CN103898178B - Enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof - Google Patents
Enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof Download PDFInfo
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- CN103898178B CN103898178B CN201410016372.3A CN201410016372A CN103898178B CN 103898178 B CN103898178 B CN 103898178B CN 201410016372 A CN201410016372 A CN 201410016372A CN 103898178 B CN103898178 B CN 103898178B
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- glucose dehydrogenase
- carbonyl acyl
- acyl reductase
- enzyme
- piperidine alcohols
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- 210000001822 immobilized cell Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001965 increasing effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- XUWHAWMETYGRKB-UHFFFAOYSA-N piperidin-2-one Chemical class O=C1CCCCN1 XUWHAWMETYGRKB-UHFFFAOYSA-N 0.000 description 1
- BIWOSRSKDCZIFM-UHFFFAOYSA-N piperidin-3-ol Chemical class OC1CCCNC1 BIWOSRSKDCZIFM-UHFFFAOYSA-N 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- LWRYDHOHXNQTSK-UHFFFAOYSA-N thiophene oxide Chemical compound O=S1C=CC=C1 LWRYDHOHXNQTSK-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229940117957 triethanolamine hydrochloride Drugs 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention provides a kind of enzyme process and prepare high chiral pure(S)3 piperidine alcohols and the method for derivant thereof, described reaction condition 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, the preparation reduction of high yield, high-purity ground(S)3 piperidine alcohols 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 and prepare high chiral pure (S)-3-piperidine alcohols and the side of derivant thereof
Method, belongs to the synthesis technical field of medicine intermediate, falls within Green Chemistry and genetic engineering neck
Territory.
Background technology
Chirality 3-piperidine alcohols is piperidine derivatives, is the key intermediate of multiple medicine, pesticide,
Always one of the focus in pharmaceutical synthesis field, is also a kind of particularly important drug matrices molecule.
Chirality 3-piperidinol derivative be found to have antidepressant, arrhythmia, antithrombus formation,
Solve spasm, calmness and reduce the effects such as cholesterolemia activity, in this external active anticancer medicine also
This class formation common.Such as, in antitumor field, the silk of CDK(mono-class dependence Cylin/
Serineprotein kinase) play an important role in the regulation and control of cell cycle, the function of CDK is lost
It is in harmonious proportion between tumor occurs and there is close relationship.Flavopiridol (Flavopiridol) is
One CDK inhibitor entering clinical trial, chemical constitution is flavonoid, is originally derived from
A kind of Indian plant Rohitukin, the most can synthetic.Containing relevant in Flavopiridol structure
(S)-3-piperidine alcohols structure of key.Additionally, also have other multi-medicaments contain (S)-3-piperidine alcohols or
(R)-3-piperidine alcohols structure.
Generally use chirality 3-piperidine alcohols time, the form of N-protected can be used, as Boc-,
The N-protected forms such as Cbz-.The most known (S) or (R)-3-piperidine alcohols and the conjunction of derivant thereof
One-tenth mode has multiple, is roughly divided into following approach:
(1) set out with the natural chiral acid such as L MALIC ACID, L-Glu or L-Asp, through being condensed,
Reduction etc. is carried out, and often step is complicated, is both needed to multistep reaction, and the chiral raw material that relates to and also
Original reagent is expensive, it is difficult to industry is amplified.
(2) with the chiral acid of 3-piperidine alcohols of racemization as (+)-dibenzoyl tartaric acid splits, and
Chiral alcohol is obtained through repeatedly recrystallization.The method is easy, but its chiral acid related to is expensive,
Yield is low, and only 24%, and chirality is relatively difficult to be guaranteed.
(3) carrying out selectivity fractionation with lipase, (S)-and (R)-type product all can obtain.But
Lipase splits the highest yield of theory only 50% obtaining (S) with (R)-3-piperidine alcohols, dividing of product
From and purification difficult, generally require silica gel column chromatography etc., it is difficult to industry amplify.It is another that enzyme splits
One big defect is to be difficult to racemization initiation material to obtain higher yield, current raw material racemization
Still without feasible short-cut method, thus the fractionation cost of lipase remains high.
(4) with microorganism or enzymatic hydroxylation mode, directly selecting property hydroxylating piperidones and
The derivant of its N-protected is corresponding (S)-or (R)-3-piperidine alcohols.This mode can be maximum
Limit ground improves substrate utilization degree, and can obtain the chiral alcohol of intimate 100%.But at present,
This approach only stays in theoretical research and laboratory stage, and concentration of substrate is less than 5g/L, does not has
Actual production meaning.
Carbonyl acyl reductase (Canbonyl reductase, EC1.1.1.184) and alcoholdehydrogenase
(Alcohol dehydrogenase, EC1.1.1.1), all can asymmetric reduction prochiral ketones molecule
The alcoholic extract hydroxyl group that ketone carbonyl is chirality, be a kind of important way introducing chirality in drug molecule.
This enzymatic asymmetric reduction generally requires reduced coenzyme Ⅰ (NADH) or codehydrogenase Ⅱ
(NADPH), reduced coenzyme Ⅰ, II in reduction reaction as hydrogen donor exist, hydrogen supply
After become oxidized coenzyme I (NAD+) or codehydrogenase Ⅱ (NADP+), oxidized coenzyme again may be used
Obtained hydrogen by other oxidase or dehydrogenase effect, again become reduced coenzyme, complete coenzyme and follow
Ring.
Carbonyl acyl reductase and alcoholdehydrogenase are mainly derived from the microorganism such as yeast, antibacterial, existing many
Plant this fermentoid gene to be in the news, such as Candida magnoliae(Genbank Acc.No.
JC7338;And Candida parapsilosis(Genbank Acc.No. GI:11360538)
BAA24528.1;GI:2815409) etc..
The most existing multiple important chirality pharmaceutical intermediate compound can use carbonyl acyl reductase and alcohol dehydrogenase
Enzymatic synthesis, including pure enzyme and the multiple synthesis mode such as microbe whole-cell or immobilized enzyme/cell.
Generally also need to add regenerating coenzyme enzyme, such as glucose dehydrogenase (Glucose in synthetic system
Dehydrogenase, GDH) and hydrogenlyase (Formate dehydrogenase, FDH)
Deng.Such 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 for he
One of key intermediate of spit of fland class medicine；The asymmetric reduction of 1-Phenylethanone. and derivant thereof (such as:
U.S.Pat.No.6,800,477)；The asymmetric reduction (WO 2005/054491) of thienone.
At medical industry circle, multiple important prochiral ketones compound is still had to need to develop enzyme reducing process.
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 prepares master-hand
Property pure (S)-3-piperidine alcohols and the method for derivant thereof.
The purpose of the present invention is achieved through the following technical solutions:
Enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof, described reacts
Journey is as follows:
P is hydrogen, tertbutyloxycarbonyl (-Boc), benzyloxycarbonyl group (-Cbz) or fluorenylmethyloxycarbonyl (-Fmoc),
Described reaction condition is pH6.0-7.5, with coexpression restructuring carbonyl acyl reductase and restructuring glucose
Dehydrogenase and coenzyme are catalyst, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase are urged
Agent is liquid solution, lyophilized powder, immobilized enzyme or immobilized cell, and described restructuring carbonyl acyl is also
The aminoacid sequence of protoenzyme is as shown in sequence table SEQ .ID NO:1, and described restructuring glucose takes off
Hydrogen enzyme amino acid sequence is as shown in sequence table SEQ .ID NO:2.
Preferably, described reaction condition is pH6.4-6.6, described restructuring carbonyl acyl reductase and weight
Group glucose dehydrogenase efficient coexpression in genetic engineering bacterium.
Preferably, described genetic engineering bacterium is the escherichia coli with recombinant vector pETDuet-1.
The method of the above-described genetic engineering bacterium of a kind of fermentation culture, including building genetic engineering
Bacterium and the further fermentation of genetic engineering bacterium, the structure of described genetic engineering bacterium comprises the following steps:
The restructuring carbonyl acyl reductase encoding gene of full genome synthesis is encoded with glucose dehydrogenase
Gene is respectively through double digestion；The difference that it is cloned into expression vector pETDuet-1 more respectively is many
Clone anti-site, after recombiant plasmid order-checking confirms, convert respectively 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 to the LB culture medium containing ampicillin
In, cultivate to OD600The fresh medium of=0.8, adds the ampicillin of filtration sterilization
Solution is cultivated to final concentration of 0.1mg/mL, 37 DEG C of 800rpm；Cultivate feed supplement after 2hr,
By strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1, as the OD of culture fluid600When reaching 25, will
Tank temperature drop, to 25 DEG C, adds final concentration of 1mmol/L IPTG, continues to control each condition of culture
Induction 14hr, last harvested by centrifugation thalline.
The beneficial effects are mainly as follows: use restructuring carbonyl acyl reductase, glucose to take off
Hydrogen enzyme is applied to background and reduces, and concentration of substrate is up to 150g/L, and productivity is high, product light
Purity 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 enzyme process and prepare high chiral pure (S)-3-piperidine alcohols and derivant thereof
Method, described course of reaction is as follows:
P is hydrogen, tertbutyloxycarbonyl (-Boc), benzyloxycarbonyl group (-Cbz) or fluorenylmethyloxycarbonyl
(-Fmoc),
Described preparation method is as follows: the compound I of a mole is dissolved in 500～2000 milliliters
In buffer solution and organic solvent, adding weight in above-mentioned solution is the 0.1～20% of compound I
Gene recombinaton carbonyl acyl reductase, glucose dehydrogenase and coenzyme, keep system at 15～45 DEG C
Between, preferentially at 25～40 DEG C.With acid/base liquid regulation pH 6.0～7.5, preferably 6.4～6.6.
Stirring 16-72h, stopped reaction, extract 3 times with the organic solvents of about 1000 milliliters, close
And organic facies, desiccant dryness, decompression is distilled off organic solvent, obtains homochiral target
Compound II.Generally compound II chiral purity is more than 98%, can be used for the preparation of medicine.
Described organic solvent selected from methanol, ethanol, propanol, butanol, the tert-butyl alcohol, isopropanol,
One in oxolane, methyl tert-butyl ether, ethyl acetate, butyl acetate and toluene.
Described buffer solution is inorganic sulphuric acid, inorganic phosphate or triethanolamine hydrochloride buffer salt
In one.
Described inorganic base is selected from sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, potassium carbonate
In one.
The described P in compound I is preferably tertbutyloxycarbonyl.
Restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase at the efficient coexpression of escherichia coli, its
Can be liquid solution, lyophilized powder, it is also possible to be immobilized enzyme or cell.
Described carbonyl acyl reductase is Evolution in vitro, utilize purification enzyme or for its express big
Enterobacteria engineering bacteria is directly catalyzed.It utilizes a kind of reddish brown shadow yeast (Sporobolomyces
The variant of carbonyl acyl reductase salmonicolor), has compared 11 amino acid differences with wild type
Not.
The sequence optimisation of carbonyl acyl reductase, round increasing activity, heat surely on the basis of wild type
Qualitative and organic solvent stability is carried out.Mainly use semi-directional based on structure to evolve and
High flux screening, the sequence of final gained has compared 11 amino acid whose differences with wild type.
Great majority sudden change concentrates on enzyme surface and subunit thereof site.Gene order is inclined according to escherichia coli
Good codon amendment, and eliminate the secondary structure that may affect expression.Carbonyl acyl after optimization is also
Protoenzyme is high efficient expression in E.coli, and enzymatic activity is more than 50 times of wild type, and stable
Property also dramatically increases.Carbonyl acyl reductase high activity variant and glucose dehydrogenase after optimization exist
After E.coli coexpression, through thick purification, can efficient catalytic 3-piperidones be (S)-3-piperidine alcohols.
Described glucose dehydrogenase is Evolution in vitro, utilizes a kind of antibacterial Burkholderia sp.
Glucose dehydrogenase modification efficiently reduces NADP+Coenzyme, has compared 3 amino with wild type
Acid difference.Its gene order is revised according to the codon of escherichia coli preference, and eliminates possible shadow
Ringing the secondary structure expressed, this sequence is at E.coli height efficient expression.
The aminoacid sequence of described restructuring carbonyl acyl reductase as shown in sequence table SEQ .ID NO:1,
Described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Individually below with restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase describe in detail its
Expression in escherichia coli 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 is through the double enzyme of Nco I and Hind III
After cutting, it is cloned into expression vector pETDuet-1(producer: Novagen, production code member:
Multiple clone site 1 71146-3), recombiant plasmid, after order-checking confirms, converts to expression strain
In E.coli BL21 (DE3), the recombinant bacterial strain of structure is named
pETDuet-KRED-BL21(DE3).Picking individual colonies on ampicillin plate, accesses
In LB culture medium containing corresponding antibiotic, 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 (16h) of final concentration of 1mmol/L.
Harvested by centrifugation thalline ,-20 DEG C frozen.SDS-PAGE detection shows, this carbonyl acyl reductase is about
37.6KDa, target protein expression can be to the 60% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM
Sodium chloride, pH7.0) resuspended to 10g/L, with the cell ultrasonic 10min of Ultrasonic Cell Disruptor ice-water bath
(800W, work 1sec stop 3sec), centrifugal (12,000rpm, 4 DEG C, 10min), bacterium
Body lysate supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM phosphoric acid
Sodium buffer (pH7.0), 5mM N-Boc-3-piperidones, 1mM NADPH(or NADH),
30 DEG C of declines measuring light absorption value at 340nm.Enzyme is lived and is defined as that oxidation 1 per minute is micro-to rub
Enzyme amount required for you NADPH is carbonyl acyl reductase enzyme unit IU alive.Protein content
Using Bradford method to be measured, result shows that this carbonyl acyl reductase enzyme is lived and is about 3.8IU/mg.
Embodiment two: glucose dehydrogenase expression in E.coli and determination of activity
Full genome synthesis glucose dehydrogenase encoding gene after Nde I and Xho I double digestion,
It is cloned into multiple clone site 2 recombiant plasmid of expression vector pETDuet-1 after order-checking confirms,
Convert to E.coli BL21(DE3In), the named pETDuet-GDH-of recombinant bacterial strain of structure
BL21(DE3).Picking individual colonies on ampicillin plate, accesses containing corresponding antibiotic
In LB culture medium, fully cultivate, to OD for 37 degree600=0.6,3% ratio is inoculated into containing ammonia benzyl
The LB culture medium of penicillin.At bacterial growth to OD600=0.7, cool the temperature to 25 degree,
Add the IPTG overnight induction of final concentration of 1mmol/L.Harvested by centrifugation thalline ,-20 DEG C freeze
Deposit.SDS-PAGE detection shows, this glucose dehydrogenase about 27.8KDa, target protein table
The amount of reaching can be to the 50% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM
Sodium chloride, pH7.0) resuspended to 10g/L, with the cell ultrasonic 10min of Ultrasonic Cell Disruptor ice-water bath
(800W, work 1sec stop 3sec), centrifugal (12,000rpm, 4 DEG C, 10min), bacterium
Body lysate supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM phosphoric acid
Sodium buffer (pH7.0), 250mM glucose, 1mM NADP+(or NAD+), 30 DEG C
The increase of light absorption value is measured at 340nm.Enzyme is lived and is defined as reduction per minute and generates 1 and micro-rub
Your NADPH(or NADH) required for enzyme amount be that a glucose dehydrogenase enzyme is lived unit
IU.Protein content uses Bradford method to be measured.Result shows 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 reality
Executing the multiple clone site 2 of recombiant plasmid pETDuet1-(MCS1) described in example one, gene is through surveying
After sequence confirms, convert to E.coli BL21(DE3In), the recombinant bacterial strain of structure is named
pETDuet-KRED-GDH-BL21(DE3).Picking individual colonies on ampicillin plate,
Access in the LB culture medium containing corresponding antibiotic, 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 1mmol/L IPTG overnight induction.Harvested by centrifugation thalline,
-20 DEG C frozen.SDS-PAGE detection shows, carbonyl acyl reductase and the table of glucose dehydrogenase
The amount of reaching is suitable, and total amount can be to the 65% of bacterial protein.
Embodiment four: prepared by fermentation and the crude enzyme liquid of restructuring carbonyl acyl reductase
In 100L fermentation tank, add following material: 1Kg peptone, 0.5Kg yeast powder and
0.5Kg sodium chloride, pH is natural.121 DEG C of sterilizing 20min.When being cooled to 37 DEG C, access
1L cultivates to OD with LB culture medium (containing ampicillin)600=0.8 fresh
pETDuet-KRED-BL21(DE3) culture fluid, add the ampicillin solution of filtration sterilization
Cultivate to final concentration of 0.1mg/mL, 37 DEG C of 800rpm.Cultivate feed supplement after 2hr, feed supplement
Culture medium is the solution 15L of 500g/L glycerol, 100g/L peptone and 50g/L yeast powder,
Strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1.OD when culture fluid600When reaching 25, by tank temperature
It is down to 25 DEG C, adds final concentration of 1mmol/L IPTG, control each condition of culture induction 14hr.
Induction terminates rear tube centrifuge maximum (top) speed harvested by centrifugation thalline, weight in wet base 4.34Kg, and 4 DEG C temporarily
Deposit standby.
By above-mentioned 4.34Kg weight in wet base pETDuet-KRED-BL21(DE3) press 1:5(v/v)
It is resuspended in 100mM sodium phosphate (+150mM sodium chloride, pH7.0) buffer, 4 DEG C
Each one time of high pressure homogenize 2 times: 800bar+600bar under low-temperature protection.In above-mentioned cracking
Add polymine to final concentration 0.5%(w/v), 4 DEG C of stirring 30min, centrifuge 10,000
Rpm is centrifuged 20min, retains supernatant and is restructuring carbonyl acyl reductase crude enzyme liquid, 4 DEG C of lucifuges
Preserve.The mensuration that carbonyl reductase enzyme is lived method as described in embodiment one measures, and is 60.5
IU/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.
In 100L fermentation tank, add following material: 1Kg peptone, 0.5Kg yeast powder and
0.5Kg sodium chloride, pH is natural.121 DEG C of sterilizing 20min.When being cooled to 37 DEG C, access
1L cultivates to OD with LB culture medium (containing ampicillin)600=0.8 fresh
pETDuet-GDH-BL21(DE3) culture fluid, add the ampicillin solution of filtration sterilization extremely
Final concentration of 0.1mg/mL, 37 DEG C of 800rpm cultivate.Cultivating feed supplement after 2hr, feed supplement is trained
Foster base is the solution 15L of 500g/L glycerol, 100g/L peptone and 50g/L yeast powder,
Strong aqua ammonia/hydrochloric acid regulation pH7.0 ± 0.1.OD when culture fluid600When reaching 25, by tank temperature
It is down to 25 DEG C, adds final concentration of 1mmol/L IPTG, control each condition of culture induction 14hr.
Induction terminates rear tube centrifuge maximum (top) speed harvested by centrifugation thalline, weight in wet base 3.52Kg, and 4 DEG C temporarily
Deposit standby.
By above-mentioned 3.52Kg weight in wet base pETDuet-KRED-BL21(DE3) press 1:5(v/v)
It is resuspended in 100mM sodium phosphate (+150mM sodium chloride, pH7.0) buffer, 4 DEG C
Each one time of high pressure homogenize 2 times: 800bar+600bar under low-temperature protection.In above-mentioned cracking
Add polymine to final concentration 0.5%(w/v), 4 DEG C of stirring 30min, centrifuge 10,000
Rpm is centrifuged 20min, retains supernatant and is recombinant glucose dehydrogenase crude enzyme liquid, keeps away for 4 DEG C
Light preserves.The mensuration that glucose dehydrogenase enzyme is lived method as described in embodiment two measures, and is 720
IU/mL, protein concentration measures with Bradford method, thick pheron concentration 31.2mg/mL.
Embodiment six: restructuring carbonyl acyl reductase is to 3-piperidones and derivant reduction thereof
By the enzyme activity determination system of embodiment one and embodiment two, to 3-piperidones and derivant thereof
Carrying out the screening of enzyme reduction vigor, result is as follows:
| Ketosubstrate | Restructuring carbonyl acyl reductase |
| N-Boc-3-piperidones | 3.85IU/mg |
| N-3-piperidones | 0.24IU/mg |
| N-Cbz-3-piperidones | 2.71IU/mg |
| N-Fmoc-3-piperidones | 0.86IU/mg |
Embodiment seven: the chiral analysis method of (S)-N-Boc-3-piperidine alcohols
Ee(chirality HPLC): Chiralpak IC150mm × 4.6mm chiral chromatographic column；Stream
Dynamic phase: normal hexane (95%)/IPA(5%)；Flow velocity: 0.6mL/min；Wavelength: 210nm；
Retention time: (S)-N-Boc-3-piperidine alcohols 30.78min, another enantiomer (R)-N-Boc-3-
Piperidine alcohols 33.66min.The reduzate of restructuring carbonyl acyl reductase is (S)-type.
Embodiment eight: the enzymatic conversion method synthesis of (S)-N-Boc-3-piperidine alcohols
(S) synthesis of-3-N-Boc-piperidine alcohols is pressed reaction equation and is carried out:
In a 250mL three-necked bottle, it is sequentially added into 100mL, 0.2mol/L
NaH2PO4·Na2HPO4(pH7.0) buffer solution, compounds I (10g), glucose (12
G) with 50mL butyl acetate, magnetic agitation 10min makes mix homogeneously, adds carbonyl acyl also
Protoenzyme (10mL), glucose dehydrogenase (5mL) and coenzyme (NADP+, 0.01g), in
At 30 DEG C stir 16 hours, control ph between 6.5～7.0, high-performance liquid chromatogram determination
Display reaction conversion ratio is more than 99.5%.Filtration adds 100mL ethyl acetate after dezymotizing,
Repeating to extract three times, organic facies is spin-dried for after drying, obtains 9.4g compound ii (N-Boc-(S)-3-
Piperidine alcohols), molar yield 93.0%.Optical purity is measured by embodiment seven, product ee
Value > 99.9%.
Embodiment nine: utilize the E.coli cell of carbonyl acyl reductase and glucose dehydrogenase coexpression
Convert and produce (S)-N-Boc-3-piperidine alcohols
In a 250mL three-necked bottle, it is sequentially added into 100mL, 0.2mol/L
NaH2PO4·Na2HPO4(pH7.0) buffer solution, compounds I (10g), glucose (15
G) with 50mL butyl acetate, magnetic agitation 10min makes mix homogeneously, adds 1.0g
Utilize and described in embodiment three, express the carbonyl acyl reductase obtained and glucose dehydrogenase coexpression
E.coli cell (pETDuet-KRED-GDH-BL21(DE3)), and coenzyme (NADP+, 0.01
G), under 30 degrees Celsius stir 16 hours, control ph between 6.5～7.0, efficient liquid phase
Chromatographic determination display reaction terminates.Filtration dezymotize with cell debris after, add 100mL acetic acid
Ethyl ester, repeats to extract three times, and organic facies is spin-dried for after drying, obtains 8.3 g of compound II
((S)-N-Boc-3-piperidine alcohols), optical purity ee value > 99.9%, molar yield 82.2%.
Embodiment ten: the feather weight enzymatic clarification of (S)-N-Boc-3-piperidine alcohols and purification
In a 10L glass reaction still, it is sequentially added into 2.0L, 0.2mol/L
NaH2PO4·Na2HPO4(pH6.5) buffer solution, compounds I (1.05Kg), Fructus Vitis viniferae
Sugar (1.15Kg) and 2.0L deionized water, stir 10 with the mechanical agitation of 700 revs/min
Min makes reaction each material mix homogeneously, adjusts the pH to 6.5 ± 0.1 of reactant liquor, and temperature is extremely
28℃±2℃.After pH with temperature stabilization, add the carbonyl of above-mentioned 1.0L embodiment four preparation
Acyl reductase enzyme liquid and the glucose dehydrogenase enzyme liquid of 0.5L embodiment five preparation, be simultaneously introduced
Codehydrogenase Ⅱ (the NADP of 0.5g+).Reaction whole-process control temperature 28 DEG C ± 2 DEG C, and control with pH
Instrument automatic dripping liquid caustic soda processed, makes reactant liquor pH between 6.3～6.5.After reaction 24hr efficiently
Liquid chromatogram measuring display reaction conversion ratio more than 99.5%, stopped reaction.
Reactant liquor pH is adjusted to 7.0, removes pH probe, add 150g kieselguhr, stirring
10min.Making kieselguhr filter cake, vacuum filtration removes the solid impurity such as enzyme and cell debris,
Filtering residue divides 3 immersion filter washes with 5L ethyl acetate.Filtrate extracts with 5L ethyl acetate simultaneously,
In triplicate, merge all organic layers, and be spin-dried for the most after drying through anhydrous sodium sulfate, obtain
The crude product ((S)-N-Boc-3-piperidine alcohols) of 1.02Kg, this is light reddish brown color grease.By it
Fully draw solvent with fine vacuum oil pump, return with 0.5% activated carbon 60 DEG C in n-heptane solution
Stream decolouring, and carry out crystallizing and recrystallization in normal heptane, recrystallisation solvent 10:1,45 DEG C of thermosols
Rear room temperature crystallize, recrystallization 4 times, finally give colourless near-white crystal 854g,
HPLC > 99.5%, optical purity ee value > 99.9%.
The present invention still has multiple specific embodiment, all employing equivalents or equivalent transformation
And all technical schemes formed, within all falling within the scope of protection of present invention.
Claims (4)
1. enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof, and it reacted
Journey is as follows:
It is characterized in that: P is hydrogen, tertbutyloxycarbonyl (-Boc), benzyloxycarbonyl group (-Cbz) or fluorenes
Methoxycarbonyl group (-Fmoc), described reaction condition is pH6.0-7.5, with coexpression restructuring carbonyl acyl
Reductase and recombinant glucose dehydrogenase and coenzyme are catalyst, described restructuring carbonyl acyl reductase and
Recombinant glucose dehydrogenase catalyst is liquid solution, lyophilized powder, immobilized enzyme or fixation cell
Born of the same parents, the aminoacid sequence of described restructuring carbonyl acyl reductase as shown in sequence table SEQ .ID NO:1,
Described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Enzyme process the most according to claim 1 is prepared high chiral pure (S)-3-piperidine alcohols and spreads out
Biological method, it is characterised in that: described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase
Efficient coexpression in genetic engineering bacterium.
Enzyme process the most according to claim 2 is prepared high chiral pure (S)-3-piperidine alcohols and spreads out
Biological method, it is characterised in that: described genetic engineering bacterium is for having recombinant vector pETDuet-1
Escherichia coli.
4. a method for fermentation culture genetic engineering bacterium as shown in claim 3, its
It is characterised by: include building the further fermentation of genetic engineering bacterium and genetic engineering bacterium, described base
Because the structure of engineering bacteria comprises the following steps:
The restructuring carbonyl acyl reductase encoding gene of full genome synthesis is encoded with glucose dehydrogenase
Gene is respectively through double digestion；The difference that it is cloned into expression vector pETDuet-1 more respectively is many
Cloning site, after recombiant plasmid order-checking confirms, converts to coli strain, builds corresponding
Recombinant bacterial strain；
The fermentation further of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded to the LB culture medium containing ampicillin
In, cultivate to OD600The fresh medium of=0.8, adds the ampicillin of filtration sterilization
Solution is cultivated to final concentration of 0.1mg/mL, 37 DEG C of 800rpm；Cultivate 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, will
Tank temperature drop, to 25 DEG C, adds final concentration of 1mmol/L IPTG, continues to control each condition of culture
Induction 14hr, last harvested by centrifugation thalline.
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| CN105420307A (en) * | 2015-12-02 | 2016-03-23 | 中国科学院成都生物研究所 | Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine |
| CN105671014A (en) * | 2016-03-09 | 2016-06-15 | 浙江工业大学 | Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof |
| CN106520855A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Method for preparing stereoscopic complementary N-heterocycle alcohol compounds by conducting biological catalysis through carbonyl reductase |
| CN107586798A (en) * | 2017-09-27 | 2018-01-16 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (S) 1 N benzene methoxycarbonyl group 3 |
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| CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
| CA2874055A1 (en) * | 2012-06-18 | 2013-12-27 | Laboratorio Chimico Internazionale S.P.A. | Enzymatic reduction |
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| CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
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