Enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol

文档序号：416615 发布日期：2021-12-21 浏览：21次中文

阅读说明：本技术 一种(s)-1-(4-氯苯基)-1,3-丙二醇的酶法生产方法 (Enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol ) 是由倪国伟尚中栋郭元勇袁培斋张数郜宪兵王咪石洪运冯尚上于 2021-09-29 设计创作，主要内容包括：本发明涉及一种(S)-1-(4-氯苯基)-1,3-丙二醇的酶法生产方法,合成路线如下：其中,R-(1),R-(2)独立地选自C1-6的烷基。本发明对羰基还原酶进行了筛选,得到了野生型的羰基还原酶WO03及其突变体,发现其对于关键医药中间体(S)-1-(4-氯苯基)-1,3-丙二醇的手性还原具有很好的催化活性,酶活性高,立体选择型好,手性纯度＞99.6％。本发明方法不需要要苛刻的低温条件,利于大规模的产业化生产。(The invention relates to aThe enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol comprises the following synthetic route: wherein R is 1 ，R 2 Independently selected from C1-6 alkyl. The invention screens carbonyl reductase to obtain wild carbonyl reductase WO03 and a mutant thereof, and finds that the wild carbonyl reductase WO03 has good catalytic activity for chiral reduction of a key medical intermediate (S) -1- (4-chlorphenyl) -1, 3-propanediol, high enzyme activity, good stereoselectivity and chiral purity of more than 99.6%. The method does not need harsh low-temperature conditions, and is beneficial to large-scale industrial production.)

1. An enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propanediol comprises the following synthetic route:

wherein R is₁，R₂Independently selected from C1-6 alkyl.

2. The production method according to claim 1, wherein the carbonyl reductase has a sequence corresponding to SEQ ID NO:2, or an amino acid sequence corresponding to SEQ ID NO:2, one or a combination of two or more of the following mutations: the V mutation at the 45 th position is I (V45I), the K mutation at the 63 th position is Q (K63Q), the G mutation at the 141 th position is A (G141A), the G mutation at the 141 th position is V (G141V), the I mutation at the 195 th position is L (I195L), and the A mutation at the 204 th position is V (A204V);

the carbonyl reductase mutants also include the following ranges: the amino acid sequence obtained by replacing, deleting, changing, inserting or increasing one or more amino acids of the amino acid sequence shown by the mutant in the range of keeping the enzyme activity; and/or one or more amino acid insertions are carried out on the amino acid sequence shown by the mutant at the N end or the C end of the sequence within the range of keeping the enzyme activity, and the number of the inserted amino acid residues is 1-20; preferably 1 to 10, more preferably 1 to 5.

3. The method of claim 2, wherein the carbonyl reductase mutant has a sequence corresponding to SEQ ID NO:2, one of the following mutations is present:

(i) the G mutation at position 141 is A (G141A), and the I mutation at position 195 is L (I195L), and the amino acid sequence is shown as SEQ ID No:4 is shown in the specification;

(ii) the G mutation at position 141 is V (G141V), and the I mutation at position 195 is L (I195L), and the amino acid sequence is shown as SEQ ID No: 6 is shown in the specification;

(iii) the V mutation at position 45 is I (V45I); the G mutation at the 141 position is V (G141V, I195L), the I mutation at the 195 position is L (I195L), and the amino acid sequence is shown as SEQ ID No: 8 is shown in the specification;

(iv) the mutation of V at position 45 is I (V45I), the mutation of G at position 141 is V (G141V, I195L), the mutation of I at position 195 is L (I195L), the mutation of A at position 204 is V (A204V), and the amino acid sequence is shown as SEQ ID No: 10 is shown in the figure;

(v) the mutation of V at position 45 is I (V45I), the mutation of K at position 63 is Q (K63Q), the mutation of L at position 118 is M (L118M), the mutation of G at position 141 is V (G141V, I195L), the mutation of A at position 204 is V (A204V), and the amino acid sequence is shown as SEQ ID No: 12 is shown in the specification;

the carbonyl reductase mutants also include the following ranges: the amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids of the amino acid sequence shown in SEQ ID No. 4, 6, 8, 10 or 12 in the range of keeping the enzyme activity; and/or

Inserting one or more amino acids into the amino acid sequence shown in SEQ ID No. 4, 6, 8, 10 or 12 at the N end or C end of the sequence within the range of keeping the enzyme activity, wherein the number of the inserted amino acid residues is 1-20; preferably 1 to 10, more preferably 1 to 5.

4. The production method according to claim 1, characterized by comprising the steps of:

(S1) reacting m-chlorobenzoic acid with an alcohol R₁Performing OH esterification reaction to obtain m-chlorobenzoic acid alkyl ester IM 01;

(S2) alkyl m-chlorobenzoate (IM01) with alkyl acetate CH under the action of organic base₃COOR₂Condensation to obtain a product, namely ketoester IM 02;

(S3) preparing a product IM03 under the catalysis of carbonyl reductase by taking ketoester IM02 as a substrate of an enzyme-catalyzed reaction;

(S4) hydrolyzing the IM03 to obtain IM 04;

(S5) reduction of IM04 gave the final product GL01, (S) -1- (4-chlorophenyl) -1, 3-propanediol.

5. The production method according to claim 4, wherein the esterification reaction in the step (S1) is carried out in the presence of an acid catalyst at a reaction temperature of 60 to 100 ℃ under heating reflux for 15 to 20 hours; preferably, m-chlorobenzoic acid and R₁The molar ratio of OH is 0.3-0.6: 1.

6. the production method according to claim 4, whichCharacterized in that in step (S2), the organic base is selected from potassium salt and/or sodium salt of organic alcohol; IM01, ester CH₃COOR₂And an organic base in a molar ratio of 1: 1.1-1.7: 1.5-2.5, preferably 1: 1.3-1.5: 1.8-2.2.

7. The process according to claim 4, wherein the ketoester IM02 of step (S3) is used as a substrate for the enzyme-catalyzed reaction at a concentration of 50 to 200 g/L; more preferably 100 to 150 g/L.

8. The production process according to claim 4, wherein the carbonyl reductase is at least one of an enzyme in a free form, an immobilized enzyme, or an enzyme in a bacterial form, and the amount of the carbonyl reductase to be used is 1 to 6 wt% based on the substrate in the reaction system.

9. The method according to claim 4, wherein in step (S3), a co-substrate and a coenzyme are further present, the co-substrate being selected from the group consisting of: at least one of isopropanol, glucose, and ammonium formate; preferably, the concentration of the co-substrate is 100-200% of the substrate concentration, more preferably, the concentration of the co-substrate is 140-170% of the substrate concentration;

the coenzyme comprises at least one of reduced coenzyme and oxidized coenzyme, and the ratio of the dosage of the coenzyme to the dosage of the substrate is 0.01 wt% -1.0 wt%, preferably 0.1 wt% -0.5 wt%;

further, an enzyme for coenzyme regeneration is also present, in particular selected from alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.

10. The production method according to claim 4, wherein the hydrolysis in the step (S4) is carried out in the presence of a base in an amount to adjust the pH of the system to 12 to 14; and/or

In the step (S5), adding a reducing agent in batches, wherein the reducing agent is sodium borohydride and/or potassium borohydride, the amount of the reducing agent is 5-10% of the mass of IM04, the reaction temperature is-5 ℃ to 5 ℃, the adding time is 1-2h, after the reducing agent is added, the low temperature is maintained, the reaction is continued for 10-30min, then boron trifluoride diethyl etherate is slowly added dropwise, the amount of the boron trifluoride diethyl etherate is 1-2 times of the mass of IM04, after the dropwise addition is completed within 1-3h, the low temperature reaction is maintained for 1-3h, then the temperature is increased to 20-30 ℃, the reaction is performed for 3-5h, then the temperature is reduced to 10-20 ℃, then methanol is added dropwise, after the dropwise addition is completed within 1-2h, the reaction is continued for 1-2h, and then the post-treatment step is performed, so that the product GL01 is obtained.

Technical Field

The invention belongs to the field of organic synthesis, and particularly relates to an enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propylene glycol.

Background

(S) -1- (4-chlorophenyl) -1, 3-propanediol is a key intermediate used in liver targeting drugs and has been used in a number of clinical and preclinical study drugs. For example, the methanesulfonic acid peradfovir, the liver targeting prodrug is developed by the Xian Xintong medicine component, Inc., and has completed the second clinical stage, and the third clinical stage is currently developed. The key double alcohol intermediate adopts a chemical synthesis route.

The preparation method is reported in patent document US20030225277, and the process route is as follows:

similar synthetic routes have also been reported in US2003/229225, and in the documents JACS,2004, vol 126, #16, p 5154-5163.

The above synthetic route has three limitations in the scale production:

(1) low-temperature operating conditions are required, particularly the second-step reaction requires ultralow-temperature conditions of-60 ℃, the requirements on production equipment are high, and large-scale production is difficult;

(2) needs to use an organic catalyst (+) -DIPCL (CAS NO:112246-73-8), has high price and complex operation of the catalytic process, and is difficult to be applied to large-scale production.

(3) The borane is used for reducing the carboxylic acid, the cost of the reaction scale production is high, and the quenching reaction is violent and exothermic, so that certain safety risk is caused.

(4) The chiral purity of the prepared diol compound 1 is 98%, and in order to meet the requirements of registration declaration, the diol compound needs to be refined, so that the production cost is further increased.

The 4-point defect causes higher production cost of the product and is difficult to meet the requirement of large-scale production. Therefore, a large-scale method for producing the (S) -1- (4-chlorphenyl) -1, 3-propylene glycol with high optical purity in a large scale is urgently needed.

Disclosure of Invention

In order to overcome the defects of high cost and low optical purity of the preparation process of the key medical intermediate (S) -1- (4-chlorphenyl) -1, 3-propanediol in the prior art, the invention uses specific carbonyl reductase and a mutant thereof as catalytic active substances to prepare the key chiral alcohol compound (IM04) with high stereoselectivity, the chiral purity is more than 99.4 percent, and then the target product (S) -1- (4-chlorphenyl) -1, 3-propanediol is obtained by continuous reduction.

In order to solve the technical problems, the invention provides the following technical scheme:

an enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propanediol comprises the following synthetic route:

wherein R is₁，R₂Independently selected from C1-6 alkyl groups such as methyl, ethyl, propyl, butyl.

The carbonyl reductase has a sequence corresponding to SEQ ID NO:2, or an amino acid sequence corresponding to SEQ ID NO:2, one or a combination of two or more of the following mutations: the V mutation at position 45 is I (V45I), the K mutation at position 63 is Q (K63Q), the G mutation at position 141 is A (G141A), the G mutation at position 141 is V (G141V), the I mutation at position 195 is L (I195L), and the A mutation at position 204 is V (A204V).

SEQ ID NO:2, WO03, and NaKRED, a carbonyl reductase derived from Novosphingobium aromaticivorans, and CN102482648, the inventors unexpectedly found a mutant obtained by mutating a specific site of WO03 carbonyl reductase to a specific amino acid by screening an enzyme library, and when IM03 was prepared using IM02 as a substrate, the enzyme activity was high, the conversion rate was high, and the stereoselectivity was good.

Preferably, the carbonyl reductase mutant is a mutant that has a sequence corresponding to SEQ ID NO:2, one of the following mutations is present:

(i) the G mutation at position 141 is A (G141A), and the I mutation at position 195 is L (I195L), and the amino acid sequence is shown as SEQ ID No:4 is shown in the specification;

(ii) the G mutation at position 141 is V (G141V), and the I mutation at position 195 is L (I195L), and the amino acid sequence is shown as SEQ ID No: and 6.

(iii) The V mutation at position 45 is I (V45I); the G mutation at the 141 position is V (G141V, I195L), the I mutation at the 195 position is L (I195L), and the amino acid sequence is shown as SEQ ID No: 8 is shown in the specification;

The carbonyl reductase mutants also include the following ranges: the amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids of the amino acid sequence shown in SEQ ID No. 4, 6, 8, 10 or 12 in the range of keeping the enzyme activity; or

The carbonyl reductase is prepared by constructing alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase for realizing coenzyme regeneration and a target gene on the same plasmid pET28a (+) vector, then introducing the vector into an expression host escherichia coli, and obtaining thalli containing the target enzyme through induction expression. The bacteria can be obtained directly by centrifugation, and crude enzyme solution or crude enzyme powder can be obtained by breaking the walls of the bacteria.

Further, the invention provides an enzymatic production method of (S) -1- (4-chlorphenyl) -1, 3-propanediol, which comprises the following steps:

(S1) reacting m-chlorobenzoic acid with an alcohol R₁Performing OH esterification reaction to obtain m-chlorobenzoic acid alkyl ester IM 01;

(S2) alkyl m-chlorobenzoate (IM01) with alkyl acetate CH under the action of organic base₃COOR₂Condensation to obtain a product, namely ketoester IM 02;

(S3) preparing a product IM03 under the catalysis of carbonyl reductase by taking ketoester IM02 as a substrate of an enzyme-catalyzed reaction;

(S4) hydrolyzing the IM03 to obtain IM 04;

(S5) reduction of IM04 gave the final product GL01, (S) -1- (4-chlorophenyl) -1, 3-propanediol.

Preferably, the esterification reaction conditions in step (S1) are well known in the art, such as in the presence of an acid catalyst, and the optional acid includes concentrated sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, phosphotungstic acid, cation exchange resin, and the like. The reaction temperature is 60-100 ℃, and the reaction is carried out for 15-20h under the condition of heating reflux. M-chlorobenzoic acid and R₁The OH proportion is not particularly restricted but is generally chosen from the alcohols R₁The OH is excessive, and the alcohol is used as a reactant and a solvent of the reaction system. In one embodiment of the invention, m-chlorobenzoic acid and R₁The molar ratio of OH is 0.3-0.6: 1.

the post-treatment after the esterification reaction in the step (S1) is well known in the art, and after the general reaction is finished, the excess alcohol is distilled off, the low-polarity solvent (n-heptane, n-hexane, petroleum ether) is added, and then the mixture is extracted and washed with water, weak base and salt solution respectively, and the product IM01 is obtained by concentrating the low-polarity solvent phase, wherein the yield is 88-93%.

Preferably, the organic base in step (S2) is selected from potassium and/or sodium salts of organic alcohols, such as at least one of potassium tert-amylate, sodium tert-amylate, potassium tert-amylate, sodium tert-butylate, sodium methoxide, potassium methoxide, sodium ethoxide, and potassium ethoxide. IM01, ester CH₃COOR₂And an organic base in a molar ratio of 1: 1.1-1.7: 1.5-2.5, preferably 1: 1.3-1.5: 1.8-2.2.

And (S2) reacting at-5 deg.C for 1-3h in THF, acetone, DMSO, DMF under water content of less than 1000 ppm. After the reaction is finished, acid is added for quenching reaction, and then post-treatment is well known in the art, and specifically, after the acid is added for quenching reaction, standing for layering, concentrating an upper layer solvent, extracting a lower layer water phase with the solvent, combining with an upper layer concentrated solution, washing with water, weak base and salt respectively and sequentially, and concentrating to obtain a product IM02, wherein the yield is 85-90%.

Preferably, the step (S3) is that the ketonic ester IM02 is used as a substrate of the enzyme-catalyzed reaction, and the concentration is 50-200 g/L; more preferably 100 to 150 g/L.

Further, the carbonyl reductase is at least one of an enzyme in a free form, an immobilized enzyme, or an enzyme in a form of bacterial cells. The dosage of the carbonyl reductase is 1 to 6 weight percent of the substrate in the reaction system. In one embodiment of the present invention, a carbonyl reductase is used in a wet form, and the mass ratio of the wet form to the substrate is 30 to 100 wt%, preferably 50 to 70 wt%.

Further, in the reaction system, a co-substrate is also present, and the co-substrate is selected from the group consisting of: at least one of isopropanol, glucose, and ammonium formate; preferably, the concentration of the co-substrate is 100-200% of the substrate concentration, preferably the concentration of the co-substrate is 140-170% of the substrate concentration.

Further, the reaction process of the step (S3) is carried out in a phosphate buffered salt system, pH is 6-8, preferably 6.5-7.5, more preferably 6.8-7.1; and/or the reaction temperature is 20 ℃ to 50 ℃, preferably 25 ℃ to 40 ℃, more preferably 30 ℃ to 35 ℃; and/or the reaction time is 3 to 20 hours, more preferably 3 to 10 hours.

Further, the coenzyme refers to a coenzyme capable of realizing electron transfer in a redox reaction; comprises at least one of reduced coenzyme and oxidized coenzyme; the reduced coenzyme is selected from NADH, NADPH, or a combination thereof; the oxidized coenzyme is selected from NAD⁺、NADP⁺Or a combination thereof; further, the ratio of the amount of the coenzyme to the amount of the substrate is 0.01 to 1.0 wt%, preferably 0.1 to 0.5 wt%.

Further, the carbonyl reductase and/or the enzyme for coenzyme regeneration are/is constructed on an expression vector.

Further, in the reaction system, an enzyme for coenzyme regeneration, specifically selected from alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof, is also present.

Preferably, the hydrolysis in step (S4) is carried out in the presence of a base, which is not particularly limited, and is generally an inorganic base such as sodium hydroxide and/or potassium hydroxide, and the amount of the base is not particularly limited, and is sufficient to completely hydrolyze, and is generally adjusted to a pH of 12 to 14. The post-treatment operation after hydrolysis is well known in the art, and specifically comprises adjusting the pH to 7-9 with an acid, adding an organic solvent for extraction to wash off organic impurities, adjusting the pH to 1-2, standing for layering, extracting with an organic solvent, washing with water, drying, filtering, and concentrating to obtain an oily substance. Redissolving the oily substance by ethyl acetate, heating to 35-50 ℃, dropwise adding a low-polarity organic solvent, cooling to-5 ℃, separating out a large amount of solid, filtering, washing and drying to obtain a needle-shaped solid which is the product IM 04. The yield of the step (S4) is 70-80%, and the purity of the product is more than 99%.

Preferably, in the step (S5), adding a reducing agent in batches, wherein the reducing agent is sodium borohydride and/or potassium borohydride, the amount of the reducing agent is 5-10% of the mass of IM04, the reaction temperature is-5 ℃ to 5 ℃, the adding time is 1-2 hours, after the reducing agent is added, maintaining the low temperature, continuing to react for 10-30min, then slowly dropwise adding boron trifluoride diethyl etherate, the amount of the boron trifluoride diethyl etherate is 1-2 times of the IM04, after dropwise adding is completed within 1-3 hours, maintaining the low temperature, reacting for 1-3 hours, then heating to 20-30 ℃, reacting for 3-5 hours, then cooling to 10-20 ℃, dropwise adding methanol, continuing to react for 1-2 hours after dropwise adding is completed within 1-2 hours, and then performing a post-treatment step to obtain the product GL 01. Said post-treatment is well known in the art, and in one embodiment of the present invention, the post-treatment is after the reaction is finished, the system is adjusted to be weakly alkaline, after extraction with an organic solvent, washing and concentration under reduced pressure are carried out to obtain a light yellow viscous liquid, which is the final product GL01, namely (S) -1- (4-chlorophenyl) -1, 3-propanediol.

The invention screens carbonyl reductase to obtain wild carbonyl reductase WO03 and mutants thereof, and finds that the wild carbonyl reductase WO03 has good catalytic activity for chiral reduction of a key medical intermediate (S) -1- (4-chlorphenyl) -1, 3-propanediol, high enzyme activity, good stereoselectivity, chiral purity of more than 99.6 percent and the preferred embodiment can reach more than 99.8 percent. The method does not need harsh low-temperature conditions, and is beneficial to large-scale industrial production.

Drawings

FIG. 1 is a chiral map of the product IM03 obtained in the step (S3) of example 2;

FIG. 2 is a diagram showing the IM03 product obtained in the step (S3) of example 2¹H NMR spectrum;

FIG. 3 shows the result of the step (S5) in example 2Of product GL01¹H NMR spectrum;

FIG. 4 is a schematic representation of GL01 as a product of step (S5) in example 2¹³A C NMR spectrum;

FIG. 5 is a HPLC chemical purity map of the product GL01 obtained in the step (S5) of example 2;

FIG. 6 is an HPLC chiral purity map of the product GL01 obtained in the step (S5) of example 2.

Detailed Description

Preparation example

Preparation exampleConstruction of carbonyl reductase engineering bacteria and carbonyl reductase homologous mutation library

Cloning 5 carbonyl reductase genes from WO03 carbonyl reductase gene and mutant genes thereof into pET28a (+) vector, then introducing into host escherichia coli BL21, and obtaining the recombinant gene engineering bacteria of carbonyl reductase through induction expression.

Using wild carbonyl reductase WO03 (the nucleic acid sequence is shown as SEQ NO.1 and the amino acid sequence is shown as SEQ NO. 2) as template, and using random point mutation kit (II Site-Directed Mutagenesis Kit) or by mutating the wild-type carbonyl reductase gene by Directed evolution to obtain a plasmid library comprising the evolved carbonyl reductase gene. The constructed plasmid library was transferred into E.coli BL21(DE3) (cat # kang century CW0809S) and cultured overnight in an LB solid medium containing 50. mu.g/mL kanamycin in an oven at 37 ℃. Single colonies were picked into 96-well plates containing 400. mu.L of LB liquid medium (containing 50. mu.g/mL kanamycin), and cultured overnight at 37 ℃ and 200rpm to obtain seed solutions. Then 10. mu.L of the seed solution was transferred to a 96-well plate containing 400. mu.L of a fermentation medium (containing 50. mu.g/mL kanamycin), and cultured at 37 ℃ to OD600 value>0.8. Then isopropyl thiogalactoside (IPTG) with the final concentration of 1mM is added, the temperature is reduced to 28 ℃ to induce the expression of the mutant, and the culture is continued for 20 h. After the fermentation was completed, the cells were collected by centrifugation at 4000g for 30min, and 200. mu.L of lysis buffer (0.1M phosphate buffer containing 1000U of lysozyme, pH 7.0) was added againThe cells were suspended and lysed at 30 ℃ for 1 h. After the cleavage, the cells were centrifuged at 4000g at 4 ℃ for 30min, and the clarified supernatant was used to determine the mutant activity. mu.L of the reaction solution (containing 0.4mM substrate, 1mM NADH, 40. mu.L dimethyl sulfoxide) was added to a new 96-well plate, 10. mu.L of the supernatant was added, the change of NADH was detected at 340nm, the consumption of NADH reflected the level of the enzyme activity of the mutant, and carbonyl reductase mutants as shown in Table 1 below were obtained by screening.

TABLE 1

Example 1

Screening the carbonyl reductase by using the compound IV as a substrate, wherein the screening method and the screening result are as follows;

the detection method of the reaction conversion rate comprises the following steps: phenomenex Gemini C184.6 x 250mm 5 μm); the mobile phase A is 10% acetonitrile, the mobile phase B is acetonitrile, and gradient elution is carried out according to the following table; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 220 nm.

The chiral monitoring method of the compound V is as follows: a chromatographic column: xylonite IB-3,5 μm,4.6 x 250 mm; mobile phase: isopropanol to n-hexane 10: 90; the flow rate is 1.0 mL/min; operating time: 20 min; the column temperature is 30 ℃; detection wavelength: 220 nm. S configuration is 10min, and R configuration is 13 min.

The results are shown in table 2 below:

TABLE 2

Enzyme numbering	Conversion rate	e.e. value	Configuration(s)
				WO03(SEQ ID No.2)^[a]	98.9％	99.80％	S
LSADH^[b]	21.3％	93.78％	R
				LK^[b]	12.4％	38.52％	S

Note: carbonyl reductase LSADH is derived from Leifsonia sp.strain S749, accession No. AB213459, carbonyl reductase LK is derived from Lactobacillus kefir, reference WO 2010/025238.

Reaction conditions (a)1g of the IV compound, 0.2g of wet cells, 0.001g of NADP +,0.1g of wet GDH cells, 1.5g of glucose and 10ml of phosphate buffer (100mM, pH 7.0) were subjected to shaking reaction at 30 ℃ and 220rpm for 24 hours; (b)1g of the IV compound, 0.2g of wet cells, 0.001g of NAD +, 20% isopropanol and 10ml of phosphate buffer (100mM, pH 7.0) were subjected to shake reaction at 30 ℃ and 220rpm for 24 hours.

It can be seen that the carbonyl reductase has the conversion rate and stereoselectivity of WO03 (the amino acid sequence is shown as SEQ ID No:2, and the coding gene is shown as SEQ ID No: 1), which can not meet the requirements of industrial production, and is not suitable for large-scale production of the key intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol, but the enzymatic activity of the carbonyl reductase WO03 is not high enough, so that the carbonyl reductase of WO03 is taken as the basis, and the carbonyl reductase mutant with excellent enzymatic activity, conversion rate and stereoselectivity is screened out.

Inoculating the recombinant genetic engineering bacteria preserved in glycerol in preparation example 1 into LB liquid culture medium containing 100ug/ml ammonia-jasmine mycin, culturing at 37℃ and 220rpm for 12-16h to obtain seed culture medium, inoculating the seed culture medium into 100ug/ml ammonia-jasmine resistant liquid culture medium at 1.5%, and culturing at 37 deg.C and 220rpm to OD₆₀₀Value of>2.0, adding lactose with the final concentration of 1.0%, cooling to 25 ℃, continuing to culture for 2 hours, adding lactose with the final concentration of 0.5%, culturing for 20 hours, canning, and centrifuging to obtain thalli which are used as a catalyst for biotransformation. Following the reaction conditions (a) of example 1, namely: compound V was prepared by shaking reaction of 0.1g of IV compound, 0.2g of wet cells, 0.001g of NADP +,0.1g of wet cells of GDH, 0.2g of glucose and 10ml of phosphate buffer (100mM, pH 7.0) at 30 ℃ and 220rpm for 24 hours. The obtained carbonyl reductase homologous mutation library was screened, and the results are shown in the following table 3:

TABLE 3

Definition of enzyme Activity: reacting in a 50ml centrifugal tube, weighing 0.5g of substrate, adding 1.5ml of isopropanol, adding 3.5ml of phosphate buffer solution with pH value of 6.0-6.5, weighing 0.025g of coenzyme NAD, preheating the system in a water bath kettle at 30 ℃ for 15min, weighing 0.5g of thallus, adding the thallus into the reaction system, putting the system in a thermostatic chamber at 30 ℃ and shaking at 150rpm, and starting timing for 1 h. After completion, 0.1ml of the reaction solution was quickly removed by a pipette gun while shaking it up, and the mixture was put into a 10ml centrifuge tube, followed by addition of 4.9ml of isopropyl alcohol and thorough mixing by a pipette gun. Centrifuge for 10min at 3500 rpm. And pouring the supernatant into a sampling tube, and detecting the conversion rate to obtain the enzyme activity.

Due to codon degeneracy, the nucleic acid sequence of the carbonyl reductase mutants described above is not limited to the nucleic acid sequences listed in Table 1. The homologues of this base sequence may be obtained by those skilled in the art by appropriately introducing substitutions, deletions, alterations, insertions or additions, and the present invention encompasses these homologues as long as the recombinant enzyme expressed therefrom retains catalytic reduction activity for the compound of IM 02. The homologue of the polynucleotide of the present invention may be prepared by substituting, deleting or adding one or more bases of the base sequence within a range in which the enzyme activity is maintained.

The invention provides a carbonyl reductase mutant with an amino acid sequence shown as SEQ ID No. 4, 6, 8, 10 or 12, which has obviously improved enzyme activity compared with wild enzyme WO03, can reduce the dosage of the enzyme, and has stereoselectivity to S configuration products of more than 99%. Therefore, the compound IM02 can be effectively reduced into the chiral alcohol compound IM03 with S configuration by virtue of WO03 carbonyl reductase and a mutant thereof, the yield, the stereoselectivity and the utilization efficiency of enzyme are all satisfactory, and the preparation of the key intermediate (S) -1- (4-chlorphenyl) -1, 3-propanediol by virtue of the catalytic activity of a specific enzyme is expected.

Example 2

(S1)：

Putting 1.5kg of SM1 into a 20L reaction kettle, adding 8.3kg of absolute ethanol, then adding sulfuric acid, heating to 80 ℃, keeping the temperature for 18h, carrying out HPLC detection until 95% of product is obtained, starting to concentrate the reaction solution, and distilling off the ethanol. Adding 4L of n-heptane into the concentrate, then adding 4L of water for washing, washing with 4L of saturated sodium bicarbonate aqueous solution after water washing, then washing with 4L of saturated saline solution, and concentrating n-heptane to obtain IM011.6 kg.

(S2)：

THF (7.6 kg) (water content < 1000ppm) was added to a 20L reactor, and potassium tert-butoxide (99.5%) 1.9kg was added at-5 ℃ and stirred until clear. Then 1.6kg of IM01 was added, 0.99kg of anhydrous EA was added dropwise at a temperature below 0 deg.C, and after 90min, the mixture was incubated at 5 deg.C for 2h and monitored by HPLC. Feed < 1% quenched by addition of 3N HCl. Followed by static stratification. The upper layer was concentrated in THF, the lower layer was extracted once with 2L EA and combined with the upper concentrate. Then diluted with EA. After washing with water, saturated sodium carbonate and saturated NaCl, IM021.67kg is obtained after concentration.

(S3)：

IM021.67kg was added to 1.6L of 0.1mol/LPBS buffer solution, 2.2kg of isopropyl alcohol and 0.53kg of WO03 wet cells were added, followed by 3.2g of NAD⁺Reacting in 35 deg.C water bath under stirring, measuring pH value of the system at intervals (0.5-1 hr) during reaction, adjusting pH value to 6.9-7.2 with saturated sodium carbonate, reacting for 15 hr, and monitoring by HPLC for the remainder of raw materials<0.5 percent of the reaction is stopped, isopropanol is concentrated, the mixture is heated to 70 ℃, 500g of diatomite is added and filtered to obtain filtrate, ethyl acetate and ethyl acetate extract an aqueous layer twice (1x2L), an organic layer is combined, saturated sodium chloride is washed by water, anhydrous sodium sulfate is dried, and the mixture is filtered and concentrated to obtain a light yellow oily product IM031.47kg, the ee value is 99.9 percent, the chiral spectrum and the chiral value are¹HNMR maps are shown in FIG. 1 and FIG. 2, respectively.¹HNMR and¹³the specific data of C NMR are: (600MHz, Chloroform-d) δ 7.39(s,1H),7.26(dq, J ═ 15.9,7.7Hz,3H), 5.24-4.94 (m,1H),4.18(q, J ═ 7.1Hz,2H),3.52(s,1H), 2.82-2.52 (m,2H), J ═ 7.2Hz,3H).¹³C NMR(151MHz,Chloroform-d)δ172.20,144.60,134.44,129.82,125.95,123.81,69.65,61.04,43.19,14.13.

(S4)：

Adding IM03(1.47kg) into a 5L four-neck bottle, cooling to 10-15 deg.C, adding alkaline water (300g sodium hydroxide dissolved in 3L water) dropwise, controlling pH to 13-14 after 2h, reacting for 3h, and detecting by HPLC<0.5%, adding concentrated hydrochloric acid to adjust pH to 8-9, adding ethyl acetate 1L to extract the water layer to wash off organic impurities, adjusting pH to 1 with concentrated hydrochloric acid, layering, extracting the water layer with ethyl acetate (1.5L × 2), combining the organic layers, washing with saturated saline (1.5L), drying with anhydrous sodium sulfate (200g), filtering, and concentrating to obtain viscous oily substance (inclusion solid). Adding ethyl acetate (900ml) into the oily substance, heating to 40 ℃, dissolving, dropwise adding 3.6L petroleum ether, cooling to 0 ℃, separating out a large amount of solid, preserving heat for 3 hours, filtering, washing with cold ethyl acetate/petroleum ether (1:4), drying to obtain needle-shaped solid IM04, 995g, the purity is 99.7%, and recovering 85.5g of mother liquor. [ alpha ] to]_D25＝-17.3°(c 1.00,MeOH).ESI-MS(m/z)：199.0[M-H]-.¹H NMR(400MHz,Chloroform-d)δ7.40(q,J＝1.4,0.9Hz,1H),7.35–7.22(m,3H),5.14(dd,J＝8.4,4.4Hz,1H),2.88–2.72(m,2H).¹³C NMR(101MHz,CDCl₃)δ176.66,144.08,134.62,129.97,128.18,125.95,123.79,69.52,42.83。

(S5)：

Adding IM 0420 g and tetrahydrofuran 160 g into a four-mouth reaction bottle under the protection of nitrogen, stirring, cooling to-5 ℃, controlling the temperature to-5 ℃ to 0 ℃, adding sodium borohydride 6.8 g in batches, controlling the time to be used for 1-1.5 hours, wherein in the batch adding process, the obvious heat release phenomenon exists, paying attention to the temperature change, simultaneously controlling the temperature of the sodium borohydride to be lower than 0 ℃ easily, reacting for 20 minutes under the condition that the temperature is controlled to be-5-0 ℃, dropwise adding boron trifluoride diethyl etherate 34 g in about 2.5 hours, and after finishing dropping, controlling the temperature to be-3- °After the reaction is carried out for 1 hour at the temperature of 0 ℃, the temperature is increased to 5 ℃ and the reaction is carried out for 1 hour at the temperature of 25 ℃, the reaction is carried out for 4 hours at the temperature of 25 ℃, the temperature is reduced to 15-20 ℃, 24 g of methanol is dripped, the time is 1-1.5 hours, the heat release phenomenon is obvious in the dripping process, the temperature control needs to be carried out, the bubbling phenomenon of reaction liquid is observed, and the temperature control reaction is carried out for 1 hour. Concentrating under reduced pressure below 30 deg.C to dry, adding water 250, stirring for dissolving, controlling the temperature to 10-12 deg.C, dropping 10% sodium hydroxide solution 50g, regulating pH value to 8.5-9.0, using about 1.5, stirring for 40 min, repeatedly measuring pH value, controlling the temperature to 12-15 deg.C, adding dichloromethane 150g, stirring for 40 min, standing for 40 min to separate lower organic phase, extracting twice with 60 g and 40 g, mixing organic phases, adding saturated salt solution 50g, regulating pH value to neutrality with 6N hydrochloric acid, stirring for 1 hr, standing for 40 min, layering, concentrating under reduced pressure below 30 deg.C until no liquid is discharged, and concentrating under reduced pressure with oil pump to obtain light yellow viscous liquid 15.5g, with HPLC chemical purity of 99.4% and ee value of 99.8%. HRMS m/z calcd for C₉H₁₁ClNaO₂：209.0340,found 209.0347[M+Na]₊.¹H NMR(600MHz,Chloroform-d)δ7.38(d,J＝2.1Hz,1H),7.33–7.18(m,3H),4.94(dd,J＝8.6,3.8Hz,1H),3.86(dt,J＝7.2,4.2Hz,2H),3.21(s,1H),1.95(dddd,J＝20.3,16.1,8.7,4.7Hz,2H).¹³C NMR(151MHz,Chloroform-d)δ146.43,134.42,129.79,127.62,125.89,123.79,73.66,61.36,40.33。

Sequence listing

<110> Shandong atlas enzyme biopharmaceutical Co., Ltd

<120> enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 780

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60

cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120

ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180

ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240

cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300

cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360

ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420

ggcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480

atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540

tccgtgcatc cgggcggcat cgataccccg atgctcggct cgatcatgga caagtacgtc 600

gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660

atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720

gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780

<210> 2

<211> 259

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala

1 5 10 15

Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala

20 25 30

Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala

35 40 45

Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala

50 55 60

Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val

65 70 75 80

His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu

85 90 95

Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile

100 105 110

Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg

115 120 125

Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg

130 135 140

Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys

145 150 155 160

Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn

165 170 175

Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu

180 185 190

Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg

195 200 205

Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met

210 215 220

Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp

225 230 235 240

Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe

245 250 255

Ser Gln Val

<210> 3

<211> 780

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60

cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120

ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180

ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240

cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300

cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360

ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420

gcgggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480

atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540

tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600

gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660

atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720

gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780

<210> 4

<211> 259

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala

1 5 10 15

Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala

20 25 30

Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala

35 40 45

Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala

50 55 60

Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val

65 70 75 80

His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu

85 90 95

Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile

100 105 110

Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg

115 120 125

Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Ala Gly Leu Arg

130 135 140

Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys

145 150 155 160

Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn

165 170 175

Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu

180 185 190

Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg

195 200 205

Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met

210 215 220

Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp

225 230 235 240

Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe

245 250 255

Ser Gln Val

<210> 5

<211> 780

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60

cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120

ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180

ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240

cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300

cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360

ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420

gtcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480

atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540

tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600

gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660

atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720

gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780

<210> 6

<211> 259

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala

1 5 10 15

Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala

20 25 30

Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala

35 40 45

Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala

50 55 60

Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val

65 70 75 80

His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu

85 90 95

Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile

100 105 110

Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg

115 120 125

Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Val Gly Leu Arg

130 135 140

Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys

145 150 155 160

Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn

165 170 175

Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu

180 185 190

Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg

195 200 205

Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met

210 215 220

Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp

225 230 235 240

Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe

245 250 255

Ser Gln Val

<210> 7

<211> 780

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60

cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120

ccctcggccg atatcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180