CN115992107B - A ketoreductase and its application - Google Patents
A ketoreductase and its applicationInfo
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- CN115992107B CN115992107B CN202310148579.5A CN202310148579A CN115992107B CN 115992107 B CN115992107 B CN 115992107B CN 202310148579 A CN202310148579 A CN 202310148579A CN 115992107 B CN115992107 B CN 115992107B
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Abstract
本发明公开了一种酮还原酶。本发明还公开了其在体外生物合成(R)‑6‑羟基‑8‑氯辛酸乙酯中的应用。其作为生物催化剂，将8‑氯‑6‑氧代辛酸乙酯转化为(R)‑6‑羟基‑8‑氯辛酸乙酯，有极佳的手性选择性，将相应手性带入合成路线，极大降低了生产成本，消除了副产物处理过程中产生的环境影响，且操作简单，条件温和，耗时较短，转化率和产物纯度均在87%以上，适合产业化应用。
The present invention discloses a ketoreductase. The present invention also discloses its application in the in vitro biosynthesis of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester. As a biocatalyst, it converts 8-chloro-6-oxooctanoic acid ethyl ester into (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester with excellent chiral selectivity, bringing the corresponding chirality into the synthesis route, greatly reducing production costs, eliminating the environmental impact generated during the by-product treatment process, and is simple to operate, mild in conditions, and short in time. The conversion rate and product purity are both above 87%, making it suitable for industrial application.
Description
Technical Field
The invention relates to the field of biochemical engineering, in particular to ketoreductase and application thereof in-vitro biosynthesis of (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester.
Background
(R) -6-hydroxy-8-chlorooctanoic acid ethyl ester is a chiral intermediate of (R) -alpha-lipoic acid. Lipoic acid (LA, commonly referred to as α -lipoic acid) is a white or pale yellow crystal insoluble in water and widely distributed in biological tissues such as animals and plants. Alpha-lipoic acid is a safe and effective powerful antioxidant and is known as a universal antioxidant. Lipoic acid has the effects of relieving body overfatigue, delaying aging, preventing memory deterioration and the like. Lipoic acid has two enantiomers, of which (R) - α -lipoic acid is much more effective than (S) - α -lipoic acid, which belongs to the vitamin B class of drugs, acts as a coenzyme in the multienzyme complex, and exhibits a critical role in tricarboxylic acid cycle and photosynthesis. Clinically, (R) -alpha-lipoic acid is increasingly used for treating diseases such as nervous diabetes, ischemia reperfusion and the like, and in addition, (R) -alpha-lipoic acid has curative effects on various diseases and can effectively inhibit the diffusion of HIV-1 in cells.
Along with the increasing application of lipoic acid in medicines, foods and health-care products, the annual demand of global lipoic acid is continuously increased, and the global lipoic acid is increased at a speed of about 10% at present, but the yield of the lipoic acid is far from the demand, and the huge supply gap enables the lipoic acid to have important market status in domestic and foreign markets.
At present, lipoic acid is mainly produced by chemical synthesis and is divided into two methods, namely an adipic acid method and a cyclohexanone method according to different raw materials. However, the steps are complicated, the process is complex, chemical raw materials are adopted, and a large amount of toxic catalysts are used in the synthesis process, so that the safety of the product is severely questioned, and the environment is severely polluted. In addition, the chemically synthesized lipoic acid is a mixture formed by equal amounts of (R) -alpha-lipoic acid and (S) -alpha-lipoic acid, the (R) -alpha-lipoic acid with biological activity can be obtained by further resolution, and the price of the racemic lipoic acid and the price of the (R) -alpha-lipoic acid are greatly different.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and the first technical problem to be solved by the invention is to provide ketoreductase.
The invention also solves the technical problem of providing an application of the ketoreductase in-vitro biosynthesis of (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester and a specific preparation method.
The invention provides a ketoreductase, wherein the amino acid sequence of the ketoreductase is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
The ketoreductase with the amino acid sequence of SEQ ID NO.1 is named SCR-1, the ketoreductase with the amino acid sequence of SEQ ID NO.2 is named SCR-2, the ketoreductase with the amino acid sequence of SEQ ID NO.3 is named SCR-3, the ketoreductase with the amino acid sequence of SEQ ID NO.4 is named SCR-4, and the ketoreductase with the amino acid sequence of SEQ ID NO.5 is named SCR-5.
The invention also provides a gene or nucleic acid encoding the ketoreductase described above.
Further, the nucleotide sequence of the ketoreductase gene or the nucleic acid is selected from SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
The invention also provides application of the ketoreductase in-vitro biosynthesis of (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester.
The invention also provides an in-vitro biosynthesis method of the (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester, which comprises the following steps of:
(1) Obtaining the above-mentioned ketoreductase gene;
(2) Constructing a recombinant vector containing the ketoreductase gene, transferring the recombinant vector into escherichia coli, and culturing to obtain recombinant escherichia coli bacterial liquid;
(3) Cracking the recombinant escherichia coli bacterial liquid to obtain crude enzyme liquid;
(4) Mixing 8-chloro-6-oxo-octanoic acid ethyl ester, phosphate buffer solution, crude enzyme solution and coenzyme factor, and obtaining (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester after the reaction.
Further, the cracking condition of the escherichia coli bacterial liquid in the step (2) is that the escherichia coli bacterial liquid is centrifuged for 30-60min at 20000-35000 g/min.
Further, the step (3) also comprises a purification treatment of the crude enzyme solution.
Further, the purification treatment of the crude enzyme solution comprises the steps of eluting the crude enzyme solution by imidazole solutions with different gradients after flowing through a Ni column, flowing the obtained Ni column eluent with the highest enzyme content through a Q column, eluting by salt solutions (mainly comprising NaCl) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution to obtain the purified enzyme solution.
Further, the step (3) further comprises preparing the enzyme solution after the purification treatment into an immobilized enzyme.
Further, the immobilized carrier in the process of preparing the immobilized enzyme can be selected from but not limited to amino resin and epoxy resin, and the pH value of the reaction system is 4-9 and the reaction time is 1-6h.
Further, the coenzyme factor in the step (4) is NADH or NADPH. Preferably NADPH, the mass ratio of the coenzyme factor to the ethyl 8-chloro-6-oxooctoate is 0.5-5:1, and the mass ratio of the crude enzyme solution to the ethyl 8-chloro-6-oxooctoate is 0.002-0.01:1.
Further, the reaction condition in the step (4) is 25-45 ℃, preferably 30 ℃, the reaction time is 2-12h, and the pH value of the reaction system is 5-9, preferably 6.5-7.5.
Further, the step (4) further comprises a purification step of adding an organic solvent of ethyl acetate, xylene or n-heptane into the (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester solution for extraction, drying the organic phase, and obtaining a white crystal product through concentration and recrystallization.
The reaction formula of the biocatalytic reaction is as follows:
Compared with the prior art, the ketoreductase disclosed by the invention has the remarkable advantages that the ketoreductase is used as a biocatalyst to convert the ethyl 8-chloro-6-oxooctoate into the ethyl (R) -6-hydroxy-8-chlorooctoate, has excellent chiral selectivity, brings corresponding chirality into a synthetic route, greatly reduces the production cost, eliminates the environmental influence generated in the by-product treatment process, has simple operation, mild condition and short time consumption, has the conversion rate and the product purity of more than 87 percent, and is suitable for industrial application.
Drawings
FIG. 1 is a high performance liquid chromatogram of ethyl 8-chloro-6-oxooctanoate.
FIG. 2 is a high performance liquid chromatogram of (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester.
FIG. 3 is a high performance liquid chromatogram during the preparation of (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester by the biocatalytic synthesis method of the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The vector pET28a, E.coli DE3 competent cells, and other biological materials used in the examples described below were all commercially available. The preparation method of the PBS buffer solution comprises the steps of weighing 40g of NaCl, 1g of KCl and 7.2g of Na 2HPO4、1.2g KH2PO4, dissolving in 800mL of distilled water, regulating the solution to 7.0 by using HCl, and finally adding distilled water to fix the volume to 1L to obtain the 50mM PBS buffer solution.
Example 1
The construction and cloning of S1 vector, namely, connecting a gene with a nucleotide sequence shown as SEQ ID NO.6 with a vector pET28a in a seamless cloning mode to obtain a recombinant vector pET28a-SCR-1, taking 10 mu l of the recombinant vector, adding 100 mu l of E.coli BL21 (DE 3) competent cells of ice bath, then carrying out ice bath for 30min, carrying out 42 ℃ heat shock for 60S, adding 300 mu l of 37 ℃ non-anti-LB culture solution into a tube, carrying out shaking table repair for 1h at 37 ℃ and 200rpm, then coating on a solid LB plate with kana resistance for cultivation at 37 ℃, picking single bacterial colony by high-pressure sterilization after bacterial colony growth, firstly carrying out line drawing and seed preservation on the LB plate with kana resistance, and carrying out corresponding marks on corresponding bacteria and line drawing areas on the plate;
S2, verifying that a 20 mu l PCR Mix system added with a T7 universal primer is uniformly stirred, and is subjected to PCR amplification under the conditions of 95 ℃ and 15min,94 ℃ denaturation and 15S,55 ℃ annealing and 15S and 72 ℃ extension for 1min, 30 cycles are performed, and finally, the temperature is kept for 5min, and electrophoresis observation results are performed after PCR amplification to obtain positive clones, namely the recombinant escherichia coli strain containing the target enzyme genes, namely pET28a-SCR-1 recombinant escherichia coli;
And S3, expressing and extracting the enzyme, namely picking pET28a-SCR-1 recombinant escherichia coli into a LB culture medium containing kana resistance, culturing OD to about 1.0 at 37 ℃, adding IPTG with a final concentration of 0.2mM, placing the cultured product at 28 ℃ for induction expression for 16 hours, centrifuging the bacterial liquid at 7000g/min for 6min, collecting bacterial cells, pouring out a supernatant culture medium, re-suspending the bacterial cells with 100mM PBS according to the ratio of the weight of the bacterial cells to PBS solution=1 g/5 ml, crushing the re-suspended bacterial cells by a high-pressure cell crusher to obtain enzyme-containing lysate, centrifuging the enzyme-containing lysate at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution of the target enzyme.
S4, purifying, namely enabling the supernatant containing the enzyme to flow through a Ni column, then eluting with imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, then eluting with salt solutions (the main component is NaCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12 hours to finally obtain a purified enzyme solution of the target enzyme;
s5 reaction, namely adding 2g of 8-chloro-6-oxo-octanoic acid ethyl ester substrate, 7.1g of NADPH and 5mL of 50mM PBS buffer solution into a 25mL glass reaction bottle, adding 5mL of ketoreductase solution (13.2 mg/mL) into the reaction bottle to start reaction, controlling the temperature of the reaction solution to be 30 ℃, controlling the pH value of the reaction solution to be 7.0, and uniformly stirring and then reacting for 9 hours.
After the reaction is finished, the reacted solution is adjusted to be alkaline (pH is more than 11), the mixture is extracted twice by using equal volume of ethyl acetate and the organic phases are combined, 10 mu l of the solution can be taken after passing through a filter membrane of 0.22 mu m, the conversion rate is more than 92 percent, the ee value is more than 99 percent by using high performance liquid chromatography detection, the organic phases are dried by using anhydrous magnesium sulfate, concentrated by rotary evaporation, and the product of 1.72g can be obtained after cooling and recrystallization, and the purity reaches 99.7 percent.
Example 2
The following reaction was carried out using the crude enzyme solution prepared in step S3 of example 1 as a reaction raw material.
In a 25mL glass reaction bottle, 2g of 8-chloro-6-oxo-octanoic acid ethyl ester substrate, 7.1g of NADPH and 5mL of 50mM PBS buffer solution are added, then 5mL of ketoreductase crude enzyme solution is added into the reaction bottle to start reaction, the temperature of the reaction solution is 30 ℃, the pH value of the reaction solution is controlled to be 7.0, and the reaction is carried out for 9 hours after uniform stirring.
After the reaction is completed, the reacted solution is adjusted to be alkaline (pH > 11), extracted twice by using equal volume of ethyl acetate, organic phases are combined, 10 mu l of the solution can be taken after passing through a filter membrane of 0.22 mu m, and the conversion rate is calculated to be more than 89% and the ee value is more than 99% by using high performance liquid chromatography detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.66g of product with the purity of 99.5 percent.
Example 3
The following reaction was carried out using the crude enzyme solution prepared in step S3 of example 1 as a reaction raw material.
2G of 8-chloro-6-oxo-octanoic acid ethyl ester substrate and 6.4g NADH,5ml 50mM PBS g of buffer solution are added into a 25mL glass reaction bottle, 5mL of ketoreductase crude enzyme solution is added into the reaction bottle to start reaction, the temperature of the reaction solution is 30 ℃, the pH value of the reaction solution is controlled to be 7.0, and the reaction is carried out for 9h after uniform stirring.
After the reaction is completed, the reacted solution is adjusted to be alkaline (pH > 11), extracted twice by using equal volume of ethyl acetate, organic phases are combined, 10 mu l of the solution can be taken after passing through a filter membrane of 0.22 mu m, and the conversion rate is calculated to be more than 81% and the ee value is more than 99% by using high performance liquid chromatography detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.54g of product with the purity of 99.2 percent.
Example 4
The following reaction was carried out using the crude enzyme solution prepared in step S3 of example 1 as a reaction raw material.
2G of 8-chloro-6-oxo-octanoic acid ethyl ester substrate and 7.1g NADPH,2ml 50mMPBS buffer solution are added into a 25mL glass reaction bottle, 8mL of ketoreductase crude enzyme solution is added into the reaction bottle to start reaction, the temperature of the reaction solution is 30 ℃, the pH value of the reaction solution is controlled to be 7.0, and the reaction is carried out for 9 hours after uniform stirring.
After the reaction is finished, the reacted solution is adjusted to be alkaline (pH is more than 11), the mixture is extracted twice by using equal volume of ethyl acetate and the organic phases are combined, 10 mu l of the solution can be taken after passing through a filter membrane of 0.22 mu m, the conversion rate is more than 93 percent, the ee value is more than 99 percent by using high performance liquid chromatography detection, the organic phases are dried by using anhydrous magnesium sulfate, concentrated by rotary evaporation, and the product of 1.69g can be obtained after cooling and recrystallization, and the purity reaches 99.7 percent.
Example 5
The following reaction was carried out using the crude enzyme solution prepared in step S3 of example 1 as a reaction raw material.
2G of substrate and 7.1g NADPH,2ml 50Mm PBS buffer solution are added into a 25mL glass reaction bottle, 8mL of ketoreductase crude enzyme solution is added into the reaction bottle to start reaction, the temperature of the reaction solution is 30 ℃, the pH value of the reaction solution is controlled to be 7.0, and the reaction is carried out for 6 hours after uniform stirring.
After the reaction is finished, the reacted solution is adjusted to be alkaline (pH is more than 11), the mixture is extracted twice by using equal volume of ethyl acetate and the organic phases are combined, 10 mu l of the solution can be taken after passing through a filter membrane of 0.22 mu m, the conversion rate is more than 92 percent, the ee value is more than 99 percent by using high performance liquid chromatography detection, the organic phases are dried by using anhydrous magnesium sulfate, concentrated by rotary evaporation, and the product with the purity of 99.7 percent can be obtained after cooling and recrystallization.
Example 6
In the step S3, the PBS buffer solution used for re-suspending the bacteria is replaced by triethanolamine buffer solution, and the rest preparation method is the same as in the example 1. The final reaction result is detected by high performance liquid chromatography to calculate the substrate conversion rate of more than 89% and the ee value of more than 99%. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.61g of product with the purity of 99.3 percent.
Example 7
In the step S1, the ketoreductase enzymes selected are respectively SCR-2, SCR-3, SCR-4, SCR-5, S2 and S3, the steps of the example 1 and the S3 are followed by the preparation method of the example 2, and the crude enzyme solution is used as a reaction raw material.
The final reaction results obtained were as follows:
| Ketoreductase enzymes | Conversion% | Ee value% | Product g | Purity% |
| SCR-2 | >95% | >99 | 1.79 | 99.4% |
| SCR-3 | >90% | >99 | 1.33 | 99.6% |
| SCR-4 | >87% | >99 | 1.22 | 99.3% |
| SCR-5 | >90% | >99 | 1.35 | 99.1% |
Example 8
The steps S1-S4 are the same as in example 1.
Immobilization of S5 enzyme by washing an amino resin with 50mM PBS buffer 3 times followed by filtration, preparing 2% glutaraldehyde solution with 50mM PBS solution, mixing an amino resin with 2% glutaraldehyde in a ratio of 2% glutaraldehyde=1:4 (mass/volume ratio), then stirring/shaking at 23℃for 1 hour, followed by filtration to obtain an amino resin, washing with 50mM PBS buffer 3 times followed by filtration, further mixing an S4 enzyme solution containing about 200mg ketoreductase with 5g of an amino resin, shaking at 23℃for 18 hours at 80rpm, and then collecting an amino resin by filtration (the filtrate can be used for measuring the immobilization rate of the enzyme), washing with 50mM PBS buffer 3 times followed by filtration to obtain an immobilized ketoreductase.
S6, filling the immobilized enzyme, namely filling the immobilized enzyme into a column reactor to obtain the column reactor containing the immobilized ketoreductase.
Preparation of S7 reaction solution 20g of ethyl 8-chloro-6-oxooctanoate substrate and 75g of NADPH were dissolved in 100ml of 50mM PBS buffer to prepare a reaction solution, and the pH of the reaction solution was controlled to 7.0.
And S8, allowing the reaction solution to flow through a column reactor containing immobilized ketoreductase, controlling the temperature at 30 ℃ in the reaction process, controlling the flowing time (namely the reaction time) to be 4 hours, and collecting the liquid flowing out of the reactor to obtain the solution containing (R) -6-hydroxy-8-chloroethyl octanoate.
The collected solution is diluted 10 times and then passes through a filter membrane with the thickness of 0.22 mu m, 10 mu L of supernatant is taken and detected by high performance liquid chromatography, and the substrate conversion rate is calculated to be more than 82% through detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.55g of product with the purity of 99.5 percent.
Example 9
The column reactor in the step S6 was replaced with a tank reactor to obtain a tank reactor containing immobilized ketoreductase, and the reaction was stirred at 100rpm for 4 hours, and the other steps were the same as in example 8.
The collected solution is diluted 10 times and then passes through a filter membrane with the thickness of 0.22 mu m, 10 mu L of supernatant is taken and detected by high performance liquid chromatography, and the substrate conversion rate is calculated to be more than 87% through detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.61g of product with the purity of 99.4 percent.
Example 10
The rest of the procedure is as in example 8, S5 as follows:
Immobilization of S5 enzyme by washing epoxy resin with 50mM PBS buffer solution for 3 times, filtering, mixing enzyme solution prepared by S4 containing about 200mg ketoreductase with 7g epoxy resin, shaking at 23 deg.C and 200rpm for 18 hours, standing for 20 hours, filtering, collecting epoxy resin (filtrate can be used for measuring immobilization rate of enzyme), washing with 50mM PBS buffer solution for 3 times, and filtering to obtain immobilized ketoreductase.
Diluting the collected solution 10 times, filtering with 0.22 μm filter membrane, collecting 10 μl supernatant, detecting with high performance liquid chromatography, and calculating substrate conversion rate >90% by detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.71g of product with the purity of 99.5 percent.
Example 11
The column reactor in the step S6 was replaced with a tank reactor to obtain a tank reactor containing immobilized ketoreductase, and the reaction was stirred at 100rpm for 4 hours, and the other steps were the same as in example 10.
The collected solution is diluted 10 times and then passes through a filter membrane with the thickness of 0.22 mu m, 10 mu L of supernatant is taken and detected by high performance liquid chromatography, and the substrate conversion rate is calculated to be more than 94% through detection. The organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, cooled and recrystallized to obtain 1.76g of product with the purity reaching 99.3 percent.
Example 12
Steps 1-5 are the same as in example 10, the remaining steps are as follows:
The immobilized enzyme is filled into a tank reactor to obtain a tank reactor containing immobilized ketoreductase, 3L of reaction liquid is prepared, 600g of substrate is added during preparation, 2.3kg NADPH,3L 50mM PBS buffer solution is added, the pH value of the reaction liquid is controlled to 7.0, the reaction liquid is poured into the tank reactor and stirred at a rotating speed of 100rpm for reaction for 4 hours, the temperature of the tank reactor is maintained at 30 ℃ during the whole reaction process, the liquid flowing out of the reactor is collected to be the solution containing (R) -6-hydroxy-8-chloroethyl octanoate, and each 100ml of reaction liquid is used as a batch for carrying out the reaction and collecting, and detection is carried out after the whole collection is completed.
The collected solution is diluted 10 times and then passes through a filter membrane with the thickness of 0.22 mu m, 10 mu L of supernatant is taken and detected by high performance liquid chromatography, the substrate conversion rate of 1-3 batches is detected to be more than 85%, the substrate conversion rate of 4-15 batches is detected to be more than 80%, and the substrate conversion rate of 15-30 batches is detected to be more than 65%. All batches of reaction liquid are collected, then the organic phase is dried by anhydrous magnesium sulfate, concentrated by rotary evaporation, and the product 435.9g can be obtained after cooling and recrystallization, and the purity reaches 98.2%.
Example 13
In the step S1, the ketoreductase selected is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively, the rest steps are the same as in example 11, and the final reaction result is as follows:
the foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
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