-11-07 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
| US20080220484A1 (en) * | 2005-09-19 | 2008-09-11 | Basf Aktiengesellschaft | Novel Dehydrogenases, the Derivatives thereof, and Method for the Production of Optically Active Alkanols |
| CN101528917A (en) * | 2006-10-02 | 2009-09-09 | 科德克希思公司 | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
| CN102676596A (en) * | 2011-03-16 | 2012-09-19 | 苏州国镝医药科技有限公司 | Bio-enzyme chiral synthesis Lipitor midbody ATS-7 |
| CN103014080A (en) * | 2012-12-19 | 2013-04-03 | 苏州汉酶生物技术有限公司 | Biological preparation method of 6-cyano-(3R, 5R)-dihydroxyhexanoate |
| CN103642765A (en) * | 2013-12-25 | 2014-03-19 | 南京工业大学 | Alcohol dehydrogenase mutant and application thereof |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
| CN104195191A (en) * | 2014-09-12 | 2014-12-10 | 尚科生物医药（上海）有限公司 | Method for preparing (3R,5R)-6-cyano-3,5-dihydroxy hexanoic acid tert-butyl ester by enzyme process |
| CN104388488A (en) * | 2014-10-28 | 2015-03-04 | 尚科生物医药（上海）有限公司 | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis |
Non-Patent Citations (4)
| Title |
|---|
| MICHAEL MULLER: "Chemoenzymatic Synthesis of Building Blocks for Statin Side Chains", 《ANGEWANDTE CHEMIE》 * |
| 孙丰来: "化学酶法制备他汀药物关键手性中间体的研究", 《中国学位论文全文数据库》 * |
| 杨频 等: "《生物无机化学原理》", 30 April 2002 * |
| 谌容 等: "醇脱氢酶不对称还原制备手性醇的研究进展", 《化工进展》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779459A (en) * | 2016-08-31 | 2018-03-09 | 安琪酵母股份有限公司 | Glucose dehydrogenase DNA molecular, carrier and bacterial strain and application |
| CN107779459B (en) * | 2016-08-31 | 2021-06-08 | 安琪酵母股份有限公司 | Glucose dehydrogenase DNA molecule, vector, strain and application |
| CN106636248A (en) * | 2016-12-21 | 2017-05-10 | 浙江海洋大学 | Method for preparing crizotinib intermediate by using carbonyl reductase |
| CN109897872A (en) * | 2017-12-11 | 2019-06-18 | 湖州颐辉生物科技有限公司 | Enzyme process prepares (2S, 3S)-N- tertbutyloxycarbonyl -3- amino -1- chlorine-2-hydroxyl -4- phenyl butane |
| CN109897872B (en) * | 2017-12-11 | 2023-12-22 | 湖州颐盛生物科技有限公司 | Enzymatic preparation of (2S, 3S) -N-t-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104830921B (en) | 2019-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106929521B (en) | Aldehyde ketone reductase gene recombination co-expression vector, engineering bacterium and application thereof | |
| CN101613672A (en) | Recombinant escherichia coli for preparing -4-chloro-3-hydroxybutanoate through asymmetric transformation and construction method thereof | |
| CN109735553B (en) | Preparation method of anti-AIDS drug atazanavir intermediate | |
| Schweiger et al. | Characterization of two aldo–keto reductases from Gluconobacter oxydans 621H capable of regio-and stereoselective α-ketocarbonyl reduction | |
| CN106995808B (en) | A kind of recombination transaminase and its application | |
| CN105543186A (en) | Alcohol dehydrogenase LC3 and gene and application thereof | |
| CN104830921A (en) | Enzymatic method for preparing statins intermediates | |
| CN103555683A (en) | Synthesis method of saxagliptin chiral intermediate | |
| CN102952814A (en) | Recombined heat-resisting short-chain dehydrogenase gene, codase, carrier, engineering bacterium and application | |
| CN110387359A (en) | Carbonyl reduction enzyme mutant and its application | |
| CN104328148A (en) | Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method | |
| Chen et al. | Cloning, expression, and characterization of an anti-Prelog stereospecific carbonyl reductase from Gluconobacter oxydans DSM2343 | |
| CN101962661B (en) | Application of carbonyl reductase in production of -4-chloro-3-hydroxy ethyl butyrate | |
| CN104561052A (en) | Recombinant formate dehydrogenase as well as preparation method and application thereof | |
| CN104388488A (en) | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis | |
| CN104928224B (en) | A kind of ferulic acid production engineering bacterial strain, construction method and bioconversion method | |
| CN109679978B (en) | Recombinant co-expression system for preparing L-2-aminobutyric acid and application thereof | |
| CN111378694A (en) | Method for preparing dapoxetine intermediate by using carbonyl reductase | |
| Goldberg et al. | Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl]-8-azaspiro [4.5] decane-7, 9-dione: Cloning and expression of reductases | |
| CN109943577A (en) | A kind of bioconversion method of anti-AIDS drug atazanavir intermediate | |
| CN110004120A (en) | A kind of recombination aldehyde Ketoreductase mutant and application | |
| CN113151131B (en) | Self-induction culture medium for producing isoeugenol monooxygenase and application thereof | |
| CN111394396B (en) | Method for producing 1, 3-propylene glycol by using glycerol fermentation by microorganisms | |
| CN102154377B (en) | Use of oxidoreductase or recombinase thereof and recombinant oxidoreductase | |
| CN106047826B (en) | Aldehyde dehydrogenase, its recombinant expression transformants and the application in the synthesis of statin precursor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |