CN103911406A - Method for synthesis of (S)-3-hydroxypyrrolidine and its derivatives by enzyme method reduction - Google Patents
Method for synthesis of (S)-3-hydroxypyrrolidine and its derivatives by enzyme method reduction Download PDFInfo
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Abstract
The invention provides a method for synthesis of (S)-3-hydroxypyrrolidine and its derivatives by enzyme method reduction. According to the method, a pH value of 6.0-7.5, recombinant carbonyl reductase and recombinant glucose dehydrogenase and coenzyme efficiently co-expressed in escherichia coli are used as catalysts, the high-purity reduced (S)-3-hydroxypyrrolidine and its high-purity derivatives are prepared at a high yield, reaction time is short and a preparation cost is low.
Description
Technical field
The present invention relates to the method for synthetic (the S)-3-hydroxyl pyrrolidine of a kind of enzyme process reduction and derivative thereof, belong to the synthesis technical field of medicine intermediate.
Background technology
(S)-3-hydroxyl pyrrolidine and derivative thereof are the key intermediates of producing multiple medicine and agricultural chemicals, if (S)-3-hydroxyl pyrrolidine is the crucial chiral raw material of synthetic darifenacin hydrobromide (Darifenacin hydrobromide), this is a kind of novel treatment urinary incontinence medicine; (S)-N-benzyl-3-hydroxyl pyrrolidine can be used for synthetic hydrochloric acid barnidipine (Barnidipine hydrochlorate), a kind for the treatment of hypertension drug of long-acting dihydropyridine calcium ion antagonist.
Conventionally in the time using (S)-3-hydroxyl pyrrolidine, can adopt the form of N-protected, as the N-protected such as Boc-, Cbz-form.The synthesis mode of known (S)-3-hydroxyl pyrrolidine and derivative thereof has multiple at present, roughly be divided into following approach: (1) sets out with natural chiral acid such as L MALIC ACID, L-Glu or L-Asp, carry out through condensation, reduction etc., often step complexity, all need polystep reaction, and the chiral raw material relating to and to go back original reagent expensive; (2) with chiral raw material (S)-4-amino-2-hydroxybutyric acid, (S)-4-chloro-3-hydroxyl fourth cyanogen and (S)-CHBE etc., carry out through approach such as reduction or cyclisation, the chiral raw material cost of these modes is high, be difficult to obtain, and synthesis step often needs multistep, complex process; (3) carry out selectivity fractionation with lipase, (S)-all can obtain with (R)-type product.But lipase splits the highest yield of theory only 50% that obtains (S)-3-hydroxyl pyrrolidine, and the separation of product and purification difficult, often need silica gel column chromatography etc., is difficult to industry and amplifies.Another large defect that enzyme splits is to be difficult to racemization starting raw material to obtain higher yield, and raw material racemization at present does not still have feasible short-cut method, thereby the fractionation cost of lipase is high.(4), with microorganism or enzymatic hydroxylation mode, directly the derivative of selectivity hydroxylation tetramethyleneimine and N-protected thereof be (S)-or (R)-3-hydroxyl pyrrolidine accordingly.At present, this approach only stays in theoretical investigation and laboratory stage, and concentration of substrate is less than 5g/L, there is no actual production meaning.(5) with the derived analogs of keto reductase (Ketoreductase) selective reduction 3-oxo-pyrrolidine, existing patent report, [JP06/141876 (1994); WO98/23768 (1998)], this patent is carried out with following approach:
Should can be directly used in synthetic Panipenan by (R)-reduzate, the powerful Broad spectrum antibiotics of a kind of New Type of Carbon penicillin alkene class.
At present existing multiple important chirality pharmaceutical intermediate compound can be synthetic with carbonyl acyl reductase, comprises multiple synthesis mode such as pure enzyme and microbe whole-cell or immobilized enzyme/cell etc.Conventionally in synthetic system, also need to add regenerating coenzyme enzyme, as Hexose phosphate dehydrogenase (Glucose dehydrogenase, GDH) and hydrogenlyase (Formate dehydrogenase, FDH) etc.For example, the asymmetric reduction of the chloro-methyl aceto acetate of 4-(as: Zhou, J.Am.Chem.Soc.1983105:5925-5926; Santaniello, J.Chem.Res. (S) 1982:132-133; U.S.Pat.No.5,891,685 etc.), its reduzate (S)-4-chloro-3-hydroxyl ethyl butyrate is one of key intermediate of statins; The asymmetric reduction (as: U.S.Pat.No.6,800,477) of methyl phenyl ketone and derivative thereof; The asymmetric reduction (WO2005/054491) of thienone.At medicine industry circle, still have multiple important prochiral ketones compound need to develop carbonyl acyl reductase technique.
Summary of the invention
The object of the invention is to solve above-mentioned technical problem, the method for synthetic (the S)-3-hydroxyl pyrrolidine of a kind of enzyme process reduction and derivative thereof is provided.
Object of the present invention is achieved through the following technical solutions:
The method of synthetic (the S)-3-hydroxyl pyrrolidine of enzyme process reduction and derivative thereof, described reaction process is as follows:
P is CO
2r, Si (R)
3or benzyl, wherein R is the C replacing
1-6alkyl, benzyl or fluorenyl; its substituting group is optionally replaced by Ar, Het or C1-6 alkyl; described reaction conditions is PH6.0-7.5; recombinate carbonyl acyl reductase and recombinant glucose dehydrogenase and coenzyme as catalyzer taking coexpression; described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell; the aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, and described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Preferably, described reaction conditions is pH6.5-7.0, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
Preferably, described genetic engineering bacterium is the intestinal bacteria with recombinant vectors pETDuet-1.
A method for fermentation culture genetic engineering bacterium as shown above, comprises the further fermentation that builds genetic engineering bacterium and genetic engineering bacterium, and the structure of described genetic engineering bacterium comprises the following steps:
To the synthetic restructuring carbonyl acyl reductase encoding gene of full gene and Hexose phosphate dehydrogenase encoding gene respectively through double digestion; It is cloned into respectively to the different Anti-TNF-αs site of expression vector pETDuet-1, recombinant plasmid order-checking is converted into respectively expression coli strain after confirming, builds corresponding recombinant bacterial strain again;
The further fermentation of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB substratum that contains penbritin, is cultured to OD
600=0.8 fresh medium, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 DEG C of 800rpm cultivate; Feed supplement after cultivation 2hr, regulates pH7.0 ± 0.1 by strong aqua/hydrochloric acid, as the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 DEG C, adding final concentration is 1mmol/L IPTG, continues to control each culture condition induction 14hr, last centrifugal results thalline.
Beneficial effect of the present invention is mainly reflected in: adopt restructuring carbonyl acyl reductase, Hexose phosphate dehydrogenase to be applied to background and to reduce, productive rate is high, and optical purity of products is high, and the reaction times is short, and preparation cost is low.
Embodiment
The present invention has disclosed a kind of with carbonyl acyl reductase and synthetic (the S)-3-hydroxyl pyrrolidine of auxiliary enzyme one-step reduction and derivative thereof, and the following reaction formula of its preparation process carries out:
In above formula, P is CO
2r, Si (R)
3or benzyl, wherein R is the C replacing
1-6alkyl, benzyl or fluorenyl, its substituting group is optionally by Ar, Het or C
1-6alkyl replaces.
Described preparation method is as follows: the Compound I of a mole is dissolved in the buffered soln of 500ml~2000ml, in above-mentioned solution, adding weight is 0.1~20% gene recombination carbonyl acyl reductase, gene recombination Hexose phosphate dehydrogenase and coenzyme of Compound I, holder ties up to 15~45 DEG C, preferentially at 25~40 DEG C; PH6.0~7.5, preferably pH6.5~7.0, stir 16-72 hour, and stopped reaction, with the organic solvent extraction of 1000ml left and right 3 times, merges organic phase, desiccant dryness, organic solvent is removed in underpressure distillation, obtains target compound II.Conventionally Compound I I chiral purity is greater than 98%, can be used for the preparation of medicine.
In described buffered soln, can add the organic solvent of 500ml-1000ml, described organic solvent is selected from methyl alcohol, ethanol, propyl alcohol, butanols, the trimethyl carbinol, propyl alcohol, tetrahydrofuran (THF), methyl tert-butyl ether, ethyl acetate, butylacetate, butylacetate and toluene.
Described buffered soln is inorganic sulfuric acid and inorganic phosphate or trolamine hydrochloric acid buffering salt.
Described mineral alkali is selected from sodium hydroxide, potassium hydroxide, ammoniacal liquor, sodium carbonate, salt of wormwood, sodium bicarbonate, saleratus.
P in described Compound I is selected from as tertbutyloxycarbonyl, carbobenzoxy-(Cbz) and fluorenylmethoxycarbonyl, is preferably tertbutyloxycarbonyl.
Restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase are at the efficient coexpression of intestinal bacteria, and it can be liquor, lyophilized powder, can be also immobilized enzyme or cell.
Described carbonyl acyl reductase is external evolution, utilizes the enzyme of purifying or the direct catalysis of colibacillus engineering for its expression.It utilizes a kind of variant of candida magnoliae Candida Mangoliae carbonyl acyl reductase, has compared 34 amino acid difference with wild-type.
The sequence optimisation of carbonyl acyl reductase is carried out round increasing activity, thermostability and organic solvent stability on wild-type basis.Main employing taking structure as basic semi-directional evolved and high flux screening.The sequence of final gained has been compared 34 amino acid whose differences with wild-type.Great majority sudden change concentrates on enzyme surface and contacts site with subunit.Gene order is according to the codon amendment of intestinal bacteria preference, and elimination may affect the secondary structure of expression.Carbonyl acyl reductase after optimization is high efficient expression in intestinal bacteria (E.coli), and enzymic activity is the more than 300 times of wild-type, and stability also significantly increases.
Described Hexose phosphate dehydrogenase is external evolution, utilizes a kind of bacterium Burkholderia sp. glucose dehydrogenase modification efficiently to reduce NADP
+coenzyme, has compared 3 amino acid difference with wild-type.Its gene order is revised according to the codon of intestinal bacteria preference, and eliminates the secondary structure that may affect expression, and this sequence is at the high efficient expression of E.coli.
Carbonyl acyl reductase high reactivity variant after optimization and Hexose phosphate dehydrogenase after E.coli coexpression, through thick purifying, reduction that can efficient catalytic 3-oxo-tetramethyleneimine.
The aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, and described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Below describe its expression and determination of activity in intestinal bacteria in detail with restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase respectively.
Embodiment mono-: expression and the determination of activity of carbonyl acyl reductase in E.coli
The synthetic restructuring carbonyl acyl reductase encoding gene of full gene, after Nco I and Hind III double digestion, is cloned into expression vector pETDuet-1(producer: Novagen
production code member: 71146-3) multiple clone site 1, recombinant plasmid through order-checking confirm after, be converted in expression strain E.coli BL21 (DE3) the recombinant bacterial strain called after pETDuet-KRED-BL21 (DE3) of structure.On penbritin flat board, select single bacterium colony, access contains in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=0.7, cool the temperature to 25 degree, adding final concentration is the IPTG induction of 1mmol/L spend the night (16h).Centrifugal results thalline ,-20 DEG C frozen.SDS-PAGE detects and shows, the about 35.2KDa of this carbonyl acyl reductase, and target protein expression amount can be to 65% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM sodium-chlor, pH7.0) resuspended to 10g/L, with the ultrasonic 10min(800W of cell Ultrasonic Cell Disruptor ice-water bath, work 1sec stops 3sec), centrifugal (12,000rpm, 4 DEG C, 10min), cellular lysate liquid supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM sodium phosphate buffer (pH7.0), 5mM N-Boc-3-oxo-pyrrolidine, 1mM NADPH(or NADH), measure the decline of light absorption value in 340nm place for 30 DEG C.Enzyme work is defined as per minute and is oxidized 1 micromole NADPH(or NADH) needed enzyme amount is a carbonyl acyl reductase IU of Mei Huo unit.Protein content adopts Bradford method to measure.Result shows that this carbonyl acyl reductase enzyme work is about 13.8IU/mg.
Embodiment bis-: expression and the determination of activity of Hexose phosphate dehydrogenase in E.coli
The synthetic Hexose phosphate dehydrogenase encoding gene of full gene is after Nde I and Xho I double digestion, be cloned into the multiple clone site 2 of expression vector pETDuet-1, recombinant plasmid is after order-checking is confirmed, be converted in E.coli BL21 (DE3) the recombinant bacterial strain called after pETDuet-GDH-BL21 (DE3) of structure.On penbritin flat board, select single bacterium colony, access is containing in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=0.7, cool the temperature to 25 degree, adding final concentration is that the IPTG induction of 1mmol/L is spent the night.Centrifugal results thalline ,-20 DEG C frozen.SDS-PAGE detects and shows, the about 27.8KDa of this Hexose phosphate dehydrogenase, and target protein expression amount can be to 60% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM sodium-chlor, pH7.0) resuspended to 10g/L, with the ultrasonic 10min(800W of cell Ultrasonic Cell Disruptor ice-water bath, work 1sec stops 3sec), centrifugal (12,000rpm, 4 DEG C, 10min), cellular lysate liquid supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM sodium phosphate buffer (pH7.0), 250mM glucose, 1mM NADP
+(or NAD
+), measure the increase of light absorption value in 340nm place for 30 DEG C.Enzyme work is defined as per minute reduction and generates 1 micromole NADPH(or NADH) needed enzyme amount is an IU of Hexose phosphate dehydrogenase Mei Huo unit.Protein content adopts Bradford method to measure.Result shows that this Hexose phosphate dehydrogenase enzyme work is about 30IU/mg.
Embodiment tri-: carbonyl acyl reductase and the Hexose phosphate dehydrogenase coexpression in E.coli
By Hexose phosphate dehydrogenase encoding gene after Nde I and Xho I double digestion, be cloned into the multiple clone site 2 of the recombinant plasmid pETDuet1-(MCS1) described in embodiment mono-, gene is after order-checking is confirmed, be converted in E.coli BL21 (DE3) the recombinant bacterial strain called after pETDuet-KRED-GDH-BL21 (DE3) of structure.On penbritin flat board, select single bacterium colony, access is containing in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=0.7, cool the temperature to 25 degree, add 1mmol/L IPTG induction to spend the night.Centrifugal results thalline ,-20 DEG C frozen.SDS-PAGE detects and shows, the expression amount of carbonyl acyl reductase and Hexose phosphate dehydrogenase is suitable, and total amount can be to 70% of bacterial protein.
Embodiment tetra-: the fermentation of restructuring carbonyl acyl reductase and crude enzyme liquid preparation
In 100L fermentor tank, add following material: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium-chlor, pH nature.121 DEG C of sterilizing 20min.While being cooled to 37 DEG C, access 1L is cultured to OD with LB substratum (containing penbritin)
600=0.8 fresh pETDuet-KRED-BL21 (DE3) nutrient solution, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 DEG C of 800rpm cultivate.Feed supplement after cultivation 2hr, supplemented medium is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L yeast powder, strong aqua/hydrochloric acid regulates pH7.0 ± 0.1.As the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 DEG C, adding final concentration is 1mmol/L IPTG, controls each culture condition induction 14hr.Induction finishes the centrifugal results thalline of rear tubular-bowl centrifuge maximum speed of revolution, weight in wet base 3.34Kg, and 4 DEG C are temporary for subsequent use.
By above-mentioned 3.34Kg weight in wet base pETDuet-KRED-BL21 (DE3) by 1:5(v/v) be resuspended in 100mM sodium phosphate (+150mM sodium-chlor, pH7.0) damping fluid under 4 DEG C of low-temperature protections high-pressure homogeneous 2 time: each one time of 800bar+600bar.In above-mentioned cracking, add polymine to final concentration 0.5%(w/v), 4 DEG C are stirred 30min, whizzer 10, the centrifugal 20min of 000rpm, retains supernatant liquor and is restructuring carbonyl acyl reductase crude enzyme liquid, and 4 DEG C keep in Dark Place.The mensuration that carbonyl reductase enzyme is lived press method described in embodiment mono-and is measured, and is 60.5IU/mL, and protein concentration is measured with Bradford method, slightly zymoprotein concentration 24.5mg/mL.
Embodiment five: the fermentation of recombinant glucose dehydrogenase and crude enzyme liquid preparation.
In 100L fermentor tank, add following material: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium-chlor, pH nature.121 DEG C of sterilizing 20min.While being cooled to 37 DEG C, access 1L is cultured to OD with LB substratum (containing penbritin)
600=0.8 fresh pETDuet-GDH-BL21 (DE3) nutrient solution, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 DEG C of 800rpm cultivate.Feed supplement after cultivation 2hr, supplemented medium is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L yeast powder, strong aqua/hydrochloric acid regulates pH7.0 ± 0.1.As the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 DEG C, adding final concentration is 1mmol/L IPTG, controls each culture condition induction 14hr.Induction finishes the centrifugal results thalline of rear tubular-bowl centrifuge maximum speed of revolution, weight in wet base 3.52Kg, and 4 DEG C are temporary for subsequent use.
By above-mentioned 3.52Kg weight in wet base pETDuet-KRED-BL21 (DE3) by 1:5(v/v) be resuspended in 100mM sodium phosphate (+150mM sodium-chlor, pH7.0) damping fluid under 4 DEG C of low-temperature protections high-pressure homogeneous 2 time: each one time of 800bar+600bar.In above-mentioned cracking, add polymine to final concentration 0.5%(w/v), 4 DEG C are stirred 30min, whizzer 10, the centrifugal 20min of 000rpm, retains supernatant liquor and is recombinant glucose dehydrogenase crude enzyme liquid, and 4 DEG C keep in Dark Place.The mensuration that Hexose phosphate dehydrogenase enzyme is lived press method described in embodiment bis-and is measured, and is 720IU/mL, and protein concentration is measured with Bradford method, slightly zymoprotein concentration 31.2mg/mL.
Embodiment six: the chiral analysis method of N-Boc-3-hydroxyl pyrrolidine.
Ee(chirality HPLC): Chiralpak IC150mm × 4.6mm chiral chromatographic column; Moving phase: normal hexane (90%)/IPA(10%); Flow velocity: 0.6mL/min; Wavelength: 210nm; Retention time: N-Boc-(S)-3-hydroxyl pyrrolidine 15.78min, N-Boc-(R)-3-hydroxyl pyrrolidine 18.66min.
The enzymatic conversion method of embodiment seven: N-Boc-(S)-3-hydroxyl pyrrolidine is synthetic.
The synthetic reaction formula of pressing of N-Boc-(S)-3-hydroxyl pyrrolidine carries out:
In a 250mL three-necked bottle, add successively 100mL, 0.2mol/LNaH
2pO
4na
2hPO
4(pH7.0) (15 g) and 50mL butylacetate, and magnetic agitation 10min makes to mix, then adds carbonyl acyl reductase (10mL), Hexose phosphate dehydrogenase (5mL) and coenzyme (NADP for buffered soln, chemical compounds I (10g), glucose
+, 0.01g), under 30 degrees Celsius, stir 16 hours, control pH6.5~7.0, high-performance liquid chromatogram determination shows that reaction finishes.Filtration adds 100mL ethyl acetate after dezymotizing, re-extract three times, after organic phase is dry, be spin-dried for, obtain 9.4 grams of compound iis (N-Boc-(S)-3-hydroxyl pyrrolidine), pressing embodiment five methods detects, its optical purity ee value >99.5%, molar yield 93.0%.
Embodiment seven: utilize the E.coli cell transformation of carbonyl acyl reductase and Hexose phosphate dehydrogenase coexpression to produce N-Boc-(S)-3-hydroxyl pyrrolidine.
In a 250mL three-necked bottle, add successively 100mL, 0.2mol/LNaH
2pO
4na
2hPO
4(pH7.0) buffered soln, chemical compounds I (10g), glucose (15g) and 50mL butylacetate; magnetic agitation 10min makes to mix; add again 0.8g to utilize to express described in embodiment tri-the carbonyl acyl reductase that obtains and the E.coli cell (pETDuet-KRED-GDH-BL21 (DE3)) of Hexose phosphate dehydrogenase coexpression, and coenzyme (NADP
+, 0.01g), stir 16 hours under 30 degrees Celsius, control pH6.5~7.0, high-performance liquid chromatogram determination shows that reaction finishes.Filtration dezymotize with cell debris after, add 100mL ethyl acetate, re-extract three times, after organic phase is dry, be spin-dried for, obtain 8.9 grams of compound iis (N-Boc-(S)-3-hydroxyl pyrrolidine), optical purity ee value >99.5%, molar yield 88.0%.
The present invention still has multiple concrete embodiment, and all employings are equal to replacement or equivalent transformation and all technical schemes of forming, within all dropping on the scope of protection of present invention.
Claims (4)
1. the method for synthetic (the S)-3-hydroxyl pyrrolidine of enzyme process reduction and derivative thereof, described reaction process is as follows:
It is characterized in that: P is CO
2r, Si (R)
3or benzyl, wherein R is the C replacing
1-6alkyl, benzyl or fluorenyl, its substituting group is optionally by Ar, Het or C
1-6alkyl replaces; described reaction conditions is pH6.0-7.5; recombinate carbonyl acyl reductase and recombinant glucose dehydrogenase and coenzyme as catalyzer taking coexpression; described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell; the aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, and described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
2. the method for synthetic (the S)-3-hydroxyl pyrrolidine of enzyme process reduction according to claim 1 and derivative thereof; it is characterized in that: described reaction conditions is pH6.5-7.0, described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
3. the method for synthetic (the S)-3-hydroxyl pyrrolidine of enzyme process reduction according to claim 1 and derivative thereof, is characterized in that: described genetic engineering bacterium is the intestinal bacteria with recombinant vectors pETDuet-1.
4. a method for the genetic engineering bacterium of fermentation culture as shown in claim 3, is characterized in that: comprise the further fermentation that builds genetic engineering bacterium and genetic engineering bacterium, the structure of described genetic engineering bacterium comprises the following steps:
To the synthetic restructuring carbonyl acyl reductase encoding gene of full gene and Hexose phosphate dehydrogenase encoding gene respectively through double digestion; It is cloned into respectively to the different Anti-TNF-αs site of expression vector pETDuet-1, recombinant plasmid order-checking is converted into respectively expression coli strain after confirming, builds corresponding recombinant bacterial strain again;
The further fermentation of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB substratum that contains penbritin, is cultured to OD
600=0.8 fresh medium, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 DEG C of 800rpm cultivate; Feed supplement after cultivation 2hr, regulates pH7.0 ± 0.1 by strong aqua/hydrochloric acid, as the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 DEG C, adding final concentration is 1mmol/L IPTG, continues to control each culture condition induction 14hr, last centrifugal results thalline.
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CN1238808A (en) * | 1996-11-26 | 1999-12-15 | 钟渊化学工业株式会社 | Process for preparation of optically active N-benzyl-3-pyrrolidinol |
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