CN117946987A - Ketoreductase and application thereof in synthesis of chiral lactone - Google Patents
Ketoreductase and application thereof in synthesis of chiral lactone Download PDFInfo
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- CN117946987A CN117946987A CN202410136548.2A CN202410136548A CN117946987A CN 117946987 A CN117946987 A CN 117946987A CN 202410136548 A CN202410136548 A CN 202410136548A CN 117946987 A CN117946987 A CN 117946987A
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- Prior art keywords
- ketoreductase
- oxo
- hbkr
- methyl ester
- decalactone
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- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 title claims abstract description 56
- 150000002596 lactones Chemical class 0.000 title claims abstract description 21
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- IMUCAHOFGPVTBF-UHFFFAOYSA-N methyl 5-oxodecanoate Chemical compound CCCCCC(=O)CCCC(=O)OC IMUCAHOFGPVTBF-UHFFFAOYSA-N 0.000 claims description 3
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to ketoreductase and application thereof in synthesizing chiral lactone. The present invention has found a ketoreductase HbKR derived from Hyphopichia burtoni by gene mining, which is capable of catalyzing 4-oxodecanoic acid and 5-oxodecanoic acid with excellent stereoselectivity to obtain (S) -gamma-decalactone and (S) -delta-decalactone, respectively, and is capable of producing chiral lactones by coupling catalysis with glucose dehydrogenase, and is also capable of performing reactions using an isopropanol cycle coenzyme system. The ketoreductase of the invention is a bifunctional enzyme with oxidation activity and reduction activity. According to the invention, through carrying out protein engineering directional design on HbKR, the catalytic property of the reduction reaction is greatly improved, and excellent stereoselectivity is maintained.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to ketoreductase and application thereof in synthesizing chiral lactone, and specifically relates to ketoreductase and a gene sequence thereof, recombinant expression plasmid and recombinant expression transformant containing the gene sequence, and application of ketoreductase and freeze-dried enzyme powder of glucose dehydrogenase in catalytic preparation of (S) -gamma-decalactone and (S) -delta-decalactone.
Background
Chiral aliphatic gamma-and delta-lactones and derivatives thereof are key chiral components of many natural products and drugs. For example, gamma-and delta-decalactones resulting from cyclization of gamma-and delta-hydroxy acids/esters are widely used fragrances and perfumes in the food industry. The chemical structure has different chiral configurations, so that the aromatic characteristics are different. These lactones find a wide variety of commercial uses in perfumery and perfumery, in particular delta-decalactone, a common perfumery compound, the (S) -enantiomer exhibiting a significantly more intense starchy taste than the (R) -enantiomer. Furthermore, gamma-decalactone is also very important in perfumery applications, with a global annual market size of hundreds of tons. (R) -gamma-decalactone has the aroma of coconut, fruit fat, while (S) -gamma-decalactone has the soft aroma of coconut, caramel, fat and fruit. Chiral lactones tend to have purer flavor profiles compared to racemic lactones. By adjusting the composition ratio of the (R) form and the (S) form, different flavor characteristics can be obtained. In apricot, peach, raspberry, strawberry, plum, mango, passion fruit, fruit juice, wine and wine, specific and characteristic enantiomer ratios of lactones have been reported. Interestingly, gamma-and delta-decalactones also possess a broad range of antimicrobial activity. For example, gamma-and delta-decalactones have been reported to inhibit the growth of certain filamentous fungi (Aspergillus niger, etc.), yeasts (Candida albicans, etc.), and bacteria (Staphylococcus aureus, etc.). The inhibitory activity of the lactone enantiomer was different. The (S) -gamma-decalactone has a longer lasting effect than (R) -gamma-decalactone, and can last for at least 24 hours.
The preparation method of chiral decalactone mainly comprises chemical method and biological method. Chemical methods mainly include noble metal and organometallic catalytic methods, such as Arai et al, which synthesize chiral aromatic lactones and diol compounds using ruthenium catalysts by changing reaction conditions; meninno et al developed an organometallic catalyst quinine for the synthesis of chiral ketoester compounds. Noble metal and organometallic catalysis, while effective methods for stereoselective synthesis of hydroxy esters, require harsh reaction conditions such as pressure, temperature, etc., and are not environmentally friendly. The use of biocatalytically synthesized optically active gamma-and delta-decalactones in the food and cosmetic industries is of increasing interest. Compared with chemical catalysis, the biocatalysis method has the advantages of high stereoselectivity, mild condition, low cost and the like. Because of the longer carbon chain, the longer carbonyl position, the stereoselective reduction of long chain alkyl gamma-/delta-keto acids/esters remains challenging. Biological processes mainly include whole-cell bioconversion and enzymatic processes, and in the early 60 s of the 20 th century, wild type s.cerevisiae was able to convert gamma-and delta-keto acids into optically pure hydroxy acids. However, cloning and expression of enzymes responsible for catalyzing reactions in Saccharomyces cerevisiae has not been successful. Whole cell bioconversion processes produce large amounts of fermentation byproducts and the product lactones are also toxic to yeast cells. In order to solve the problems of the above methods, the enzymatic synthesis of decalactone is an effective approach, and has been receiving more and more attention.
(1) As early as the 60 s of the 20 th century, the conversion of gamma-and delta-keto acids into optically pure hydroxy acids by wild-type Saccharomyces cerevisiae has been shown to give R-type chiral lactones at substrate concentrations of 6mM. (NATURE 1962,194,995-996)
(2) Oscar et al carried out kinetic resolution of racemic delta-hydroxy esters by lipase-catalyzed transesterification, combining the enzymatic kinetic resolution with ruthenium-catalyzed alcohol racemization. (JOURNALOF ORGANICCHEMISTRY,2002,67 (4): 1261-1265)
(3) In 2014, diaz-Rodriguez, alba et al prepared optically pure short chain gamma-and delta-hydroxy esters and lactones by a chemical-enzymatic cascade, with a product configuration of S, an ee of 67% and a substrate concentration of 50mM. (CHEMCATCHEM, 2014,6 (4): 977-980)
(4) In 2016, boratynski, F.et al synthesized enantiomerically enriched gamma-and delta-decalactones from the corresponding racemic primary-secondary 1, 4-and 1, 5-diols under catalytic oxidation by various alcohol dehydrogenases. (PLOS ONE,2016.11 (1))
(5) In 2017 ChaoZhang et al, from Serratiamarces, found a first carbonyl reductase SmCR, such as (R) -gamma-/delta-decalactones, which was capable of converting a linear long-chain gamma/delta-keto acid to the corresponding gamma-/delta-lactone, with an enantioselectivity of up to 99% (R). (CATALYSIS COMMUNICATION,2017, 102:35-39)
(6) In 2019, mengChen et al modified carbonyl reductase SmCR to obtain mutant SmCR v4, which can effectively and stereoselectively synthesize various gamma-and delta-lactones, wherein the product configuration is R, the ee value of gamma-decalactone is >99%, the ee value of delta-decalactone is 95%, the substrate concentration is up to 1M, and the space-time yield of (R) -gamma-decalactone is 1175g/L/d. (CHEMCATCHEM, 2019,11 (11): 2600-2606)
(7) 2021, TaoWang et al obtained carbonyl reductase variant SmCR M5 by directed evolution of SmCR v4 structure orientation. The mutant has improved specific activity to 16 detected substrates, and has stereoselectivity as high as 99% in asymmetric synthesis of 13 gamma-/delta-lactones. In particular, the enzyme activity of the model substrate 5-oxo capric acid is improved by 13.8 times, and the (R) -delta-decalactone is obtained with the stereoselectivity of 99 percent. (CHEMICALCOMMUNICATIONS, 2021.57 (81): p.10584-10587)
(8) 2021, ChunleiRen et al successfully found two carbonyl reductases in Saccharomyces cerevisiae, odCR and OdCR, which produced optically pure (R) -gamma and (R) -delta-lactones by intramolecular cyclization from asymmetric reduction of chiral hydroxy acids from gamma and delta-keto acids/esters. (CHINESEJOURNALOF CHEMICALENGINEERING, 2021.29:305-310)
(9) 2023, Chu et al isolated a novel olefin reductase (SsER) from Swingsiasamuensis, combined with carbonyl reductase SsCR, and synthesized optically pure tobacco lactones and whiskey lactones from α, β -unsaturated γ -ketoesters. (CHINESECHEMICALLETTERS, 2023:108896)
Although the whole cell and enzyme preparation methods reported above can be used for preparing chiral decalactone, most of researches have the stereoselectivity of R-decalactone, the chiral synthesis of R-decalactone has relatively more researches, and the current asymmetric enzyme synthesis of S-decalactone has lower selectivity, and the related researches of the high-selectivity enzyme synthesis of S-decalactone are also relatively less.
Disclosure of Invention
In order to solve the technical problems, the invention provides ketoreductase and application thereof in synthesizing chiral lactone. Aiming at the fact that the production modes of (S) -decalactone in the prior art are few, the invention provides an enzyme catalysis preparation method of (S) -decalactone, and solves the problem of providing a novel enzyme catalysis method capable of generating (S) -decalactone with high chiral purity quality.
The invention is realized by the following technical scheme:
A first object of the present invention is to provide the use of a ketoreductase enzyme in the preparation of an oxidizing agent or a reducing agent, said ketoreductase enzyme being a ketoreductase enzyme of the following (a) or (b);
(a) Ketoreductase with an amino acid sequence shown as SEQ ID No. 2;
(b) Ketoreductase derived from (a) having one or more amino acids substituted, deleted or added in the amino acid sequence of (a) and having ketoreductase activity.
Further, the nucleotide sequence of the gene encoding the ketoreductase in (a) is shown as SEQ ID No. 1.
Further, the application uses keto acid ester compounds or alcohol compounds as substrates;
The ketoacid ester compound is selected from one or more of 4-oxo octanoic acid, 4-oxo octanoic acid methyl ester, 5-oxo octanoic acid methyl ester, 4-oxo decanoic acid methyl ester, 5-oxo decanoic acid methyl ester, 4-oxo dodecanoic acid methyl ester, 5-oxo dodecanoic acid and 5-oxo dodecanoic acid methyl ester;
The alcohol compound is selected from one or more of R-phenethyl alcohol, S-phenethyl alcohol, n-propanol, isopropanol, 2-butanol, 3-methylcyclohexanol, 4-ethylcyclohexanol and 2, 3-butanediol.
The second object of the present invention is to provide a method for synthesizing chiral lactone, comprising the steps of: under the combined action of a coenzyme and a coenzyme regeneration system, a ketoester compound is used as a substrate, and the ketoreductase or an expression system containing the ketoreductase is used as a catalyst, so that the substrate is converted into chiral lactone through an asymmetric reduction reaction.
Further, the ketoreductase is used in an amount of 1g/L to 20g/L; the concentration of the ketoacid ester compound is 5mM-100mM; the temperature of the reaction is 25-30 ℃.
Further, the reaction system also comprises glucose dehydrogenase; the mass ratio of the ketoreductase to the glucose dehydrogenase is 1-2:1.
A third object of the present invention is to provide a ketoreductase mutant having an amino acid sequence shown in SEQ ID No.3 (HbKR V1 amino acid sequence) or SEQ ID No.4 (HbKR V2 amino acid sequence).
A fourth object of the present invention is to provide a gene encoding the ketoreductase mutant.
A fifth object of the present invention is to provide an expression vector carrying the gene.
A sixth object of the present invention is to provide a recombinant bacterium expressing the ketoreductase mutant.
The invention also provides a preparation method of the ketoreductase, which comprises the following steps: e.coli containing expression vector and alcohol dehydrogenase is inoculated into LB culture medium, cultured at 37 ℃ until OD 600 = 0.6-0.8, added with IPTG with final concentration of 0.2mM, the temperature is continuously controlled at 16 ℃, corresponding fermentation broth is obtained after fermentation culture is finished, and thalli are collected by centrifugation.
Further, the invention provides an application of a recombinant expression transformant of co-expression of ketoreductase and glucose dehydrogenase in catalyzing asymmetric reduction of 4-oxo-decanoic acid to prepare (S) -gamma-decalactone, which comprises the following steps: the recombinant expression transformant co-expressed by the ketoreductase and the glucose dehydrogenase is used as a catalyst to catalyze the asymmetric reduction of the 4-oxo-decanoic acid, and then a conventional chemical separation method is adopted to extract and refine the optically pure chiral (S) -gamma-decalactone generated by the reaction from the reaction liquid.
The dosage of the ketoreductase is 10g/L-20g/L, the concentration of the 4-oxo-decanoic acid is 5mM-10mM, and the reaction temperature is 25 ℃ to 30 ℃.
Further, the invention provides an application of a recombinant expression transformant of carbonyl reductase and glucose dehydrogenase co-expression in catalyzing asymmetric reduction of 5-oxo-decanoic acid to prepare (S) -delta-decalactone, which comprises the following steps: the recombinant expression transformant co-expressed by the ketoreductase and the glucose dehydrogenase is used as a catalyst to catalyze the asymmetric reduction of the 5-oxo-decanoic acid, and then a conventional chemical separation method is adopted to extract and refine the optical pure chiral (S) -delta-decalactone generated by the reaction from the reaction liquid.
The dosage of the ketoreductase is 1g/L-10g/L, the concentration of the 5-oxo-decanoic acid is 10mM-100mM, and the reaction temperature is 25 ℃ to 30 ℃.
Further, the invention provides an enzyme catalysis preparation method of (S) -decalactone, which comprises the following steps:
Under the combined action of a coenzyme and a coenzyme regeneration system, the substrates 4-oxo-decanoic acid and 5-oxo-decanoic acid are respectively converted into (S) -4-hydroxydecanoic acid and (S) -5-hydroxydecanoic acid by enzyme catalytic reduction reaction under the action of a catalyst containing carbonyl reductase, then the (S) -4-hydroxydecanoic acid and the (S) -5-hydroxydecanoic acid are respectively cyclized into (S) -gamma-decalactone and (S) -delta-decalactone by acidification and heating methods, and the ketoreductase has an amino acid sequence shown in SEQ ID No. 2.
The ketoreductase with the amino acid sequence shown as SEQ ID No.2 finally screened by a large number of researches can effectively convert carbonyl in a substrate into a chiral product, and the chiral purity can reach more than 99%. That is, the development of the present invention provides a new biocatalytic enzyme catalyst, so that a new enzyme catalytic synthesis process can be effectively provided, and the ketoreductase HbKR having the amino acid sequence shown in SEQ id No.2 of the present invention may be derived from Hyphopichiaburtoni, which also provides better ketoreductase selectivity for providing carbonyl conversion into highly chiral hydroxyl groups, and is beneficial to industrial production requirements. The invention modifies the selected ketoreductase by a mutation strategy of directional design, and the related mutation sites are H79,S82,P83,V84,L85,F86,E94,L95,P98,A99,I100,T123,S124,F126,A127,A128,V129,N145,R157,L158,R161,G162,S163,S190,F191,N206,F207,S208,A209,I211,G230.
In the above-mentioned method for preparing (S) -gamma-decalactone and (S) -delta-decalactone by enzyme catalysis, the ketoreductase can be added into the enzyme catalysis reaction system in different forms, and is preferably obtained by fermenting and culturing genetically engineered bacteria, and can be recombinant escherichia coli containing T7 promoter and expressing corresponding recombinant HbKR.
Further, the chiral lactone intermediates are (S) -4-hydroxydecanoic acid and (S) -5-hydroxydecanoic acid.
Further, glucose is added in the coupling catalysis process, and the molar mass ratio of the glucose to the 4-oxo-decanoic acid or the 5-oxo-decanoic acid is 1.5:1.
Further, the addition amount of the ketoreductase HbKR is 54% of the mass of the 4-oxo-decanoic acid or the 5-oxo-decanoic acid.
Further, the loading of 5-oxo-decanoic acid is 18.6g.L -1.
Further, the nucleotide sequence of the ketoreductase is shown as SEQ ID No. 1.
Further, the temperature of the coupling catalytic reaction is 30 ℃, and the pH is 6.0.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the ketoreductase HbKR of the invention can asymmetrically reduce 4-oxo-decanoic acid and 5-oxo-decanoic acid with excellent stereoselectivity, and can respectively obtain (S) -4-hydroxy-decanoic acid and (S) -5-hydroxy-decanoic acid, and the stereoselectivity can reach more than 99%. Through HbKR protein directional design engineering, a mutant HbKR V2 with obviously improved enzyme activity is obtained, the enzyme activity of 4-oxo-decanoic acid is improved to 2.14U/mg, the activity of the mutant is improved by 6.2 times compared with that of WT, the activity of 5-oxo-decanoic acid is improved to 8.37U/mg, and the activity of the mutant is improved by 9.7 times compared with that of WT. Apart from 4-oxo-decanoic acid and 5-oxo-decanoic acid, hbKR V2 have different degrees of improvement in the viability of a variety of 4/5-oxo-ketoesters of different chain lengths. Coupling ketoreductase optimal mutant HbKR V2 with glucose dehydrogenase BmGDH, under optimal reaction conditions, 100mM of 5-oxo-decanoic acid can be converted into 90.4% in 1h, and the e.e. value of the final product (S) -delta-decalactone reaches 99.9%, and the product purity is 99.4%.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
Enzyme activity determination method activity determination principle: depending on the characteristic absorbance peak of NADPH at 340nm, ketoreductases produce or consume NADPH during the reaction where oxidation or reduction occurs. Therefore, the enzyme activity can be calculated indirectly using the change in NADPH at 340 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1. Mu. Mol of NADPH per minute under the above-mentioned assay conditions.
Determination of reduction Activity System:
the measuring and activating process comprises the following steps: the measured temperature was set at 30℃and all buffers were preheated to within 30 ℃. And respectively adding the substrate, the coenzyme and the buffer solution into the clean ELISA plate.
The protein concentration of the crude enzyme solution was measured by the Bradford method, and the protein-pigment conjugate was measured at 595nm according to the color change of coomassie brilliant blue G-250 after the protein was bound, and the absorption value was proportional to the protein concentration. Adopting BSA bovine serum standard protein with the concentration of 5 mg.L -1 as mother liquor, and preparing protein concentration standard curve with the concentration interval of 0.01-0.12 mg.L -1 by gradient dilution. The protein to be tested is diluted to the range of the standard curve of the protein concentration, 20 mu L of protein solution is sucked, 180 mu L of Coomassie brilliant blue is added, the mixture is stood for 5min at 30 ℃, and the detection is carried out at 595 nm. In order to reduce errors, the measured samples are measured together with the protein standard curve, a standard protein concentration curve is drawn, and the protein concentration of the measured samples is calculated according to the curve. 3 replicates were measured for each sample.
The concentration of pure enzyme protein is determined based on the fact that most proteins have a maximum absorption peak at 280nm, so that concentration data can be directly obtained by a Nanodrop instrument. After the purified protein was concentrated and desalted, 5. Mu.L of pure enzyme was dripped on the instrument using the website https:// web. Expasy. Org/protparam/finding the molar extinction coefficient of the protein and the protein molecular weight, and the protein concentration was read according to the molar extinction coefficient and the protein molecular weight setting. The proteins are diluted in sequence by different times, and the determination results under different dilution times are verified to have good linear relationship, so that the protein concentration of the pure enzyme can be obtained.
The conversion and chirality were analyzed by Gas Chromatography (GC). The sample to be tested is extracted by ethyl acetate, dried by anhydrous sodium sulfate, volatilized by a vacuum concentrator, and finally dissolved in a proper amount of ethyl acetate. The chiral detection chromatographic column is a CP 7502-chirail-DEXCB chiral gas phase column, the detector is a hydrogen ion flame detector, the split ratio is 1:50, the temperature of the sample inlet is 280 ℃, the temperature of the detector is 280 ℃, and the temperature rising program is as follows: the initial temperature is 80 ℃ for 10min, 1 ℃/min to 100 ℃,30 min, 0.5 ℃/min to 130 ℃, 10min, 10 ℃/min to 180 ℃, 10min and total time 145min. The conversion rate detection analysis column is an HP-5 column, the split ratio is 1:50, the temperature of a sample inlet is 280 ℃, the temperature of a detector is 280 ℃, and the temperature rising program is as follows: the initial temperature was 80℃and increased to 100℃at 5℃per minute and to 125℃at 0.5℃per minute for a total time of 54 minutes.
Example 1:
preparation of LB medium: 5g of yeast extract, 10g of peptone, 10g of sodium chloride and 1L of water are added into a 1L measuring cylinder, sterilized at 121 ℃ for 20min, and cooled to 37 ℃ to obtain a corresponding LB medium.
Recombinant plasmid containing T7 promoter and expressed recombinant carbonyl reductase gene is introduced into colibacillus to construct HbKR recombinant colibacillus strain. Inoculating recombinant escherichia coli strains into an LB culture medium from a flat plate, culturing for 8 hours at 37 ℃, transferring 400 mu L to 40mL of LB culture medium, culturing until OD 600 is between 0.6 and 0.8, reducing the temperature to 16 ℃, adding IPTG with the final concentration of 0.2mM, continuously controlling the temperature at 16 ℃, and obtaining corresponding fermentation broth after fermentation culture is finished. And centrifugally collecting thalli, and preserving at-20 ℃ for standby. And (3) taking the obtained thalli, re-suspending the thalli by using a sodium phosphate buffer solution with the concentration of 10mmol/LpH of 7.0, performing ultrasonic crushing to obtain corresponding wall-broken enzyme solution, centrifuging at 10000rpm for 20min to obtain standby enzyme solution, standing the enzyme solution at the temperature of minus 80 ℃ for overnight freezing, and then putting the enzyme solution into a vacuum freeze dryer for freeze drying for 48h to obtain freeze-dried enzyme powder.
Example 2: the effect of reaction pH on the viability of ketoreductase HbKR.
The optimal reaction pH for the reductive and oxidative activities of ketoreductase HbKR was investigated. The enzyme activity of the substrate is measured by an enzyme-labeling instrument under different pH conditions by taking 5-oxo-decanoic acid as a mode substrate of a reduction reaction and isopropyl alcohol as a mode substrate of an oxidation reaction. The highest enzyme activity measured was used as a 100% control and the enzyme activities measured at other pH values were calculated as a percentage of the control enzyme activity.
As can be seen from Table 1, the optimal pH for HbKR reduction is 6.0, and as can be seen from Table 2, the optimal pH for HbKR oxidation is 9.0.
TABLE 1 influence of pH on HbKR reduction Activity
TABLE 2 influence of pH on HbKR Oxidation Activity
Example 3: influence of the reaction temperature on HbKR reduction reactions.
After incubation of the metal bath at different temperatures (25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃) for 2min, the enzyme activity was measured on an enzyme-labeled instrument, with the highest enzyme activity measured as 100% control, and the enzyme activities measured at the other temperatures were calculated as percentages of the control enzyme activity. As can be seen from Table 3, hbKR has an optimum temperature for the reduction reaction of 30 ℃.
TABLE 3 optimization of reaction temperature
Example 4:
In order to improve the enzymatic activity of HbKR-oxodecanoic acid and other larger steric hindrance substrates and maintain excellent stereoselectivity, 5-oxodecanoic acid is selected as a model substrate, the protein steric structure of HbKR is predicted by AlphaFold2, and then 5-oxodecanoic acid is docked into the active pocket of HbKR by DiscoveryStudio, although 5-oxodecanoic acid can be accommodated in the substrate binding pocket of HbKR, but due to its structural curvature and distance from the catalytic residue Tyr160 Is unfavorable for high-efficiency catalysis.
To expand the binding pocket of the substrate, enhance the interaction between the keto acid substrate and surrounding residues, active hydroxyl groups are present at Y160Amino acid residues that interact with cofactors and non-spatial amino acids are excluded from the surrounding, and 31 amino acid residues are selected for scanning mutations of alanine and positively charged amino acids. Among the mutation results of alanine scanning, the single mutant F207A with the most remarkable improvement effect, designated HbKR V1, was found to have 6.2-fold improvement in activity against 5-oxo-decanoic acid and a stereoselectivity of 99.9%. In the mutation result of positive amino acid scanning, the activity of F86H on 5-oxo-decanoic acid is improved by 1.4 times, and excellent stereoselectivity is still maintained. Then, by taking F207A as a parent and carrying out iterative saturation mutation at the F86 site, the optimal double mutant F207A/F86M, named HbKR V2, is screened, the activity of the double mutant F207A/F86M on 5-oxo-decanoic acid is improved by 9.7 times, and the double mutant F207A/F86M has stereoselectivity of 99.9 percent.
Example 5:
To explore the reduction substrate spectra of HbKR and HbKR V2, ketoacid substrates such as 4-oxooctanoic acid, 4-oxooctanoic acid methyl ester, 5-oxooctanoic acid methyl ester, 4-oxodecanoic acid methyl ester, 5-oxodecanoic acid methyl ester, 4-oxododecanoic acid methyl ester, 5-oxododecanoic acid methyl ester and the like were selected, and the activities of HbKR and HbKR V2 on the ketoacid ester substrates with different carbon chain lengths were measured. As shown in Table 4, hbKR was active on all substrates, and HbKR V2 was improved on all keto ester substrates.
Table 4: reduction substrate profiling of HbKR and HbKR V2
Example 6:
In order to explore the oxidation substrate spectra of HbKR and HbKR V2, alcohol substrates such as R-phenethyl alcohol, S-phenethyl alcohol, n-propanol, isopropanol, 2-butanol, 3-methylcyclohexanol, 4-ethylcyclohexanol and 2, 3-butanediol were selected, and the oxidation activities of HbKR and HbKR V2 on the alcohol substrates were measured, respectively. As shown in Table 5, hbKR was active on all substrates and HbKR V2 was less active on all ketoester substrates.
Table 5: oxidation substrate profiling of HbKR and HbKR V2
Note ND: notactivitywasdetected A
Example 7: asymmetric synthesis of (S) -gamma-decalactone was performed by HbKR and mutants thereof.
In the GDH coupling cofactor regeneration system, the reaction system was 10mL, including HbKR and its mutant lyophilized enzyme powder, GDH lyophilized enzyme powder, glucose, PBS buffer (100 mm, pH 7.0), coenzyme NADP +, and substrate 4-oxo-decanoic acid, the reaction temperature was 30deg.C, and the pH was 6.0. Reacting for 1h, adding 20% sulfuric acid solution to make pH 1-2, heating at 90 deg.C for 2h to complete lactonization reaction, treating sample, and performing gas phase detection.
As can be seen from Table 6 HbKR is able to asymmetrically catalyze 4-oxodecanoic acid with a stereoselectivity of 99% (S), at 1h the conversion of 5mM 4-oxodecanoic acid was 99% and the conversion of 10mM 4-oxodecanoic acid was 92%.
TABLE 6 asymmetric Synthesis of (S) -delta-decalactone
Example 8: asymmetric Synthesis of (S) -delta-decalactone by HbKR and mutants thereof
Two different cofactor regeneration systems were tried, in which the reaction system was 1mL, including HbKR and its mutants in pure enzyme solution, GDH lyophilized enzyme powder, glucose, PBS buffer (100 mM, pH 7.0), coenzyme NADP + and substrate 5-oxodecanoic acid, at a reaction temperature of 30℃and pH 6.0. In an IPA coupled self-sufficient cofactor regeneration system, the reaction system was 1mL, including HbKR and its mutant pure enzyme solution, appropriate amount of isopropyl alcohol, tris-HCl buffer (100 mM, pH 8.5), coenzyme NADP +, and substrate 5-oxo-decanoic acid, the reaction temperature was 30deg.C, pH 8.5. After the reaction, a 20% sulfuric acid solution was added to adjust the pH to 1-2, and the mixture was heated at 90℃for 2 hours to complete the lactonization reaction, and after the sample was treated, gas phase detection was performed.
From Table 7, it can be seen that the mutants had greater conversion than WT and HbKRRT had 89.0% conversion of 10mM 5-oxodecanoic acid in the IPA-coupled self-sufficient cofactor regeneration system, whereas HbKR V1 and HbKR V2 were capable of fully converting 10mM 5-oxodecanoic acid in the IPA-coupled self-sufficient cofactor regeneration system. In the GDH coupled cofactor regeneration system, hbKR V2 to 100mM conversion of 5-oxo-decanoic acid was able to reach 90.4% in 1h, with a stereoselectivity greater than 99% (S).
Table 7: asymmetric synthesis of (S) -delta-decalactone
a GDH coupled cofactor regeneration system: 1mL of the reaction system, PBS buffer (100 mM, pH 7.0)
b IPA coupled self-sufficient cofactor regeneration System 1mL Tris-HCl buffer (100 mM, pH 8.5) in the reaction System
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. Use of a ketoreductase in the preparation of an oxidising or reducing agent, characterised in that the ketoreductase is a ketoreductase of (a) or (b) as follows;
(a) Ketoreductase with an amino acid sequence shown as SEQ ID No. 2;
(b) Ketoreductase derived from (a) having one or more amino acids substituted, deleted or added in the amino acid sequence of (a) and having ketoreductase activity.
2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the ketoreductase of (a) is shown in SEQ ID No. 1.
3. The use according to claim 1, characterized in that the use is based on keto-acid ester compounds or alcohol compounds;
The ketoacid ester compound is selected from one or more of 4-oxo octanoic acid, 4-oxo octanoic acid methyl ester, 5-oxo octanoic acid methyl ester, 4-oxo decanoic acid methyl ester, 5-oxo decanoic acid methyl ester, 4-oxo dodecanoic acid methyl ester, 5-oxo dodecanoic acid and 5-oxo dodecanoic acid methyl ester;
The alcohol compound is selected from one or more of R-phenethyl alcohol, S-phenethyl alcohol, n-propanol, isopropanol, 2-butanol, 3-methylcyclohexanol, 4-ethylcyclohexanol and 2, 3-butanediol.
4. A method of synthesizing a chiral lactone, comprising the steps of: under the combined action of a coenzyme and a coenzyme regeneration system, a ketoester compound is used as a substrate, the ketoreductase of claim 1 or an expression system containing the ketoreductase of claim 1 is used as a catalyst, and the substrate is converted into chiral lactone through asymmetric reduction reaction.
5. The method of claim 4, wherein the ketoreductase is used in an amount of 1g/L to 20g/L; the concentration of the ketoacid ester compound is 5mM-100mM; the temperature of the reaction is 25-30 ℃.
6. The method according to claim 4, wherein the reaction system further comprises glucose dehydrogenase; the mass ratio of the ketoreductase to the glucose dehydrogenase is 1-2:1.
7. A ketoreductase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID No.3 or SEQ ID No. 4.
8. A gene encoding the ketoreductase mutant of claim 7.
9. An expression vector carrying the gene of claim 8.
10. A recombinant bacterium expressing the ketoreductase mutant of claim 7.
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