CN104988189A - Method for preparing (S)-6-hydroxy-8-chlorine ethyl caprylate through reductase - Google Patents
Method for preparing (S)-6-hydroxy-8-chlorine ethyl caprylate through reductase Download PDFInfo
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Abstract
The invention discloses a method for preparing (S)-6-hydroxy-8-chlorine ethyl caprylate through reductase and belongs to the technical field of biological catalysis. The method comprises the steps that mixed spinning 6-carbonyl-8-chlorine ethyl caprylate, ethanol, a buffer agent and glucose are sequentially added into a reaction container provided with a stirring device, the stirring device is turned on to conduct stirring until the glucose is dissolved fully, lactic dehydrogenase and coenzyme are added into the reaction container to conduct a reaction, the reaction temperature, the reaction time and the pH value of the buffer agent of a reaction system in the reaction process are controlled, extraction is conducted after the reaction is finished, vacuum concentration is conducted, and then the (S)-6-hydroxy-8-chlorine ethyl caprylate is obtained. The conversion rate of the target product of the (S)-6-hydroxy-8-chlorine ethyl caprylate obtained through reduction reaches 100 percent, the yield is 94 percent or more, the ee value is 99 percent or more, the value of industrially amplifying production is achieved, and an economical basis capable of meeting the requirement for environment protection is laid for preparation of (R)-lipoic acid.
Description
Technical field
The invention belongs to biocatalysis technology field, be specifically related to the method for a kind of reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, the method for the asymmetric reduction DL 6-carbonyl of carbonyl reductase-8-chloroctanoic acid ethyl ester preparation (R)-Thioctic Acid chiral intermediate (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester.
Background technology
The English of 6-hydroxyl-8-chloroctanoic acid ethyl ester is called: 6-hydroxy-8-chlorine octylic acid ethyl ester, molecular formula is: C
10h
19o
3cl; Molecular weight is: 222.71, and its structure is shown below, and is a kind of chemical reaction intermediate, and for the synthesis of 6,8-dichloro-octanoic acid ethyl ester, and 6,8-dichloro-octanoic acid ethyl ester is the important intermediate of synthesis Thioctic Acid.
LA (Lipoic acid) belongs to vitamin(e) B group compound, is a kind of coenzyme be present in human body, also containing LA in many food such as Cauliflower and red meat.Thioctic Acid is as the extremely strong antioxidant of occurring in nature, and free radical that can be nearly all in purged body, regenerates other endogenic antioxidant, as vitamin-E, ascorbate salt and Coenzyme Q10 99.0 etc. by redox couple.Because Thioctic Acid has very strong resistance of oxidation, make it obtain in clinical application and study widely.Thioctic Acid can strengthen the skeletal muscle of non insulin dependent diabetes animal and erythrocyte to the absorption of glucose, and then reduces blood-sugar content, also can improve the ratio of insulin receptor kinase simultaneously, thus enhance the metabolism of glucose.The metal ions such as iron, chromium, copper and manganese in all right chelating organism of Thioctic Acid, avoid its catalysis Fenton to react, thus metal ion can be suppressed to generate the oncogenic pathways of active oxygen.Research shows, in food, antioxidant and derivative thereof can the differentiation of inducing tumor cell and apoptosis when heavy dose, suppress it to breed.Except this, American-European countries starts to be applied to food, healthcare products and makeup in recent years, and its market growth that expands to of range of application provides wide space.
The Thioctic Acid of chemosynthesis is R/S Thioctic Acid, and LA is the natural form of Thioctic Acid in human body, and S-Thioctic Acid does not have activity in human body.Current Thioctic Acid product is in and is replaced in the process of DL Thioctic Acid by LA, and the preparation of chirality LA is still focus.
The method preparing chirality LA in existing Industrialized processing technique is mainly raw material with monoethyl adipatee, pass through chloride, 6-carbonyl-8-chloroctanoic acid ethyl ester is generated again through Addition on ethylene, then DL-6-hydroxyl-8-chloroctanoic acid ethyl ester is reduced into, DL-6 is obtained through superchlorination, 8-dicloro caprylate ethyl ester, last condensation generates Thioctic Acid, recycling phenylethylamine splits it as resolving agent, the racemic thioctic acid phenylethylamine salt obtained, (R)-Thioctic Acid crude product is obtained through sour desalination, qualified (R)-Thioctic Acid (US06670484) can be obtained after refining.Due to this method complex process, need repeatedly crystallization, yield is low, cost is high, and resolution yield is no more than 50%, and (S)-Thioctic Acid can not effectively utilize simultaneously, causes the pollution of the wasting of resources and environment.
Paper (biological processing, 6 (4), 13-18 that Chinese patent Authorization Notice No. CN100558905C and Yang Qian delivers; 2008) be all that studying enzyme splits the method for preparation (R)-6-hydroxyl-8-chloroctanoic acid ethyl ester, its total recovery, in 46.7%, ee value 93.8%, is not all very desirable, could not improve yield, (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester fails effectively to utilize simultaneously.
" a strain rhodococcus and the purposes for the preparation of optical purity (R)-6-hydroxyl-8-chloroctanoic acid ester and other optical activity chirality alcohol " that patent of invention publication No. CN103451124.A recommends only have studied the preparation of R-6-hydroxyl-8-chloroctanoic acid ethyl ester as aforementioned CN100558905C, because R-6-hydroxyl-8-chloroctanoic acid ethyl ester is difficult to obtain highly purified LA, more easily obtain S-Thioctic Acid, thus do not possess the basis of industrialized production.
Summary of the invention
Task of the present invention is to provide a kind of reductase enzyme to prepare the method for (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, and the method utilizes the carbonyl reductase reduction 6-carbonyl-8-chloroctanoic acid ethyl ester with highly selective and uses (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester of obtaining high-optical-purity and ideal recovery and have economy and environmental protection effect.
Task of the present invention has been come like this, a kind of method of reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, comprise the following steps: first in the reaction vessel being furnished with whipping appts, add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, buffer reagent and glucose and open whipping appts and be stirred to glucose and fully dissolve, serum lactic dehydrogenase is added again and coenzyme reacts in reaction vessel, and control temperature of reaction, control the pH value of the buffer reagent of reaction system in reaction times and control reaction process, extract after the completion of reaction, then concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester.
In a specific embodiment of the present invention, the weight ratio of described DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, buffer reagent and ethanol is 1: 1-4: 5-50: < 5; The weight ratio of described DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and coenzyme is 10: 0.5-2: 0.004-0.01.
In another specific embodiment of the present invention, phosphoric acid salt or three (methylol) aminomethane hydrochloric acid (Tris-HCl) of described buffer reagent to be pH value be 6-8.
In another specific embodiment of the present invention, described serum lactic dehydrogenase is pulverous serum lactic dehydrogenase lyophilized powder.
In another specific embodiment of the present invention, described coenzyme is NADP
+.
Also have in a specific embodiment of the present invention, described control temperature of reaction temperature of reaction is controlled as 20-40 DEG C; The described control reaction times controls the reaction times for 5-40h.
More of the present invention and in a specific embodiment, in described control reaction process, the pH value of the buffer reagent of reaction system refers to that with alkaline solution, the pH value of the buffer reagent of reaction system being adjusted to pH is 6-8.
In an of the present invention and then specific embodiment, the potassium hydroxide solution of described alkaline solution to be mol concentration be 0.1-2mol/L, sodium hydroxide solution or ammoniacal liquor.
Of the present invention again more and in a specific embodiment, described extraction refers to extraction organic solvent.
In an again of the present invention and then specific embodiment, described organic solvent is toluene, methylene dichloride, ethylene dichloride, butanols or ethyl acetate.
Technical scheme provided by the invention is owing to make use of the highly selective of carbonyl reductase, the target product that thus reduction can be made to obtain is that the transformation efficiency of (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester reaches 100%, yield is more than 94%, ee value more than 99%, possessed the value of industrial amplification production, the preparation for (R)-Thioctic Acid is established relatively cheap and can be met the basis of environment protection requirement.
Embodiment
The following examples are only the preferred examples of the applicant, thus can not be interpreted as the restriction to side of the present invention.
Embodiment 1:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 7 three (methylol) aminomethane hydrochloric acid (i.e. Tris-HCl, also be Lewis acid, as follows) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 2: 5: 2, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+(NADP, as follows) reacts, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 0.8: 0.006, and controlling temperature of reaction is 20 DEG C, the control reaction times is 40h, controlling at pH with 0.1mol/L sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 7, namely toluene is adopted repeatedly to extract with organic solvent after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 96.8%, detecting GC is 94.53%, ee% is 99.21%, optically-active is-20.99 ° of (C=1%, ethanol), it was Genzyme serum lactic dehydrogenase lyophilized powder (25mg at the trade mark of market sale that serum lactic dehydrogenase described in the present embodiment adopts by abundant Techcom Imp Exp GmbH of Fujian province Xiamen City before the application proposes, 1500u < u represents enzyme activity international unit >)
Embodiment 2:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 8 three (methylol) aminomethane hydrochloric acid (Tris-HCl) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 4: 20: 5, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 0.9: 0.004, and control that temperature of reaction is 40 DEG C, the control reaction times is 5h, controlling at pH with 2mol/l potassium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 8, repeatedly extract with organic solvent toluene after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 95.6%, detecting GC is 94.76%, ee% is 99.73%, and optically-active is-22.53 ° (C=1%, ethanol).In the present embodiment, NM content is all with the description to embodiment 1.
Embodiment 3:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 6 three (methylol) aminomethane hydrochloric acid (Tris-HCl) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 2: 25: 3, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 1.5: 0.008, and control that temperature of reaction is 30 DEG C, the control reaction times is 24h, controlling at pH with ammonia soln by the pH value of the buffer reagent of reaction system in reaction process is 6, repeatedly extract by organic solvent ethyl acetate after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 95.3%, detecting GC is 94.88%, ee% is 99.44%, and optically-active is-21.87 ° (C=1%, ethanol).In the present embodiment, NM content is all with the description to embodiment 1.
Embodiment 4:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 7 phosphoric acid salt and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, phosphoric acid salt and ethanol is 1: 1: 50: 2, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 2: 0.004, and control that temperature of reaction is 25 DEG C, the control reaction times is 35h, controlling at pH with 1.8mol/L sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 7, repeatedly extract with organic solvent ethylene dichloride after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 95.9%, detecting GC is 94.62%, ee% is 99.25%, and optically-active is-21.03 ° (C=1%, ethanol).All the other are all with the description to embodiment 1.
Embodiment 5:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 6.8 phosphoric acid salt and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, phosphoric acid salt and ethanol is 1: 2.4: 20: 5, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 2: 0.01, and control that temperature of reaction is 35 DEG C, the control reaction times is 10h, controlling at pH with 0.5mol/L sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 6.8, repeatedly extract with organic solvent butanols after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 94.2%, detecting GC is 94.29%, ee% is 99.13%, and optically-active is-20.96 ° (C=1%, ethanol).All the other are all with the description to embodiment 1.
Embodiment 6:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 6 phosphoric acid salt and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, phosphoric acid salt and ethanol is 1: 2: 50: 1, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 1.5: 0.004, and control that temperature of reaction is 20 DEG C, the control reaction times is 40h, with ammonia soln, the pH value of the buffer reagent of reaction system is controlled at pH in reaction process and be 6, repeatedly extract with organic solvent butanols after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtains (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 94.8%, detecting GC is 94.32%, ee% is 99.18%, and optically-active is-20.98 ° (C=1%, ethanol).All the other are with the description to embodiment 1.
Embodiment 7:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 7.5 three (methylol) aminomethane hydrochloric acid (Tris-HCl) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 3: 40: 0.1, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, the weight ratio of serum lactic dehydrogenase and NADP+ is 10: 1: 0.006, and controlling temperature of reaction is 25 DEG C, the control reaction times is 18h, controlling at pH with 1mol/l potassium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 7.5, repeatedly extract by organic solvent dichloromethane after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 96.7%, detecting GC is 94.42%, ee% is 99.53%, optically-active is-21.35 ° of (C=1%, ethanol).All the other are with the description to embodiment 1.
Embodiment 8:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 8 phosphoric acid salt and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, phosphoric acid salt and ethanol is 1: 4: 50: 2.5, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 1: 0.01, and control that temperature of reaction is 35 DEG C, the control reaction times is 16h, controlling at pH with 1.5mol/L sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 8, repeatedly extract with organic solvent ethylene dichloride after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 96.9%, detecting GC is 94.42%, ee% is 99.53%, and optically-active is-21.35 ° (C=1%, ethanol).All the other are with the description to embodiment 1.
Embodiment 9:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 7 three (methylol) aminomethane hydrochloric acid (Tris-HCl) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 2: 10: 2, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 1: 0.008, and control that temperature of reaction is 25 DEG C, the control reaction times is 35h, controlling at pH with 1.0mol/l sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 7, repeatedly extract with organic solvent toluene after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 96.2%, detecting GC is 94.55%, ee% is 99.58%, and optically-active is-21.48 ° (C=1%, ethanol).All the other are with the description to embodiment 1.
Embodiment 10:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 7.5 three (methylol) aminomethane hydrochloric acid (Tris-HCl) and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, three (methylol) aminomethane hydrochloric acid and ethanol is 1: 1: 40: 5, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, the weight ratio of serum lactic dehydrogenase and NADP+ is 10: 0.5: 0.006, and controlling temperature of reaction is 25 DEG C, the control reaction times is 18h, controlling at pH with 1mol/l potassium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 7.5, repeatedly extract by organic solvent dichloromethane after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 96.4%, detecting GC is 94.18%, ee% is 99.33%, optically-active is-21.65 ° of (C=1%, ethanol).All the other are with the description to embodiment 1.
Embodiment 11:
In the reaction vessel being furnished with whipping appts, first add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, pH be 8 phosphoric acid salt and glucose, the weight ratio of DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, phosphoric acid salt and ethanol is 1: 4: 50: 0.5, and open whipping appts to be stirred to glucose and fully to dissolve, then add serum lactic dehydrogenase and NADP in reaction vessel
+react, DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and NADP
+weight ratio be 10: 2: 0.008, and control that temperature of reaction is 35 DEG C, the control reaction times is 16h, controlling at pH with 1.5mol/L sodium hydroxide solution by the pH value of the buffer reagent of reaction system in reaction process is 8, repeatedly extract with organic solvent ethylene dichloride after the completion of reaction, merge organic phase, concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, yield 95.4%, detecting GC is 94.67%, ee% is 99.33%, and optically-active is-21.15 ° (C=1%, ethanol).All the other are with the description to embodiment 1.
In sum, the present invention passes through a large amount of Select to use serum lactic dehydrogenase as reductase enzyme, under the condition of additionally not adding expensive coenzyme, chloro-for 8-6-carbonyl ethyl octylate Restore All can be (S)-8-chloro-6-Hydroxyoctanoic acid ethyl ester by this enzyme, solve (S)-Thioctic Acid in split process to be difficult to be converted into useful product and wasting problem, and theoretical maximum yield is only 50%; And raw material can all be converted by this enzyme process asymmetric synthesis, theoretical maximum yield is 100%; Significantly improve product yield.
Claims (10)
1. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, it is characterized in that comprising the following steps: first in the reaction vessel being furnished with whipping appts, add DL 6-carbonyl-8-chloroctanoic acid ethyl ester successively, ethanol, buffer reagent and glucose and open whipping appts and be stirred to glucose and fully dissolve, serum lactic dehydrogenase is added again and coenzyme reacts in reaction vessel, and control temperature of reaction, control the pH value of the buffer reagent of reaction system in reaction times and control reaction process, extract after the completion of reaction, then concentrating under reduced pressure, obtain (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester.
2. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1, is characterized in that the weight ratio of described DL 6-carbonyl-8-chloroctanoic acid ethyl ester, glucose, buffer reagent and ethanol is 1: 1-4: 5-50: < 5; The weight ratio of described DL 6-carbonyl-8-chloroctanoic acid ethyl ester, serum lactic dehydrogenase and coenzyme is 10: 0.5-2: 0.004-0.01.
3. the method for reductase enzyme according to claim 1 and 2 preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, is characterized in that described buffer reagent to be pH value is phosphoric acid salt or three (methylol) aminomethane hydrochloric acid of 6-8.
4. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1 and 2, is characterized in that described serum lactic dehydrogenase is pulverous serum lactic dehydrogenase lyophilized powder.
5. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1 and 2, is characterized in that described coenzyme is NADP
+.
6. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1, is characterized in that described control temperature of reaction temperature of reaction is controlled as 20-40 DEG C; The described control reaction times controls the reaction times for 5-40h.
7. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1, is characterized in that the pH value of the buffer reagent of reaction system in described control reaction process refers to that with alkaline solution, the pH value of the buffer reagent of reaction system being adjusted to pH is 6-8.
8. the method for reductase enzyme according to claim 7 preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester, is characterized in that described alkaline solution to be mol concentration is the potassium hydroxide solution of 0.1-2mol/L, sodium hydroxide solution or ammoniacal liquor.
9. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 1, is characterized in that described extraction refers to extraction organic solvent.
10. the method for reductase enzyme preparation (S)-6-hydroxyl-8-chloroctanoic acid ethyl ester according to claim 9, is characterized in that described organic solvent is toluene, methylene dichloride, ethylene dichloride, butanols or ethyl acetate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063156A (en) * | 2007-04-30 | 2007-10-31 | 江南大学 | Method for preparing (R)-6-hydroxy-8-chlorine octanoic acid ethyl by enzyme resolution |
| CN103451124A (en) * | 2013-06-14 | 2013-12-18 | 华东理工常熟研究院有限公司 | Rhodococcus baikonurensis and application thereof in preparing optically pure (R)-6-hydroxy-8-chlorocaprylate and other optically active chiral alcohol |
-
2015
- 2015-07-07 CN CN201510393726.0A patent/CN104988189A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063156A (en) * | 2007-04-30 | 2007-10-31 | 江南大学 | Method for preparing (R)-6-hydroxy-8-chlorine octanoic acid ethyl by enzyme resolution |
| CN103451124A (en) * | 2013-06-14 | 2013-12-18 | 华东理工常熟研究院有限公司 | Rhodococcus baikonurensis and application thereof in preparing optically pure (R)-6-hydroxy-8-chlorocaprylate and other optically active chiral alcohol |
Non-Patent Citations (2)
| Title |
|---|
| KATJA GOLDBERG ET AL: "Biocatalytic ketone reduction-a powerful tool for the production of chiral alcohols-part I:processes with isolated enzymes", 《APPL MICROBIOL BIOTECHNOL》 * |
| QI YE ET AL: "Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate", 《BIORESOURCE TECHNOLOGY》 * |
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