CN103911406B - Enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof - Google Patents
Enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof Download PDFInfo
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Abstract
The invention provides a kind of enzyme process reduction synthesis(S)Reaction condition described in the method for 3 hydroxyl pyrrolidines and derivant thereof is pH6.0 7.5, with the restructuring carbonyl acyl reductase of efficient coexpression in escherichia coli and recombinant glucose dehydrogenase and coenzyme as catalyst, and the preparation reduction of high yield, high-purity ground(S)3 hydroxyl pyrrolidines and derivant thereof, its response time is short, and preparation cost is low.
Description
Technical field
The present invention relates to a kind of enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof, belong in medicine
The synthesis technical field of mesosome.
Background technology
(S)-3-hydroxyl pyrrolidine and derivant thereof are to produce multiple medicine and the key intermediate of pesticide, such as (S)-3-hydroxyl
Base pyrrolidine is the crucial chiral raw material of synthesis darifenacin hydrobromide (Darifenacin hydrobromide), and 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), the treatment hypertension drug of a kind of long-acting dihydropyridine calcium ion antagonist.
Generally when using (S)-3-hydroxyl pyrrolidine, the form of N-protected can be used, such as N-protected shapes such as Boc-, Cbz-
Formula.The most known (S)-3-hydroxyl pyrrolidine and the synthesis mode of derivant thereof have multiple, are roughly divided into following way
Footpath: (1) sets out with the natural chiral acid such as L MALIC ACID, L-Glu or L-Asp, is carried out through condensation, reduction etc., and often step is complicated,
It is both needed to multistep reaction, and the chiral raw material that relates to and to go back original reagent expensive；(2) with chiral raw material (S)-4-amino-2-hydroxyl
Base butanoic acid, (S)-4-chloro-3-hydroxyl fourth cyanogen and (S)-4-chloro-3-hydroxyl ethyl n-butyrate. etc., carried out through approach such as reduction or cyclisation,
The chiral raw material cost of these modes is high, it is difficult to obtain, and synthesis step generally requires multistep, complex process；(3) with lipase
Carrying out selectivity fractionation, (S)-and (R)-type product all can obtain.But lipase splits the reason obtaining (S)-3-hydroxyl pyrrolidine
Opinion the highest yield only 50%, the separation of product and purification difficult, generally require silica gel column chromatography etc., it is difficult to industry is amplified.Enzyme is torn open
Another the big defect divided is to be difficult to racemization initiation material to obtain higher yield, and current raw material racemization is still without feasible
Short-cut method, thus the fractionation cost of lipase remains high.(4) with microorganism or enzymatic hydroxylation mode, directly selecting property
The derivant of hydroxylating pyrrolidine and N-protected thereof is corresponding (S)-or (R)-3-hydroxyl pyrrolidine.At present, this approach only stops
Staying in theoretical research and laboratory stage, concentration of substrate is less than 5g/L, does not has actual production meaning.(5) with keto reductase
(Ketoreductase) derived analogs of 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 for synthesizing Panipenan by (R)-reduzate, a kind of New Type of Carbon penicillin alkenes strength wide spectrum resists
Raw element.
The most existing multiple important chirality pharmaceutical intermediate compound can synthesize with carbonyl acyl reductase, complete including pure enzyme and microorganism
The multiple synthesis modes such as cell or immobilized enzyme/cell.Generally also need to add regenerating coenzyme enzyme, such as glucose in synthetic system
Dehydrogenase (Glucose dehydrogenase, GDH) and hydrogenlyase (Formate dehydrogenase, FDH) etc..Example
As, the asymmetric reduction of the chloro-ethyl acetoacetate of 4-is (such 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 n-butyrate. is one of key intermediate of statins；1-Phenylethanone. and the asymmetric reduction of derivant thereof
(such as: U.S.Pat.No.6,800,477)；The asymmetric reduction (WO2005/054491) of thienone.At medical industry circle, still have
Multiple important prochiral ketones compound needs to develop carbonyl acyl reductase technique.
Summary of the invention
It is an object of the invention to solve above-mentioned technical problem, it is provided that a kind of enzyme process reduction synthesis (S)-3-hydroxypyrrole
Alkane and the method for derivant thereof.
The purpose of the present invention is achieved through the following technical solutions:
Enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof, described course of reaction is as follows:
P is CO2R、Si(R)3Or benzyl, wherein R is substituted C1-6Alkyl, benzyl or fluorenyl, its substituent group optionally by Ar,
Het or C1-6Alkyl replaces, and described reaction condition is PH6.0-7.5, takes off with coexpression restructuring carbonyl acyl reductase and restructuring glucose
Hydrogen enzyme and coenzyme are catalyst, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyst are liquid solution, lyophilizing
Powder, immobilized enzyme or immobilized cell, the aminoacid sequence such as sequence table SEQ .ID NO:1 institute of described restructuring carbonyl acyl reductase
Showing, described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Preferably, described reaction condition is pH6.5-7.0, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase exist
Efficient coexpression in genetic engineering bacterium.
Preferably, described genetic engineering bacterium is the escherichia coli with recombinant vector pETDuet-1.
The method of a kind of fermentation culture genetic engineering bacterium as shown above, including building genetic engineering bacterium and genetic engineering
The further fermentation of bacterium, the structure of described genetic engineering bacterium comprises the following steps:
Restructuring carbonyl acyl reductase encoding gene and the glucose dehydrogenase encoding gene double enzymes of warp respectively to full genome synthesis
Cut；It is cloned into respectively the different Anti-TNF-α sites of expression vector pETDuet-1 again, after recombiant plasmid order-checking confirms, point
Do not convert to expressing coli strain, build corresponding recombinant bacterial strain；
The fermentation further of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB culture medium containing ampicillin, cultivates to OD600=
The fresh medium of 0.8, adds the ampicillin solution of filtration sterilization to final concentration of 0.1mg/mL, 37 DEG C of 800rpm trainings
Support；Cultivate feed supplement after 2hr, by strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1, as the OD of culture fluid600When reaching 25, by tank temperature
It is down to 25 DEG C, adds final concentration of 1mmol/L IPTG, continue to control each condition of culture induction 14hr, last harvested by centrifugation bacterium
Body.
The beneficial effects are mainly as follows: use restructuring carbonyl acyl reductase, glucose dehydrogenase to be applied to background
Thing reduces, and productivity is high, and optical purity of products is high, and the response time is short, and preparation cost is low.
Detailed description of the invention
Present invention is disclosed a kind of with carbonyl acyl reductase and auxiliary enzyme one-step reduction synthesis (S)-3-hydroxyl pyrrolidine thereof and
Derivant, the following reaction equation of its preparation process is carried out:
In above formula, P is CO2R、Si(R)3Or benzyl, wherein R is substituted C1-6Alkyl, benzyl or fluorenyl, its substituent group is appointed
Choosing is by Ar, Het or C1-6Alkyl replaces.
Described preparation method is as follows: be dissolved in the buffer solution of 500ml～2000ml by the compound I of a mole, upper
State and solution adds 0.1～the gene recombinaton carbonyl acyl reductase of 20%, the gene recombinaton glucose dehydrogenase that weight is compound I
And coenzyme, holding system is at 15～45 DEG C, preferentially at 25～40 DEG C；PH6.0～7.5, preferably pH6.5～7.0, stir 16-72
Hour, stopped reaction, extract 3 times with the organic solvent of about 1000ml, merge organic facies, desiccant dryness, decompression distillation removes
Remove organic solvent, obtain target compound II.Generally compound II chiral purity is more than 98%, can be used for the preparation of medicine.
Can add the organic solvent of 500ml-1000ml in described buffer solution, described organic solvent is selected from methanol, second
Alcohol, propanol, butanol, the tert-butyl alcohol, propanol, oxolane, methyl tert-butyl ether, ethyl acetate, butyl acetate, butyl acetate and first
Benzene.
Described buffer solution is inorganic sulphuric acid and inorganic phosphate or triethanolamine hydrochloride buffer salt.
Described inorganic base is selected from sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, potassium carbonate, sodium bicarbonate, bicarbonate
Potassium.
The described P in compound I is selected from as tertbutyloxycarbonyl, benzyloxycarbonyl group and fluorenylmethoxycarbonyl, preferably tertiary butyloxycarbonyl
Base.
Restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase are at the efficient coexpression of escherichia coli, and it can be that liquid is molten
Liquid, lyophilized powder, it is also possible to be immobilized enzyme or cell.
Described carbonyl acyl reductase is Evolution in vitro, utilizes the enzyme of purification or straight for its colibacillus engineering expressed
Connect catalysis.It utilizes the variant of a kind of candida magnoliae Candida Mangoliae carbonyl acyl reductase, has compared with wild type
34 aminoacid difference.
The sequence optimisation of carbonyl acyl reductase, round increasing activity, heat stability and organic solvent on the basis of wild type
Stability is carried out.The semi-directional based on structure is mainly used to evolve and high flux screening.The sequence of final gained and wild
34 amino acid whose differences compared by type.Great majority sudden change concentrates on enzyme surface and subunit thereof site.Gene order is according to greatly
The codon amendment of enterobacteria preference, and eliminate the secondary structure that may affect expression.Carbonyl acyl reductase after optimization is at large intestine
High efficient expression in bacillus (E.coli), enzymatic activity is more than 300 times of wild type, and stability also dramatically increases.
Described glucose dehydrogenase is Evolution in vitro, utilizes a kind of antibacterial Burkholderia sp. glucose dehydrogenase
Variant efficiently reduces NADP+Coenzyme, has compared 3 aminoacid difference with wild type.Its gene order is according to escherichia coli preference
Codon amendment, and eliminate may impact express secondary structure, this sequence is at E.coli height efficient expression.
Carbonyl acyl reductase high activity variant after optimization and glucose dehydrogenase are after E.coli coexpression, through the purest
Change, can the reduction of efficient catalytic 3-oxo-pyrroli.
The aminoacid sequence of described restructuring carbonyl acyl reductase as shown in sequence table SEQ .ID NO:1, described restructuring glucose
Dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Its table in escherichia coli is described in detail individually below with restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase
Reach and determination of activity.
Embodiment one: the expression in E.coli of the carbonyl acyl reductase and determination of activity
The restructuring carbonyl acyl reductase encoding gene of full genome synthesis, after Nco I and Hind III double digestion, is cloned into table
Reach carrier pETDuet-1 (producer:Production code member: 71146-3) multiple clone site 1, recombiant plasmid through survey
After sequence confirms, convert to expression strain E.coli BL21 (DE3), the named pETDuet-KRED-of recombinant bacterial strain of structure
BL21(DE3).Picking individual colonies on ampicillin plate, accesses in the LB culture medium containing corresponding antibiotic, and 37 degree are filled
Divide and cultivate, to OD600=0.6,3% ratio is inoculated into the LB culture medium containing ampicillin.At bacterial growth to OD600=0.7,
Cool the temperature to 25 degree, add the IPTG overnight induction (16h) of final concentration of 1mmol/L.Harvested by centrifugation thalline ,-20 DEG C frozen.
SDS-PAGE detection shows, this carbonyl acyl reductase about 35.2KDa, target protein expression can be to the 65% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, resuspended extremely with 100mM sodium phosphate buffer (+150mM sodium chloride, pH7.0)
10g/L, with the cell ultrasonic 10min of Ultrasonic Cell Disruptor ice-water bath (800W, work 1sec stop 3sec), is centrifuged (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), 30 DEG C measure under light absorption value at 340nm
Fall.Enzyme is lived, and to be that carbonyl acyl reductase enzyme is lived single for the enzyme amount required for being defined as oxidation 1 micromole NADPH (or NADH) per minute
Position IU.Protein content uses Bradford method to be measured.Result shows that this carbonyl acyl reductase enzyme is lived and is about 13.8IU/mg.
Embodiment two: glucose dehydrogenase expression in E.coli and determination of activity
The glucose dehydrogenase encoding gene of full genome synthesis, after Nde I and Xho I double digestion, is cloned into expression vector
The multiple clone site 2 of pETDuet-1, recombiant plasmid, after order-checking confirms, converts to E.coli BL21 (DE3), the weight of structure
Group Strain Designation is pETDuet-GDH-BL21 (DE3).Picking individual colonies on ampicillin plate, accesses containing corresponding antibiosis
In the LB culture medium of element, fully cultivate, to OD for 37 degree600=0.6,3% ratio is inoculated into the LB culture medium containing ampicillin.
At bacterial growth to OD600=0.7, cool the temperature to 25 degree, add the IPTG overnight induction of final concentration of 1mmol/L.Centrifugal receipts
Obtaining thalline ,-20 DEG C frozen.SDS-PAGE detection shows, this glucose dehydrogenase about 27.8KDa, target protein expression can be extremely
The 60% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, resuspended extremely with 100mM sodium phosphate buffer (+150mM sodium chloride, pH7.0)
10g/L, with the cell ultrasonic 10min of Ultrasonic Cell Disruptor ice-water bath (800W, work 1sec stop 3sec), is centrifuged (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+), 30 DEG C of increases measuring light absorption value at 340nm.Enzyme is lived and is defined as
Enzyme amount required for reduction generation 1 micromole NADPH (or NADH) per minute is glucose dehydrogenase enzyme unit IU alive.Egg
Bai Hanliang uses Bradford method to be measured.Result shows that this glucose dehydrogenase enzyme is lived and is about 30IU/mg.
Embodiment three: carbonyl acyl reductase and the glucose dehydrogenase coexpression in E.coli
By glucose dehydrogenase encoding gene after Nde I and Xho I double digestion, it is cloned into the restructuring described in embodiment one
The multiple clone site 2 of plasmid pETDuet1-(MCS1), gene, after order-checking confirms, converts to E.coli BL21 (DE3), structure
The named pETDuet-KRED-GDH-BL21 of the recombinant bacterial strain (DE3) built.Picking individual colonies on ampicillin plate, connects
Enter in the LB culture medium containing corresponding antibiotic, fully cultivate, to OD for 37 degree600=0.6,3% ratio is inoculated into containing ampicillin
LB culture medium.At bacterial growth to OD600=0.7, cool the temperature to 25 degree, add 1mmol/L IPTG overnight induction.Centrifugal
Results thalline ,-20 DEG C frozen.SDS-PAGE detection shows, carbonyl acyl reductase is suitable with the expression of glucose dehydrogenase, total amount
Can be to the 70% of bacterial protein.
Embodiment four: prepared by fermentation and the crude enzyme liquid of restructuring carbonyl acyl reductase
Following material is added: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium chloride, pH is natural in 100L fermentation tank.
121 DEG C of sterilizing 20min.When being cooled to 37 DEG C, access 1L and cultivate to OD with LB culture medium (containing ampicillin)600=0.8 new
Fresh pETDuet-KRED-BL21 (DE3) culture fluid, adds the ampicillin solution extremely final concentration of 0.1mg/ of filtration sterilization
ML, 37 DEG C of 800rpm cultivate.Cultivating feed supplement after 2hr, supplemented medium is 500g/L glycerol, 100g/L peptone and 50g/L ferment
The solution 15L of female powder, strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1.OD when culture fluid600When reaching 25, by tank temperature drop to 25
DEG C, add final concentration of 1mmol/L IPTG, control each condition of culture induction 14hr.Induction terminates rear tube centrifuge maximum and turns
Speed harvested by centrifugation thalline, weight in wet base 3.34Kg, 4 DEG C temporary standby.
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 chloride, pH7.0) in buffer, each a time of high pressure homogenize 2 times: 800bar+600bar under 4 DEG C of low-temperature protections.Upwards
Stating and add polymine in cracking to final concentration 0.5% (w/v), 4 DEG C of stirring 30min, centrifuge 10,000rpm is centrifuged
20min, retains supernatant 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 is by real
Method described in example one of executing measures, and for 60.5IU/mL, protein concentration measures with Bradford method, thick pheron concentration 24.5mg/
mL。
Embodiment five: prepared by fermentation and the crude enzyme liquid of recombinant glucose dehydrogenase.
Following material is added: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium chloride, pH is natural in 100L fermentation tank.
121 DEG C of sterilizing 20min.When being cooled to 37 DEG C, access 1L and cultivate to OD with LB culture medium (containing ampicillin)600=0.8 new
Fresh pETDuet-GDH-BL21 (DE3) culture fluid, adds the ampicillin solution extremely final concentration of 0.1mg/mL of filtration sterilization,
37 DEG C of 800rpm cultivate.Cultivating feed supplement after 2hr, supplemented medium is 500g/L glycerol, 100g/L peptone and 50g/L yeast powder
Solution 15L, strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1.OD when culture fluid600When reaching 25, by tank temperature drop to 25 DEG C, add
Enter final concentration of 1mmol/L IPTG, control each condition of culture induction 14hr.Induction terminate rear tube centrifuge maximum (top) speed from
Heart results thalline, weight in wet base 3.52Kg, 4 DEG C temporary standby.
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 chloride, pH7.0) in buffer, each a time of high pressure homogenize 2 times: 800bar+600bar under 4 DEG C of low-temperature protections.Upwards
Stating and add polymine in cracking to final concentration 0.5% (w/v), 4 DEG C of stirring 30min, centrifuge 10,000rpm is centrifuged
20min, retains supernatant and is recombinant glucose dehydrogenase crude enzyme liquid, and 4 DEG C keep in Dark Place.The mensuration that glucose dehydrogenase enzyme is lived
As described in embodiment two, method measures, and for 720IU/mL, protein concentration measures with Bradford method, thick pheron concentration
31.2mg/mL。
The chiral analysis method of embodiment six: N-Boc-3-hydroxyl pyrrolidine.
Ee (chirality HPLC): Chiralpak IC150mm × 4.6mm chiral chromatographic column；Flowing 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 synthesis of embodiment seven: N-Boc-(S)-3-hydroxyl pyrrolidine.
The synthesis of N-Boc-(S)-3-hydroxyl pyrrolidine is pressed reaction equation and is carried out:
In a 250mL three-necked bottle, it is sequentially added into 100mL, 0.2mol/LNaH2PO4·Na2HPO4(pH7.0) buffering
Solution, compounds I (10g), glucose (15g) and 50mL butyl acetate, magnetic agitation 10min makes mix homogeneously, adds carbonyl
Acyl reductase (10mL), glucose dehydrogenase (5mL) and coenzyme (NADP+, 0.01g), stir 16 hours under 30 degrees Celsius, control
PH6.5 processed～7.0, high-performance liquid chromatogram determination display reaction terminates.Filtration adds 100mL ethyl acetate after dezymotizing, and repeats extraction
Taking three times, organic facies is spin-dried for after drying, obtains 9.4 g of compound II (N-Boc-(S)-3-hydroxyl pyrrolidine), by embodiment five
Method 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 glucose dehydrogenase coexpression to produce N-Boc-
(S)-3-hydroxyl pyrrolidine.
In a 250mL three-necked bottle, it is sequentially added into 100mL, 0.2mol/LNaH2PO4·Na2HPO4(pH7.0) buffering
Solution, compounds I (10g), glucose (15g) and 50mL butyl acetate, magnetic agitation 10min makes mix homogeneously, adds
0.8g utilizes the E.coli cell expressing carbonyl acyl reductase and the glucose dehydrogenase coexpression obtained described in embodiment three
(pETDuet-KRED-GDH-BL21 (DE3)), and coenzyme (NADP+, 0.01g), stir 16 hours under 30 degrees Celsius, control
PH6.5～7.0, high-performance liquid chromatogram determination display reaction terminates.Filtration dezymotize with cell debris after, add 100mL acetic acid second
Ester, repeats to extract three times, and organic facies is spin-dried for after drying, obtains 8.9 g of compound II (N-Boc-(S)-3-hydroxyl pyrrolidine), light
Learn purity ee value > 99.5%, molar yield 88.0%.
The present invention still has multiple specific embodiment, all employing equivalents or equivalent transformation and all skills of being formed
Art scheme, within all falling within the scope of protection of present invention.
<110>Suzhou Guodie Pharmaceuticals Science & Technology Co., Ltd
<120>enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof
<140> 201410015913.0
<160> 2
<210> 1
<211> 331
<212> PRT
<213> Candida Mangoliae
<220> 0
<221> VARIANT
<400> 1
Val Gly Thr Thr Thr Leu Asn Thr Gly Ala Ser Glu Leu Val Gly
1 5 10 15
Tyr Gly Thr Trp Gln Ala Ala Pro Gly Glu Val Gly Gln Gly Val
20 25 30
Lys Val Ala Ile Glu Thr Gly Tyr Arg His Leu Asp Leu Ala Lys
35 40 45
Val Tyr Ser Asn Gln Pro Glu Val Gly Ala Ala Ile Lys Glu Ala
50 55 60
Gly Val Lys Arg Glu Asp Leu Phe Ile Thr Ser Lys Ser Leu Trp
75 80 85
Asn Asn Ser His Pro Glu Gln Val Lys Glu Pro Ala Leu Asp Asp
90 95 100
Thr Leu Lys Glu Leu Gly Leu Glu Tyr Leu Asp Leu Tyr Leu Ile
105 110 115
His Trp Pro Val Ala Phe Pro Pro Glu Gly Asp Ile Thr Asn Leu
120 125 130
Phe Pro Lys Ala Asn Asp Leu Glu Val Leu Asp Leu Ala Glu Val
135 140 145
Ser Leu Val Asp Thr Trp Lys Ala Pro Met Val Lys Leu Leu Asp
150 155 160
Thr Gly Lys Val Lys Ala Ile Gly Val Ser Asn Phe Asp Ala Lys
165 170 175
Met Val Ile Asp Ala Ile Ile Ala Thr Gly Val Thr Pro Ser Val
180 185 190
Asn Gln Ile Glu His Arg His Pro Leu Leu Gln Pro Glu Leu Ile
195 200 205
Ala His Lys Ala Lys Asn His Ile Thr Ala Tyr Ser Pro Leu Gly
210 215 220
Asn Asn Thr Val Gly Ala Pro Asn Leu Leu Val Gln His Pro Glu
225 230 235
Ile Lys Arg Ala Glu Lys Asn Gly Cys Thr Pro Ala Gln Val Leu
240 245 250
Ile Ala Trp Ala Ile Val Gly Gly His Ser Val Ile Pro Leu Lys
255 260 265
Ser Val Thr Pro Ser Arg Ile Gly Glu Asn Phe Lys Lys Gln Val
270 275 280
Ser Leu Ser Gln Glu Asp Val Asp Ala Val Ser Lys Leu Gly Glu
285 290 295
Gly Ser Gly Arg Arg Arg Tyr Asn Ile Pro Cys Thr Tyr Ser Pro
300 305 310
Lys Trp Asp Ile Asn Phe Gly Glu Glu Asp Glu Lys Ser Cys Lys
315 320 325
Asn Ala Val Lys Ile Lys
330
<210> 2
<211> 246
<212> PRT
<213>thermoplasma acidophilum bacterium (Thermoplasma acidophilum)
<220> 0
<221> VARIANT
<400> 2
Met Lys Arg Asn Ser Ala Leu val Ile Val Ile Gly Val Gly Ala
1 5 10 15
Glu Leu Gly Leu Gly Ala Ala Leu Cys Arg Ile Ala Ala Asn Gly
20 25 30
Tyr His Val Tyr Val Ala Gly Arg Thr Gln Ala Lys Leu Asp Ile
35 40 45
Val Thr Asn Gly Ile Ala Gly Gly Gly Ser Ala Glu Ser Phe Ala
50 55 60
Met Asp Gly Thr Ser Glu Ala Asp Ile Met Arg Leu Phe Asp Arg
65 70 75
Ala Met Ser Pro Pro Asp Lys Ile Asp Val Pro Ser Leu Val Ile
80 85 90
Tyr Asn Val Gly Asn Asn Arg His Val Pro Phr Arg Asp Leu Thr
95 100 105
Glu Ala Gln Met Gln Asp Phe Leu Arg Ser Gly Pro Val Gly Gly
110 115 120
Phe Leu Val Gly Arg Glu Ala Ala Arg Arg Leu Ala Pro Leu Gly
125 130 135
Arg Gly Thr Val Ile Phe Thr Gly Ala Tyr Ala Ser Leu Arg Gly
140 145 150
Lys Pro Gly Phe Ala His Phe Ala Ala Ala Lys Ala Gly Leu Arg
155 160 165
Met Val Ala Gln Ser Met Ala Arg Glu Phe Gly Pro Leu Gly Leu
170 175 180
His Val Ala His Val Val Ile Asp Gly Gly Ile Asp Gly Glu Arg
185 190 195
Leu His Val Ser Arg Pro Gln Ala Ala Ala Glu Arg Gly Glu Asn
200 205 210
Gly Leu Leu Asn Val Asp Leu Ile Ala Glu Ala Tyr Trp Gln Leu
215 220 225
His Leu Gln His Pro Ser Ala Trp Thr His Glu Ile Asp Leu Arg
230 235 240
Pro Phe Lys Glu Pro Phe
245
Claims (2)
1. enzyme process reduction synthesis (S)-3-hydroxyl pyrrolidine and the method for derivant thereof, described course of reaction is as follows:
It is characterized in that: P is tertbutyloxycarbonyl, described reaction condition is pH6.0-7.5, with coexpression restructuring carbonyl acyl reductase and
Recombinant glucose dehydrogenase and coenzyme are catalyst, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyst are liquid
Liquid solution, lyophilized powder, immobilized enzyme or immobilized cell, the aminoacid sequence such as sequence table of described restructuring carbonyl acyl reductase
Shown in SEQ.ID NO:1, described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2；
Described reaction condition is pH6.5-7.0, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase are at genetic engineering bacterium
In efficient coexpression；
Described genetic engineering bacterium is the escherichia coli with recombinant vector pETDuet-1.
2. the method for the fermentation culture genetic engineering bacterium as shown in claim 1, it is characterised in that: include building gene work
Journey bacterium and the further fermentation of genetic engineering bacterium, the structure of described genetic engineering bacterium comprises the following steps:
Restructuring carbonyl acyl reductase encoding gene and glucose dehydrogenase encoding gene to full genome synthesis are respectively through double digestion；Again
It is cloned into respectively the different Anti-TNF-α sites of expression vector pETDuet-1, after recombiant plasmid order-checking confirms, converts respectively
To expressing coli strain, build corresponding recombinant bacterial strain；The aminoacid sequence such as sequence table of described restructuring carbonyl acyl reductase
Shown in SEQ.ID NO:1, described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2；
The fermentation further of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB culture medium containing ampicillin, cultivates to OD600=0.8
Fresh medium, add the ampicillin solution of filtration sterilization to final concentration of 0.1mg/mL, 37 DEG C of 800rpm cultivate；Training
Support feed supplement after 2hr, by strong aqua ammonia/salt acid for adjusting pH 7.0 ± 0.1, as the OD of culture fluid600When reaching 25, by tank temperature drop extremely
25 DEG C, add final concentration of 1mmol/L IPTG, continue to control each condition of culture induction 14hr, last harvested by centrifugation thalline.
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| CA2311649A1 (en) * | 1998-02-18 | 1999-08-26 | Susanne Thoni | Method for producing trifluoro-3(r)-hydroxybutyric acid derivatives |
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