CN103898177A - Method for preparing high-chiral purity (R)-3-haloperidol and derivatives of high-chiral purity (R)-3-haloperidol - Google Patents
Method for preparing high-chiral purity (R)-3-haloperidol and derivatives of high-chiral purity (R)-3-haloperidol Download PDFInfo
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- CN103898177A CN103898177A CN201410016356.4A CN201410016356A CN103898177A CN 103898177 A CN103898177 A CN 103898177A CN 201410016356 A CN201410016356 A CN 201410016356A CN 103898177 A CN103898177 A CN 103898177A
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Abstract
The invention provides a method for preparing high-chiral purity (R)-3-haloperidol and derivatives of high-chiral purity (R)-3-haloperidol through an enzymatic method. Reduced (R)-3-haloperidol and the derivatives thereof with high yield and high purity are prepared by using recombinant alcohol dehydrogenase, recombinant glucose dehydrogenase and coenzyme which are efficiently co-expressed in escherichia coli as catalysts under the reaction condition that the pH value is 6.0-7.5. The method is short in reaction time and low in preparation cost.
Description
Technical field
The present invention relates to one and prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof, belong to the synthesis technical field of medicine intermediate, also belong to Green Chemistry and genetically engineered field.
Background technology
Chirality 3-piperidine alcohols is piperidine derivatives, is the key intermediate of multiple medicine, agricultural chemicals, is one of the focus in the synthetic field of medicine always, is also a kind of very important medicine molecule of the skeleton.Chirality 3-piperidine alcohols derivative has been found to have antidepressant, anti-arrhythmia, antithrombotic and has formed, separated the effects such as spasm, calmness and reduction blood cholesterol activity, also common this class formation in this external antitumour activity medicine.For example, in antitumor field, CDK(mono-class relies on the protein serine/threonine of Cylin) in the regulation and control of cell cycle, play an important role, the functional disorder of CDK and tumour exist close relationship between occurring.Flavopiridol (Flavopiridol) is first CDK inhibitor that enters clinical trial, and chemical structure is flavonoid, derives from first a kind of Indian plant Rohitukin, at present can synthetic.In Flavopiridol structure, contain crucial (S)-3-piperidine alcohols structure.In addition, also have other multi-medicaments to contain (S)-3-piperidine alcohols or (R)-3-piperidine alcohols structure.
Conventionally in the time using chirality 3-piperidine alcohols, can adopt the form of N-protected, as the N-protected such as Boc-, Cbz-form.The synthesis mode of known (S) or (R)-3-piperidine alcohols and derivative thereof has multiplely at present, is roughly divided into following approach:
(1) set 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 needs polystep reaction, and the chiral raw material relating to and to go back original reagent expensive, is difficult to industry and amplifies.
(2) split as (+)-dibenzoyl tartaric acid through chiral acid with the 3-piperidine alcohols of racemization, and obtain chiral alcohol through recrystallization repeatedly.This method is easy, but its chiral acid relating to is expensive, and yield is low, is only 24%, and chirality is difficult to be guaranteed relatively.
(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) and (R)-3-piperidine alcohols, 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 piperidone and N-protected thereof be (S)-or (R)-3-piperidine alcohols accordingly.This mode can improve substrate utilization degree to greatest extent, and can obtain the chiral alcohol that is close to 100%.But 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.
Carbonyl acyl reductase (Canbonyl reductase; EC1.1.1.184) with alcoholdehydrogenase (Alcohol dehydrogenase; EC1.1.1.1), the alcoholic extract hydroxyl group that ketone carbonyl that all can asymmetric reduction prochiral ketones molecule is chirality, is a kind of important way of introducing chirality in drug molecule.This enzymatic asymmetric reduction often needs reduced coenzyme Ⅰ (NADH) or codehydrogenase Ⅱ (NADPH), and reduced coenzyme Ⅰ, II exist as hydrogen donor in reduction reaction, become oxidized coenzyme I (NAD after hydrogen supply
+) or codehydrogenase Ⅱ (NADP
+), oxidized coenzyme can be obtained hydrogen by other oxydase or desaturase effect again, again becomes reduced coenzyme, completes coenzyme circulation.
Carbonyl acyl reductase and alcoholdehydrogenase are mainly derived from the microorganism such as yeast, bacterium, and existing multiple this fermentoid gene is in the news, as Candida magnoliae(Genbank Acc.No.JC7338; And Candida parapsilosis(Genbank Acc.No.BAA24528.1 GI:11360538); GI:2815409) etc.
At present existing multiple important chirality pharmaceutical intermediate compound can be synthetic with carbonyl acyl reductase and alcoholdehydrogenase, 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 enzyme reducing process.
Summary of the invention
The object of the invention is to solve above-mentioned technical problem, provide one to prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof.
Object of the present invention is achieved through the following technical solutions:
The method of preparing high chiral purity (R)-3-piperidine alcohols and derivative thereof, described reaction process is as follows:
P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc), described reaction conditions is pH6.0-7.5, recombinate alcoholdehydrogenase and recombinant glucose dehydrogenase and coenzyme as catalyzer taking coexpression, described restructuring alcoholdehydrogenase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell, the aminoacid sequence of described restructuring alcoholdehydrogenase 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 pH6.4-6.6, and described restructuring alcoholdehydrogenase 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 the above-described genetic engineering bacterium of fermentation culture, 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 alcoholdehydrogenase 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 alcoholdehydrogenase, Hexose phosphate dehydrogenase to be applied to background and to reduce, concentration of substrate is up to 150g/L, and 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 the method for the high chiral purity of preparation (R)-3-piperidine alcohols and derivative thereof, and described reaction process is as follows:
P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc),
Described preparation method is as follows: the Compound I of a mole is dissolved in the buffered soln and organic solvent of 500～2000ml, in above-mentioned solution, adding weight is 0.1～20% gene recombination alcoholdehydrogenase, Hexose phosphate dehydrogenase and coenzyme of Compound I, holder ties up between 15～45 DEG C, preferentially at 25～40 DEG C.Regulate pH 6.0～7.5 with acid/alkali lye, preferably 6.4～6.6.Stir 16-72h, stopped reaction, with the organic solvent extraction of 1000 milliliters of left and right 3 times, merging organic phase, desiccant dryness, organic solvent is removed in underpressure distillation, obtains homochiral target compound III.Conventionally compound III chiral purity is greater than 98%, can be used for the preparation of medicine.
Described organic solvent is selected from the one in methyl alcohol, ethanol, propyl alcohol, butanols, the trimethyl carbinol, Virahol, tetrahydrofuran (THF), methyl tert-butyl ether, ethyl acetate, butylacetate and toluene.
Described buffered soln is the one in inorganic sulfuric acid, inorganic phosphate or trolamine hydrochloric acid buffering salt.
Described mineral alkali is selected from the one in sodium hydroxide, potassium hydroxide, ammoniacal liquor, sodium carbonate, salt of wormwood.
P in described Compound I is preferably tertbutyloxycarbonyl.
Restructuring alcoholdehydrogenase 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 alcoholdehydrogenase is external evolution, utilizes the enzyme of purifying or the direct catalysis of colibacillus engineering for its expression.It utilizes the variant of the alcoholdehydrogenase of a kind of hot water high temperature coccus (Thermococcus hydrothermalis), and variant has been compared 1 amino acid difference with wild-type.
The sequence optimisation of alcoholdehydrogenase 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 1 amino acid whose difference 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.Alcoholdehydrogenase after optimization is high efficient expression in E.coli, and enzymic activity is the more than 2 times of wild-type, and stability also significantly increases.Alcoholdehydrogenase high reactivity variant after optimization and Hexose phosphate dehydrogenase, after E.coli coexpression, through thick purifying, can efficient catalytic 3-piperidone be (S)-3-piperidine alcohols.
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.
The aminoacid sequence of described restructuring alcoholdehydrogenase 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 alcoholdehydrogenase and recombinant glucose dehydrogenase respectively.
Embodiment mono-: expression and the determination of activity of alcoholdehydrogenase in E.coli
The synthetic restructuring alcoholdehydrogenase 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 into expression strain E.coliBL
21(DE
3) in, the recombinant bacterial strain called after pETDuet-ADH-BL of structure
21(DE
3).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 44.5KDa of this alcoholdehydrogenase, and target protein expression amount can be to 55% 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-piperidone, 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 to be oxidized the needed enzyme amount of 1 micromole NADPH be alcoholdehydrogenase reductase enzyme IU of unit that lives.Protein content adopts Bradford method to measure, and result shows that this alcoholdehydrogenase enzyme work is about 5.6IU/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, after Nde I and Xho I double digestion, is cloned into the multiple clone site 2 of expression vector pETDuet-1, and recombinant plasmid, after order-checking is confirmed, is converted into E.coli BL
21(DE
3) in, 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 50% 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-: alcoholdehydrogenase and the Hexose phosphate dehydrogenase coexpression in E.coli
Hexose phosphate dehydrogenase encoding gene, after Nde I and Xho I double digestion, is cloned into the multiple clone site 2 of the recombinant plasmid pETDuet1-(MCS1) described in embodiment mono-, and gene, after order-checking is confirmed, is converted into E.coli BL
21(DE
3) in, the recombinant bacterial strain called after pETDuet-ADH-GDH-BL of structure
21(DE
3).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 alcoholdehydrogenase and Hexose phosphate dehydrogenase is suitable, and total amount can be to 60% of bacterial protein.
Embodiment tetra-: the fermentation of restructuring alcoholdehydrogenase 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-ADH-BL
21(DE
3) 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 4.21Kg, and 4 DEG C are temporary for subsequent use.
By above-mentioned 4.21Kg weight in wet base pETDuet-ADH-BL
21(DE
3) 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 alcoholdehydrogenase crude enzyme liquid, and 4 DEG C keep in Dark Place.The mensuration that alcoholdehydrogenase enzyme is lived press method described in embodiment mono-and is measured, and is 120.5IU/mL, and protein concentration is measured with Bradford method, slightly zymoprotein concentration 27.5mg/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-BL
21(DE
3) 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-BL
21(DE
3) 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: restructuring alcoholdehydrogenase is to 3-piperidone and derivative reductive action thereof
Press the enzyme activity determination system of embodiment mono-and embodiment bis-, 3-piperidone and derivative thereof are carried out to the screening of enzyme reduction vigor, result is as follows:
| Ketosubstrate | Restructuring alcoholdehydrogenase |
| N-Boc-3-piperidone | 5.63IU/mg |
| N-3-piperidone | 0.45IU/mg |
| N-Cbz-3-piperidone | 3.84IU/mg |
| N-Fmoc-3-piperidone | 0.53IU/mg |
Embodiment seven: the chiral analysis method of (R)-N-Boc-3-piperidine alcohols
Ee(chirality HPLC): Chiralpak IC150mm × 4.6mm chiral chromatographic column; Moving phase: normal hexane (95%)/IPA(5%); Flow velocity: 0.6mL/min; Wavelength: 210nm; Retention time: (R)-N-Boc-3-piperidine alcohols 33.66min, another enantiomorph (S)-N-Boc-3-piperidine alcohols 30.78min.The reduzate of restructuring alcoholdehydrogenase is (R)-type.
Embodiment eight: the enzymatic conversion method of (R)-N-Boc-3-piperidine alcohols is synthetic
(R) the synthetic reaction formula of pressing of-3-N-Boc-piperidine alcohols carries out:
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 (12g) and 50mL butylacetate, magnetic agitation 10min makes to mix, then adds alcoholdehydrogenase (8mL), Hexose phosphate dehydrogenase (5mL) and coenzyme (NADP
+, 0.01g), under 30 degrees Celsius, stir 16 hours, control pH value between 6.5～7.0, high-performance liquid chromatogram determination shows that reaction conversion ratio is more than 99.5%.Filtration adds 100mL ethyl acetate after dezymotizing, re-extract three times is spin-dried for after organic phase is dry, obtains 9.5 and digests compound III ((R)-N-Boc-3-piperidine alcohols), molar yield 94.6%.Optical purity is pressed method described in embodiment seven and is measured, product ee value >99.4%.
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 the high chiral purity of preparation (R)-3-piperidine alcohols and derivative thereof, described reaction process is as follows:
It is characterized in that: P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc), described reaction conditions is pH6.0-7.5, recombinate alcoholdehydrogenase and recombinant glucose dehydrogenase and coenzyme as catalyzer taking coexpression, described restructuring alcoholdehydrogenase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell, the aminoacid sequence of described restructuring alcoholdehydrogenase 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 the high chiral purity of preparation according to claim 1 (R)-3-piperidine alcohols and derivative thereof, it is characterized in that: described reaction conditions is PH6.4-6.6, described restructuring alcoholdehydrogenase and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
3. the method for the high chiral purity of preparation according to claim 1 (R)-3-piperidine alcohols 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 alcoholdehydrogenase 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN107574194A (en) * | 2017-09-27 | 2018-01-12 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (R) 1 N benzene methoxycarbonyl group 3 |
| CN107630054A (en) * | 2017-04-13 | 2018-01-26 | 绍兴百茵生物技术有限公司 | The bioconversion method of penem-like pharmaceutical intermediate |
| CN110618202A (en) * | 2018-06-20 | 2019-12-27 | 成都康弘生物科技有限公司 | Method for detecting protein purity |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107630054A (en) * | 2017-04-13 | 2018-01-26 | 绍兴百茵生物技术有限公司 | The bioconversion method of penem-like pharmaceutical intermediate |
| CN107574194A (en) * | 2017-09-27 | 2018-01-12 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (R) 1 N benzene methoxycarbonyl group 3 |
| CN110618202A (en) * | 2018-06-20 | 2019-12-27 | 成都康弘生物科技有限公司 | Method for detecting protein purity |
| CN110618202B (en) * | 2018-06-20 | 2022-07-08 | 成都康弘生物科技有限公司 | Method for detecting protein purity |
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