CN103911403A - Method for preparing chiral intermediate of atorvastatin - Google Patents
Method for preparing chiral intermediate of atorvastatin Download PDFInfo
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Abstract
The invention discloses a method for preparing 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate by using genetically engineered bacteria as a whole-cell biocatalyst. The method specifically comprises the following steps of designing and optimizing a tandem co-expression policy of a carbonyl reductase and a glucose dehydrogenase according to the expression characteristics of the carbonyl reductase and the glucose dehydrogenase, establishing a brand-new biological catalysis system in which cyclic regeneration of coenzymes is matched with the reduction of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate, thus realizing in-situ biological synthesis of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate, namely a chiral side chain synthesis precursor of atorvastatin. By optimizing expression conditions and reaction conditions and under the conditions that a cosolvent is 5% dimethyl sulfoxide, a reaction solution pH is 7.0, the temperature is 20 DEG C, and a ratio of glucose to a substrate is 1.2: 1, the concentration of the substrate for the in-situ biological synthesis of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate can be 35g/L, and meanwhile, the addition of an exogenous coenzyme is completely avoided, and therefore, the method has wide application prospect.
Description
Technical field
The invention belongs to chiral medicinal intermediate preparation field, relate to the method for a kind of biological catalysis unilateral system for chiral medicinal intermediate, be specifically related to a kind of take 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester as substrate, using the intestinal bacteria of coexpression carbonyl reductase and Hexose phosphate dehydrogenase as biological catalyst, realize the processing method of 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester of single optical purity.
Background technology
Statins (Statins) is found in the secondary metabolite of Mycophyta, and its molecular structure is made up of the hydrophobic parent nucleus of a rigidity and the side chain of (3R, 5S/R)-dihydroxy ester structure.Because this side-chain structure and hydroxymethyl glutaryl base have structural similarity, therefore statins is to the competitive violently property of 3-hydroxy-3-methylglutaryl coenzyme A reductase restraining effect.This restraining effect can be blocked hydroxyl first valeric acid pathways metabolism in cell, make the synthetic minimizing of cell inner cholesterol, thereby feedback irritation cell film surface (being mainly liver cell) low density lipoprotein receptor, make its quantity and active increasing, reduce serum cholesterol level thereby increase serum cholesterol clearance rate.Because the tune fat effect of Statins is strong, better tolerance, be used as clinically the choice drug of the hyperlipidaemias such as treatment atherosclerosis, coronary heart disease.And along with the going deep into of correlative study, the new purposes of statins is also developed, such as preventing osteoporosis and alzheimer's disease etc.According to the difference in source, statins can be divided into natural compounds (as lovastatin, Simvastatin, Pravastatin, mevastatin) and artificial-synthetic compound's (as fluvastatin, atorvastatin, Cerivastatin, rosuvastatin) two classes, and wherein synthetic class statins feature efficient with it, low side effect becomes the primary categories of statins.
Because statins chiral side chain exists diol structure between special chirality, make traditional chemical synthesis process exist chiral selectivity not high, severe reaction conditions, the defects such as reactions steps length consuming time are (referring to Tetrahedron.Lett.2002,43,2569-2571 and Tetrahedron.Lett.2002,43,2679-82).The energy consumption that has greatly increased whole reaction process techniques such as low-temp reaction (60 ℃), has reduced economy.
The biocatalysis technology that last century end starts development has the features such as very strong Substratspezifitaet, higher catalytic efficiency, gentle reaction conditions, easy reactions steps and environmental pollution be little, has been widely used in preparing Statins chiral side chain precursor.Existing multiple enzyme is applied to the biosynthesis process of his spit of fland chiral side chain, such as alcoholdehydrogenase, esterase, dehalogenase, carbonyl reductase and zymohexase etc., and substrate kind and the reaction process of every kind of enzyme use are all not quite similar.A kind of carbonyl reductase from Saccharomyces cerevisiae of existing report is (referring to Biosci.Biotechnol.Biochem.2004,68,2306-2312), the carbonyl that can reduce in alpha-carbonyl lipid substrate, this process has very high chiral selectivity and substrate specificity.For example having this enzyme of report can be 6-cyano group-(3R by 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester asymmetric reduction under the environment that has NADPH, 5R)-dihydroxyl hecanoic acid t-butyl ester, reaction ee value reaches 99% (referring to Biochim.Biophys.Acta.2007,1773,321-329), this reaction product can be used as the important as precursors of synthetic atorvastatin chiral side chain.But in actual suitability for industrialized production, this Catalytic processes all exists many obstacles, for example, owing to need to adding expensive nicotinamide class coenzyme NADP 11, the reasons such as the lower and crude enzyme liquid preparation technology of crude enzyme liquid catalytic efficiency is comparatively complicated, have caused the problem of a series of obstruction scale operation such as less economical, production efficiency is low.
Therefore set up a kind of biocatalysis technique of efficiently preparing Statins chiral side chain precursor, reach the requirement of high concentration of substrate, zero coenzyme input and concise production process, there is practical value and significance widely to improving economy and feasibility in suitability for industrialized production.
Summary of the invention
Goal of the invention of the present invention is by the sequencing of the series connection coexpression of preferred carbonyl reductase and Hexose phosphate dehydrogenase, set up a coenzyme cyclic regeneration and 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and reduce the brand-new biocatalysis system matching, provide one to prepare 6-cyano group-(3R, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester, on the basis guaranteeing without the interpolation of external source coenzyme, highly-solid selectively, high conversion and high yield, effectively improve concentration of substrate, simplify production technique and reduce production costs.
The technical scheme that to achieve the above object of the invention, the present invention has adopted is: a kind of method of preparing atorvastatin chiral intermediate, comprises the following steps:
(1) described fermentation thalline is the colibacillus engineering containing recombinant co-expression plasmid; Concentration of substrate is 1～55g/L, preferably 35g/L; The volume fraction of solubility promoter dimethyl sulfoxide (DMSO) is 0%-10%, preferably 5%, and the mol ratio of cosubstrate glucose and substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester is 20:1～0.5:1, preferably 1.2:1;
(2) step (1) gained reaction system completes in vibration or agitation condition, and reaction solution pH scope is 5.0～9.0, and preferably 7.0, temperature of reaction is 15～35 ℃, preferably 20 ℃.After having reacted rear ethyl acetate extraction, obtain 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester crude product.
In technique scheme, in step (1), described gene recombination E.Coli specifically refers to through gene recombination, the carbonyl reductase that coexpression comes from Saccharomyces cerevisiae and the one coming from two kinds of intestinal bacteria of Hexose phosphate dehydrogenase of Bacillus megaterium.
In technique scheme, according to reorganization scheme difference, described carbonyl reductase gene order can be SEQ ID NO:1 and SEQ ID NO:2.
In technique scheme, according to reorganization scheme difference, described glucose dehydrogenase gene sequence can be SEQ ID NO:3 and SEQ ID NO:4.
In technique scheme, in step (1), the preparation of described fermented liquid also comprises step (3): gene recombination E.Coli is inoculated in aseptic LB substratum, vibration condition under, with 37 ℃ be cultured to nutrient solution OD
600after=0.8 ± 0.1, adding final concentration is the inductor IPTG of 0.5mM, at 15～35 ℃, preferably 25 ℃, continues to cultivate 16h.
In further technical scheme, in the activity to fermented liquid is investigated, also comprise step (4): after step (3) finishes, obtain after wet thallus centrifugal fermented liquid, add potassium primary phosphate-dipotassium hydrogen phosphate damping fluid of appropriate pH=7.0, even thalline resuspended rear use high pressure cracker is carried out to fragmentation, after centrifugal the thalline after fragmentation, obtain crude enzyme liquid.Utilize SDS-PAGE to investigate the coexpression situation of two kinds of enzymes in crude enzyme liquid, and build 1mL reaction system simultaneously, and with coenzyme consumption or generation in ultraviolet spectrophotometry detection reaction system, investigate respectively the ratio vigor of two kinds of enzymes in crude enzyme liquid to determine preferred inducing temperature.
In technique scheme, in step (1), the compound method of the LB substratum that described fermented liquid uses is prior art.
In technique scheme, in step (1), described substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester structural formula is:
In technique scheme, in step (1), the order of addition of each reagent is any random order, without strict sequence requirement.
In technique scheme, in step (2), regulate the reagent of reaction system pH to be selected from formic acid, acetic acid, hydrochloric acid, ammoniacal liquor, aqueous sodium hydroxide solution or potassium hydroxide aqueous solution.
Further in technical scheme, described preparation 6-cyano group-(3R, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester also comprises: step (2) is mixed reaction solution after having reacted with isopyknic ethyl acetate, make protein denaturation and extraction product from aqueous phase system, the centrifugal metaprotein of removing, after desolventizing, obtain 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester oily matter.
Due to the utilization of technique scheme, the present invention compared with prior art has following characteristics:
1, because the present invention has used coexpression NADPH dependent form carbonyl reductase and NADP
+the gene recombined escherichia coli of dependent form Hexose phosphate dehydrogenase is as biological catalyst, and these two kinds of enzymes have formed internal cell coenzyme cyclic regeneration system.Utilize the endogenous coenzyme of intestinal bacteria to carry out efficient coenzyme circulation, in reaction, do not need ectogenic coenzyme to add completely, greatly reduced process costs.
2, by the tandem expression sequence of preferred carbonyl reductase and Hexose phosphate dehydrogenase, set up that carbonyl reductase catalyzes and synthesizes that 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and glucose-Hexose phosphate dehydrogenase NADPH regeneration system rapidly match biosynthesis system.
3, because reaction adopts escherichia coli fermented broth as catalyzer, do not need thalline to carry out separated and collected, more need to not prepare crude enzyme liquid by thalline, greatly simplified technique.Technique of the present invention can realize the whole process of fermentation-reaction in fermentor tank simultaneously, is convenient to very much industry's enlarging production, has higher economic worth.Adopt fermentor tank to carry out can further improving fermented liquid density after high density fermentation simultaneously, thereby further promote the maximum substrate carrying capacity of reaction.
4, the coenzyme reducing power donor that the present invention adopts is glucose, cheap, stable in properties, easy handling in reaction.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates that comprises expression plasmid in two kinds of gene recombination E.Coli described in embodiment mono-.
Fig. 2 implements the coenzyme circulating system coupling carbonyl reductase being mediated by Hexose phosphate dehydrogenase described in five examples to catalyze and synthesize 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester reaction process schematic diagram.
Embodiment
By reference to the accompanying drawings, list following embodiment for the present invention is described, but should not be considered as limitation of the present invention.
Carbonyl reductase gene order in p-gdh-cr1 plasmid is as shown in SEQ ID NO:1.
Carbonyl reductase gene order in p-cr1-gdh plasmid is as shown in SEQ ID NO:2.
Grape dehydrogenase gene sequence in p-cr1-gdh plasmid is as shown in SEQ ID NO:3.
Grape dehydrogenase gene sequence in p-gdh-cr1 plasmid is as shown in SEQ ID NO:4.
Embodiment mono-
The structure of gene recombination E.Coli
Complete synthesis from the carbonyl reductase gene cr1 of Saccharomyces cerevisiae with from the glucose dehydrogenase gene gdh of Bacillus megaterium, and insert respectively Restriction Enzyme inscribe site at gene order upstream and downstream: No. 1 multiple clone site (MCS1) of co-expression carrier pETDeut-1 is upper, and to insert the restriction enzyme site that foreign gene adopts be Nco I and BamH I site; The restriction enzyme site that the upper insertion of No. 2 multiple clone site (MCS2) foreign gene adopts is Nde I and Xho I.Cut-method of attachment of the enzyme that utilization those skilled in the art are afamiliar with, inserts carbonyl reductase gene and glucose dehydrogenase gene respectively respectively No. 1 and No. 2 multiple clone site (MCS) of prokaryotic organism co-expression carrier.After empirical tests and evaluation, according to the difference of gene insertion point, positive plasmid is divided into 2 kinds of schemes, scheme 1: carbonyl reductase gene is positioned at MCS1, and glucose dehydrogenase gene is positioned at MCS2, recombinant plasmid is p-cr1-gdh; Scheme 2: carbonyl reductase gene is positioned at MCS2, and glucose dehydrogenase gene is positioned at MCS1, recombinant plasmid is p-gdh-cr1.These two kinds of recombinant plasmids are proceeded to respectively in E.Coli BL21 expressive host, obtain two kinds of gene recombination E.Coli, be called pGC bacterium (carbonyl reductase gene is at MCS2) and pCG bacterium (carbonyl reductase gene is at MCS1) according to the difference of gene insertion sequence in expression vector.
Embodiment bis-
The preparation of gene recombination E.Coli fermented liquid
By two kinds of gene recombination E.Coli that obtain in embodiment mono-respectively in the LB of 50mL substratum (containing 100 μ g/mL penbritins), 37 ℃ of jolting incubated overnight.Bacterium liquid after 20mL incubated overnight is joined in the LB substratum (containing 100 μ g/mL penbritins) of 250mL, 37 ℃ of joltings are cultured to nutrient solution OD
600=0.8 ± 0.1 o'clock, adding final concentration was the IPTG of 0.5mM, continues jolting and cultivate after 16h at 15～35 ℃, obtained gene recombination E.Coli fermented liquid.
Embodiment tri-
The investigation of carbonyl reductase and Hexose phosphate dehydrogenase coexpression situation under differing temps
Thalline fermented liquid in embodiment bis-is collected to thalline through the centrifugal 15min of 5000*g, and wherein 15 ℃ obtain wet bacterium 8g/L, and 25 ℃ obtain wet bacterium 11g/L, and 20 ℃ obtain wet bacterium 13g/L.The resuspended thalline of potassium primary phosphate-dipotassium hydrogen phosphate damping fluid adding after microorganism collection, through the broken thalline of 35kpsi high pressure.To after the centrifugal 20min of thalline 15000*g after fragmentation, obtain crude enzyme liquid.Use 15% SDS-PAGE to detect crude enzyme liquid, equal normal 2 kinds of enzymes of coexpression in pGC and pCG, the wherein about 35.5kDa of carbonyl reductase CR1 monomer molecule amount, Hexose phosphate dehydrogenase GDH monomer molecule amount is about 28.3kDa.
Embodiment tetra-
The vigor matching of carbonyl reductase and Hexose phosphate dehydrogenase is preferred
The crude enzyme liquid obtaining in embodiment bis-is carried out to enzyme activity determination, wherein carbonyl reduction enzyme activity determination system is that pH is the crude enzyme liquid of the suitable dilution of 7.0 0.1M potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, 0.1 μ M NADPH, 5 μ M6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 10 μ L, and cumulative volume is 1mL.Hexose phosphate dehydrogenase vitality test system is that pH is 7.0 0.1M potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, 0.6 μ M NADP
+, 5 μ M glucose and 10 μ L the crude enzyme liquid of suitable dilution, cumulative volume is 1mL.Utilize ultraviolet spectrophotometer, under the constant temperature of 37 ℃ in assaying reaction system the absorbancy of 340nm wavelength change can reflect system in the increase and decrease of coenzyme, and then calculate the ratio vigor of certain enzyme in system.In the present invention, the enzyme activity of 1 unit is defined as enzyme per minute oxidation NADPH/ reduction NADP contained in every liter of fresh fermented liquid
+micromole's number.Table 1 is the ratio live data of two kinds of enzymes in two kinds of different gene recombination E.coli crude enzyme liquids.According to vigor data, containing in the recombination bacillus coli of pGC plasmid, the vigor matching of carbonyl reductase and Hexose phosphate dehydrogenase is higher.
Two kinds of specific activity of enzyme in crude enzyme liquid under the different inducing temperatures of table 1
| ? | 15 | 20 | 25 | 30 | 35 |
| In pGC, CR1 is than vigor (U/L) | 4.496 | 6.386 | 13.798 | 12.572 | 7.209 |
| In pCG, CR1 is than vigor (U/L) | 2.874 | 2.896 | 6.312 | 4.414 | 4.340 |
| In pGC, GDH is than vigor (U*10 2/L) | 1.010 | 1.949 | 8.392 | 12.051 | 9.550 |
| In pGC, GDH is than vigor (U*10 2/L) | 9.522 | 13.958 | 40.045 | 78.050 | 66.712 |
Embodiment five
The feasibility study of biosynthesizing 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester
Get any one gene recombination E.Coli fermented liquid 30mL obtaining in embodiment bis-, add the glucose of 1g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 20 times of substrate molar equivalents, jolting reaction 7h regulate reaction pH to 7.0 at 25 ℃.Reaction finishes rear sampling and adds isopyknic acetonitrile to make protein denaturation termination reaction, gets supernatant after centrifugal to obtain reaction solution extract after 0.22 μ M membrane filtration.
Respectively substrate standard substance, product fiducial mark product and reaction solution extract are detected, use reverse C18 post (5 μ m, 250 × 4.6mm, Thermo, USA) carry out high performance liquid phase (Shimadzu2010A HT, Japan) stratographic analysis, moving phase is: A is water (containing 0.25% acetic acid), and B is acetonitrile.Wash-out adopts 25% Mobile phase B, flow velocity 1mL/min, wash-out 15 minutes.Column temperature is 30 ℃, and detector is UV detector, and detection wavelength is 220nm.Appearance time is respectively: product 6.9min, substrate 10.1min.In HPLC-UV atlas analysis reaction solution extract, substrate approaches completely and transforms.
Embodiment six
The matching of the catalysis of carbonyl reductase and Hexose phosphate dehydrogenase is preferred
Get two kinds of each 30mL of gene recombination E.Coli fermented liquid that obtain in embodiment bis-, add the glucose of 5g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 20 times of substrate molar equivalents, jolting reaction 7h regulate reaction pH to 7.0 at 25 ℃.Separately get two kinds of each 30mL of gene recombination E.Coli fermented liquid that obtain in embodiment bis-, add the glucose of 50g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 20 times of substrate molar equivalents, jolting reaction 7h regulate reaction pH to 7.0 at 25 ℃.Reaction finishes sample respectively and add isopyknic acetonitrile to make protein denaturation termination reaction afterwards, gets supernatant after centrifugal to obtain reaction solution extract after 0.22 μ M membrane filtration.
Adopt the HPLC analytical procedure in embodiment five to investigate the substrate conversion efficiency in reaction system.HPLC-UV atlas analysis result shows: in the different types of gene recombination E.Coli fermented liquid of use, 5g/L concentration of substrate reaction system, substrate all approaches completely and transforms; In the reaction system of the high concentration of substrate of 50g/L, containing pGC plasmid and realize carbonyl reductase and the transformation efficiency of the recombination bacillus coli biological catalyst catalytic substrate of Hexose phosphate dehydrogenase coexpression is 72.4%, apparently higher than the recombination bacillus coli (10.5%) that contains pCG plasmid.Illustrate that carbonyl reductase gene has higher catalytic efficiency at pETDuet-1 carrier MCS2 while glucose dehydrogenase gene at the gene recombination E.Coli of MCS1, can realize carbonyl reductase and express the compatibility of mating of vigor and catalysis with Hexose phosphate dehydrogenase.In conjunction with the embodiments one with the result of embodiment bis-, will be at 25 ℃, through the pGC fermented liquid of 0.5mM IPTG induction 16h as preferred fermented liquid.
Embodiment seven
Under differential responses pH, the catalytic efficiency of gene recombination E.Coli is investigated
Get the preferred fermented liquid 30mL obtaining in embodiment six, add the glucose of 50g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 2 times of substrate molar equivalents, jolting reaction 7h regulate reaction pH to 5.0,6.0,7.0,8.0 or 9.0 at 25 ℃.Obtain reaction solution extract according to the method in embodiment five after completion of the reaction, go out through HPLC-UV detection computations that substrate conversion efficiency is as shown in table 2 is:
Substrate conversion efficiency under table 2 differential responses system pH
| Reaction system pH | 5.0 | 6.0 | 7.0 | 8.0 | 9.0 |
| Substrate conversion efficiency (%) | 2.31 | 52.69 | 73.05 | 49.75 | 47.03 |
Can find out that preferred reaction liquid pH is 7.0.
At embodiment eight differential responses temperature, the catalytic efficiency of gene recombination E.Coli is investigated
Get the preferred fermented liquid 30mL obtaining in embodiment six, add the glucose of 50g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 2 times of substrate molar equivalents, jolting reaction 7h adjusting reaction pH to 7.0 at 15 ℃, 20 ℃, 25 ℃, 30 ℃ or 35 ℃.Obtain reaction solution extract according to the method in embodiment five after completion of the reaction, go out through HPLC-UV detection computations that substrate conversion efficiency is as shown in table 3 is:
Substrate conversion efficiency at table 3 differential responses temperature
| Temperature of reaction (℃) | 15 | 20 | 25 | 30 | 35 |
| Substrate conversion efficiency (%) | 56.49 | 75.68 | 64.17 | 18.90 | 5.49 |
Can find out that 20 ℃ for preferable reaction temperature.
Under embodiment nine preferred reaction conditions, substrate carrying capacity is investigated
Get the preferred fermented liquid 30mL obtaining in embodiment six, add the glucose of 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L or 55g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 2 times of substrate molar equivalents, jolting reaction 7h adjusting reaction pH to 7.0 at 20 ℃.Obtain reaction solution extract according to the method in embodiment five after completion of the reaction, go out substrate conversion efficiency through HPLC-UV detection computations as shown in table 4.
Different concentration of substrate reaction system substrate conversion efficiencies under table 4 preferred reaction conditions
Can find out under preferred reaction conditions, the preferred substrate carrying capacity of reaction system is 35g/L.
Under embodiment ten preferred reaction conditions, cosubstrate add-on is investigated
Get the preferred fermented liquid 30mL obtaining in embodiment six, add the glucose of 35g/L substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 1.5mL dimethyl sulfoxide (DMSO), 20 times, 15 times, 10 times, 5 times, 2 times, 1.8 times, 1.6 times, 1.4 times, 1.2,1 times, 0.8 times, 0.5 times substrate molar equivalents, jolting reaction 7h adjusting reaction pH to 7.0 at 20 ℃.Obtain reaction solution extract according to the method in embodiment five after completion of the reaction, go out substrate conversion efficiency through HPLC-UV detection computations as shown in table 5.
Different cosubstrate consumption reaction system substrate conversion efficiencies under table 5 preferred reaction conditions
Can find out under preferred reaction conditions, the preferred cosubstrate add-on of reaction system is 1.2 times of substrate molar equivalents.
Embodiment 11
Get the preferred fermented liquid 500mL obtaining in embodiment six, add 17.519g substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 25mL dimethyl sulfoxide (DMSO), 14.188g glucose, jolting reaction 7h adjusting reaction pH to 7.0 at 20 ℃.Add after completion of the reaction 500mL ethyl acetate, mix the rear centrifugal 10min of 10000*g of vibration, draw organic phase, except desolventizing, obtain 14.440g crude product through rotary evaporation method, yield is 82.4%.Crude product HPLC-UV in embodiment five detects, and result display substrate transformation efficiency is 99.50%.
Claims (8)
1. a method of preparing 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester, is characterized in that, comprises the following steps:
(1) in Recombinant E. coli Fermentation Broth, add substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, solubility promoter dimethyl sulfoxide (DMSO), cosubstrate glucose, build biology in situ synthetic system;
Described fermentation thalline is the colibacillus engineering containing recombinant co-expression plasmid; Concentration of substrate is 1～55g/L, preferably 35g/L; The volume fraction of solubility promoter dimethyl sulfoxide (DMSO) is 0%-10%, preferably 5%, and the mol ratio of cosubstrate glucose and substrate 6-cyano group-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester is 20:1～0.5:1, preferably 1.2:1;
(2) step (1) gained reaction system completes in vibration or agitation condition, and reaction solution pH scope is 5.0～9.0, and preferably 7.0, temperature of reaction is 15～35 ℃, preferably 20 ℃.
2. after having reacted rear ethyl acetate extraction, obtain 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester sterling.
3. preparation 6-cyano group-(3R according to claim 1, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester, it is characterized in that, the recombinant plasmid described in step (1) is the recombinant co-expression carrier containing carbonyl reductase and glucose dehydrogenase gene.
4. can be SEQ ID NO:1 and SEQ ID NO:2 according to the different carbonyl reductase gene orders of reorganization scheme; Glucose dehydrogenase gene sequence can be SEQ ID NO:3 and SEQ ID NO:4.
5. recombinant plasmid according to claim 2, is characterized in that, described expression vector is prokaryotic organism co-expression carrier, contains more than 2 or 2 multiple clone site (MCS); Recombinant co-expression plasmid construction scheme is characterised in that, scheme 1: carbonyl reductase gene can be positioned at MCS1, and glucose dehydrogenase gene is positioned at MCS2, and recombinant plasmid is p-gdh-cr1; Scheme 2: carbonyl reductase gene is at expression vector MCS2, and glucose dehydrogenase gene inserts MCS1, and recombinant plasmid is p-cr1-gdh.
6. the colibacillus engineering containing recombinant co-expression plasmid according to claim 1, it is characterized in that, contained recombinant expression plasmid is preferably pGC, the endogenous coenzyme recycle system of carbonyl reduction enzyme catalysis 6-cyano group in reaction process-(5R)-hydroxyl-3-carbonyl hecanoic acid t-butyl ester reduction reaction and Hexose phosphate dehydrogenase mediation is mated completely, without utilizing exogenous coenzyme;
Preparation 6-cyano group-(3R claimed in claim 1, in the method for 5R)-dihydroxyl hecanoic acid t-butyl ester, the preparation method of the escherichia coli fermented broth described in step (1), it is characterized in that, containing the engineering bacteria of recombinant co-expression plasmid, in LB substratum, jolting is cultured to nutrient solution OD at 37 ℃
600after=0.8 ± 0.1, add the inductor IPTG of 0.5mM, at 15～35 ℃, continue to cultivate 16h.
7. preparation 6-cyano group-(3R according to claim 1, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester, it is characterized in that, described preparation 6-cyano group-(3R, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester also comprises: step (2) after having reacted extracts the isopyknic ethyl acetate of reaction solution, obtain 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester oily matter.
8. preparation 6-cyano group-(3R according to claim 1, the method of 5R)-dihydroxyl hecanoic acid t-butyl ester, it is characterized in that, in step (2), regulate the reagent of reaction system pH to be selected from formic acid, acetic acid, hydrochloric acid, ammoniacal liquor, aqueous sodium hydroxide solution or potassium hydroxide aqueous solution.
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| CN104357502A (en) * | 2014-10-16 | 2015-02-18 | 尚科生物医药（上海）有限公司 | Biological preparation method of (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoate |
| 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 |
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