Ketoreductase mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a ketoreductase mutant as well as a preparation method and application thereof. The ketoreductase cgKR1 derived from candida glabrata is mutated to obtain a ketoreductase mutant, and the ketoreductase mutant is applied to preparation of (R)-3-chloro-1-phenyl-propanol by reducing 3-chloropropiophenone, and compared with wild type ketoreductase cgKR1, the yield of the (R)-3-chloro-1-phenyl-propanol is increased, and the yield of the (R)-3-chloro-1-phenyl-propanol is increased. The ketoreductase mutant provided by the invention has higher enzyme activity and stereoselectivity on the reduction reaction of 3-chloropropiophenone, the isomer purity of a reaction product is greatly improved, and industrial production requirements can be better met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a ketoreductase mutant and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application, and should not necessarily be regarded as acknowledging or implying in any form that the information constitutes prior art known to those skilled in the art.

[0003] ( R )-3-chloro-1-phenyl-propanol is an important chiral intermediate that can be used to prepare drugs such as tomoxetine hydrochloride and dapoxetine hydrochloride. The existing synthesis methods mainly include: (1) reacting 3-chloropropiophenone with NaBH4 or KBH4, and using a chiral amino acid derivative as a chiral inducer to produce (-)-3-chloro-1-phenyl-propanol, wherein the chiral amino acid derivative includes a levorotatory tryptophan derivative, a levorotatory isoleucine derivative, etc. The cost of the amino acid derivative is high, and this method has certain limitations in actual production application. (2) reacting 3-chloropropiophenone with diisopinocampylchloroborane in an organic phase solvent at 10-80℃ to obtain compound (-)-3-chloro-1-phenyl-propanol. This method uses a large amount of organic solvent, which causes great pollution to the environment, and the reaction conditions are harsh. The reaction route is as follows: R R

[0004] Keto reductase (Keto Reductase, KRED for short) is a kind of key biological catalyst that can efficiently catalyze the asymmetric reduction reaction of chiral ketones, and plays an important role in the synthesis of chiral alcohols. Keto reductase has mild catalytic conditions, does not need high temperature, high pressure or heavy metal participation, significantly reduces energy consumption and environmental burden; at the same time, the enzyme shows excellent stereoselectivity, can generate optically pure chiral alcohol with high enantioselectivity, effectively avoids the formation of racemates and subsequent resolution steps. Based on these outstanding advantages, keto reductase has become an indispensable tool in the green biological manufacturing of chiral drugs, fine chemicals and intermediates, and has realized large-scale industrial application in multiple fields.

[0005] In the current field of biological catalysis research, Candida rugosa (Candida rugosa) derived ketoreductase has been widely used in the synthesis of chiral alcohols, and has shown excellent catalytic performance. Candida glabrata ​​Ketoreductases (KRs) have attracted increasing attention. The enzyme belongs to the short-chain dehydrogenase / reductase (SDR) superfamily and exhibits catalytic activity on ketone substrates containing aryl and heteroaryl structures in the natural state. When catalyzing large steric hindrance ketone compounds (such as diaryl ketones), it exhibits different substrate recognition and binding characteristics from traditional ketoreductases. Based on this catalytic property, it can be used to efficiently and greenly synthesize chiral alcohol intermediates with high optical purity, thereby avoiding the cumbersome chiral resolution step in the traditional chemical synthesis path, not only reducing production costs, but also significantly reducing environmental pollution. However, natural ketoreductases still have certain limitations in industrial applications, mainly in the following two aspects: (1) The stereoselectivity needs to be improved: The catalytic product of some drug intermediate precursors has an enantiomeric excess (ee) value of only 70%-85%, which is difficult to meet the requirements of pharmaceutical-grade chiral purity. ee

[0006] (2) The catalytic efficiency is low: For long-chain alkyl-substituted ketone substrates, the turnover number (TON) is usually less than 500, which restricts the industrial application of the enzyme.

[0007] Therefore, structural modification and performance optimization of ketoreductases to improve their stereoselectivity and catalytic efficiency and expand their application range have become a key research direction for efficient synthesis of large steric hindrance chiral alcohols, and have significant industrial application value and scientific research significance. SUMMARY

[0008] Therefore, the present application provides a ketoreductase mutant, a preparation method and application thereof.

[0009] In order to achieve the above-mentioned purpose, the present application is realized by the following technical solutions: In a first aspect, the present application provides a ketoreductase mutant, which comprises an amino acid sequence as shown in SEQ ID NO: 1 and at least one of the following mutations (a)-(h): (a) the 136th amino acid A is mutated to F or Y (denoted as M1 or M2); (b) the 144th amino acid M is mutated to I (denoted as M3); (c) the 136th amino acid A is mutated to F, and the 144th amino acid M is mutated to I (denoted as M4); (d) the 136th amino acid A is mutated to F, and the 138th amino acid N is mutated to L or V (denoted as M5 or M6); (e) the 136th amino acid A is mutated to F or Y, and the 208th amino acid Y is mutated to F (denoted as M7 or M8); (f) the 136th amino acid A is mutated to Y, the 138th amino acid N is mutated to L, and the 208th amino acid Y is mutated to F (denoted as M9);​ (g) the amino acid at position 136 is mutated to Y, the amino acid at position 208 is mutated to F, and the amino acid at position 229 is mutated to Y (denoted as M10); (h) the amino acid at position 136 is mutated to Y, the amino acid at position 138 is mutated to L, the amino acid at position 208 is mutated to F, and the amino acid at position 229 is mutated to Y (denoted as M11).

[0010] The present application screens a ketoreductase cgKR1 from Candida glabrata, which can catalyze 3-chloropropiophenone to generate 3-chloro-1-phenyl-propanol. Candida glabrata Compared with the wild-type enzyme, the ketoreductase mutant has higher enzyme activity and selectivity in catalyzing 3-chloropropiophenone, and the purity of the reaction product isomers is greatly improved, which can better adapt to the needs of industrial development.

[0011] Further, the amino acid sequence (SEQ ID NO: 1) is: VPRGSHMTTFVVSGATGFIAQHVVRQLLDQNYKVIGSVRSAEKGDHLKNVIFKGGDFNYEIVKDISDPTAFDHVFEKHGKDIKVVLHTASPFHFNTTDIEKDLLIPAVNGTKGILESIKKYAAQTVERVVVTSSFAANTSTVDMFYAKDSSKTITEESWNQDTWESCQSDPIRGYCGSKKFAEKAAWDFYNANKDSVKFKLSIINPVYVFGPQNYVEPGKKILNTSSEVINSLVHLKKDDPLPEFAGGHIDVRDVAKAHILAFQKDELIEQRLMLHAGLFTTQTLLDIINEQFPELKGKIPAGKPGTGNPDDALTPVDNSKTKKLLGFEFIDLKKDLYDTISQILEAEKNSN In a second aspect, the present application provides a composition comprising a coenzyme regenerating enzyme and the ketoreductase mutant of the first aspect.

[0012] Further, the coenzyme regenerating enzyme is glucose dehydrogenase.

[0013] In a third aspect, the present application provides a biological material comprising at least one of the following A) to D): A), a nucleic acid encoding the ketoreductase variant of the present application or the composition of the present application; B), a recombinant vector containing the nucleic acid of A); C) host cells transformed or transfected with the recombinant vector as described in B); D) a mixture obtained by culturing the host cells as described in C).

[0014] The nucleic acid of the present application can be DNA, RNA, cDNA or PNA. In the embodiments of the present application, the nucleic acid is in the form of DNA; the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.

[0015] The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing desired coding sequences and suitable nucleic acid sequences or elements necessary for the expression of operably linked coding genes in a specific host organism. The nucleic acid sequences or elements necessary for expression in prokaryotic cells include promoters, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to use promoters, enhancers and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome.

[0016] The host cells of the present application are transformed or transfected with the recombinant vector, and the host cells are transformed or transfected with the vector constructed using recombinant DNA technology, so that the transformed host cells have the ability to replicate the vector encoding the protein or express the desired protein.

[0017] In a fourth aspect, the present application provides the use of the ketoreductase mutant of the first aspect or the composition of the second aspect or the biological material of the third aspect in the reduction of 3-chloropropiophenone to prepare ( R )-3-chloro-1-phenyl-propanol.

[0018] The present application screens a ketoreductase cgKR1 derived from Candida glabrata, which can catalyze 3-chloropropiophenone to generate ( Candida glabrata )-3-chloro-1-phenyl-propanol. R The proportion of (-)-3-chloro-1-phenyl-propanol: (+)-3-chloro-1-phenyl-propanol in the product is 11: 89, and the modified enzyme catalyzing the generation of R )-3-chloro-1-phenyl-propanol with a value of > 99% is obtained through modification and screening. S R ee

[0019] Catalytic principle: the ketoreductase (cgKR1) gene of Candida glabrata is recombinantly expressed in Escherichia coli, and the recombinant cgKR1 catalyzes asymmetric reduction reaction to prepare R chiral alcohol with R configuration. The catalytic reaction is as follows: ​​​

[0020] In a fifth aspect, the present application provides a method for preparing (-)-3-chloro-1-phenyl-propanol, comprising reducing 3-chloropropiophenone by using the ketoreductase mutant of the first aspect or the composition of the second aspect or the biological material of the third aspect. R R In a fifth aspect, the present application provides a method for preparing (-)-3-chloro-1-phenyl-propanol, comprising reducing 3-chloropropiophenone by using the ketoreductase mutant of the first aspect or the composition of the second aspect or the biological material of the third aspect.

[0021] Further, the method further comprises a co-substrate, a coenzyme and a coenzyme regenerating enzyme; the co-substrate is glucose, the coenzyme is NADP + , and the coenzyme regenerating enzyme is glucose dehydrogenase (GDH).

[0022] Further, the concentration of the ketoreductase mutant of the first aspect or the composition of the second aspect or the biological material of the third aspect is 0.5-5 mg / mL.

[0023] Further, the concentration of the glucose is 1-10 mg / mL.

[0024] Further, the concentration of the NADP + In the reaction system, the NADP + solution with a mass fraction of 5% is used in an amount of 0.5-5 mg / mL.

[0025] Further, the concentration of the coenzyme regenerating enzyme is 5-50 U / mL.

[0026] Further, the reaction temperature is 20-40℃.

[0027] Further, the buffer solution of the reaction is 20-200 mmol / L phosphate buffer solution with pH = 6.0-8.0.

[0028] Further, the 3-chloropropiophenone is dissolved in an organic solvent before the reaction, and the concentration of the 3-chloropropiophenone in the organic solvent is 1-15%; the organic solvent is at least one selected from the group consisting of ethanol, diethyl ether, isopropyl alcohol, n-heptane, n-pentane and tetrahydrofuran.

[0029] Compared with the prior art, the present application has the following beneficial effects: The present application mutates the ketoreductase cgKR1 derived from Candida glabrata to obtain a ketoreductase mutant, and applies the ketoreductase mutant to reduce 3-chloropropiophenone to prepare (-)-3-chloro-1-phenyl-propanol. R ​)-3-chloro-1-phenyl-propanol, the ketoreductase mutant provided by the present application has higher enzyme activity and stereoselectivity in the reduction reaction of 3-chloro-1-phenyl-propanone compared with the wild-type ketoreductase cgKR1, the isomer purity of the reaction product is greatly improved, and the industrial production demand can be better met. BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings constituting a part of the specification of the present application serve to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute improper limitation on the present application.

[0031] Figure 1 is the pET-28a (+) plasmid map containing the cgKR1 gene sequence of the present application; Figure 2 is the original cgKR1 and substrate docking effect diagram and ligand-receptor binding mode diagram of the present application; Figure 3 is the modified cgKR1 and substrate docking effect diagram and ligand-receptor binding mode diagram of the present application; Figure 4 is the original cgKR1 and substrate docking effect diagram and ligand-receptor binding mode diagram of the present application; R )-3-chloro-1-phenyl-propanol, S is the liquid chromatography identification diagram of the mixed standard solution of (-)-3-chloro-1-phenyl-propanol; Figure 5 is the liquid chromatography identification diagram of the product after the original cgKR1 catalytic reaction; Figure 6 is the liquid chromatography identification diagram of the product after the modified cgKR1 catalytic reaction. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0033] The technical solutions of the present application will be further described below in combination with specific embodiments.

[0034] Embodiment 1 The present embodiment provides a preparation method of the ketoreductase used, specifically: (1) Preparation of a strain of recombinant ketoreductase The wild-type ketoreductase is derived from Candida glabrata (ATCC 34874), and its amino acid sequence is shown in SEQ ID NO: 1. In the preparation of the recombinant ketoreductase, the ketoreductase expression vector is pET-28a (+); the ketoreductase expression cell is BL21 (DE3). Candida glabrata E. coli ​​

[0035] The cgKR1 gene sequence was synthesized by using the whole gene synthesis method, and was connected to the pET-28a (+) plasmid to obtain a recombinant plasmid, and the recombinant plasmid was transformed into E. coli BL21 (DE3) to obtain a recombinant E. coli expressing the cgKR1 enzyme.

[0036] (2) Point mutation method: The plasmid containing the cgKR1 gene was extracted, and the mutation primer was designed. The mutation site was taken as the center, and 12-18 bases were extended on both sides to make the total length reach 25-40 bp. The mutation base was placed in the middle region of the primer, and the GC content was ensured to be 40%-60%, the Tm value was ≥55°C, and the Tm difference between the upstream and downstream primers was ≤2°C. At the same time, self-hairpin structure, primer dimer and continuous same base were avoided. After the design, the accuracy of the mutation site and the binding specificity needed to be verified. The PCR site mutation experiment was carried out, the PCR product was purified, and after demethylation treatment, the E. coli DH5α, and single colonies were picked for preservation and sequencing. The plasmid with correct sequencing was transformed into E. coli BL21 (DE3) strain. The PCR reaction system and reaction program are shown in Tables 1 and 2, respectively.

[0037] Table 1 PCR reaction system

[0038] Table 2 PCR reaction program

[0039] (3) Induction expression of enzyme protein: single colonies were picked into LB liquid medium containing kanamycin, and were cultured overnight at 37°C and 220 r / min. Then, the seed liquid after expansion was inoculated into TB liquid medium containing antibiotics at a 2% inoculation amount, and was continued to be cultured at 37°C and 220 r / min. When the OD 600 absorbance value reached 0.8-1, 0.5 mmol / L IPTG solution was added, and the culture was cultured at 18°C to induce the expression of the target protein.

[0040] (4) Collection of crude enzyme liquid: the overnight induced bacterial liquid was centrifuged at 8000 r / min for 5 min, and the bacterial sediment was collected. According to the mass ratio of bacterial sediment to 1×PBS buffer of 1:4, 1×PBS buffer was added to resuspend the bacterial body. The bacterial liquid was broken by using an ultrahigh pressure cell disrupter, the breaking pressure was set to 1100-1300 bar, the temperature was controlled at 4°C, and the continuous breaking was carried out for three times to obtain the cell broken liquid. Then, the cell broken liquid was centrifuged at 12000 r / min for 10 min to remove the cell debris sediment, and the obtained supernatant was the crude enzyme liquid.

[0041] The pET-28a (+) plasmid containing the cgKR1 gene sequence of this invention is shown in the image below. Figure 1 As shown; the docking effects of the original cgKR1 and the modified cgKR1 with the substrate and the ligand-receptor binding mode diagram are shown in the figure. Figures 2-3 As shown.

[0042] Example 2 Using wild-type ketone reductase cgKR1 enzyme solution and the ketone reductase mutant enzyme solution obtained in Example 1 as catalysts, screening was conducted using the reaction system shown in Table 3. The enzyme solution specifically generating [the desired product] was selected in the liquid phase. R Ketone reductase with specific configuration.

[0043] Table 3 Crude enzyme screening system

[0044] The enzyme activity reaction and detection method are as follows: NADP is added sequentially according to the above reaction system. + Glucose, GDH, phosphate buffer, a heptane solution of 3-chlorophenylacetone, and crude enzyme were added to an EP tube. The tube was placed in a shaker and incubated at 30°C and 200 r / min for 2 h. The reaction was then terminated. The entire upper organic phase was removed, dried, and reconstituted with 1 mL of mobile phase. Liquid chromatography analysis was performed, and the results are shown in Table 4.

[0045] Chiral detection method: Yuexu Ultimate Cell-D 4.6×250mm 5μm, mobile phase n-hexane:ethanol=95:5, flow rate 1.0 mL / min, detection wavelength 210 nm, column temperature 30℃, injection volume 10 μL, detection time 20 min.

[0046] ( R )-3-chloro-1-phenyl-propanol, ( S The liquid chromatography identification chromatogram of the mixed standard of 3-chloro-1-phenyl-propanol is shown below. Figure 4 As shown; the liquid phase identification diagram of the product after the original cgKR1 catalytic reaction is shown below. Figure 5 As shown; the liquid phase identification diagram of the product after the modified cgKR1 catalytic reaction is shown below. Figure 6 As shown.

[0047] Table 4 Results of screening for ketone reductase mutants

[0048] This invention uses only biological methods, with 3-chlorophenylacetone as a substrate, to directly obtain ( ) during the enzymatic reduction stage. R 3-Chloro-1-phenyl-propanol, the operation method is simple, the reaction conditions are mild, it is environmentally friendly, and the photochemical purity is high, reaching over 99%. eeThe value can be up to 99% or more.

[0049] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A ketoreductase mutant, characterized in that, The amino acid sequence as shown in SEQ ID NO: 1 has at least one of the following (a)-(h) mutations: (a) the amino acid at position 136 is mutated from A to F or Y; (b) the amino acid at position 144 is mutated from M to I; (c) the amino acid at position 136 is mutated from A to F and the amino acid at position 144 is mutated from M to I; (d) the amino acid at position 136 is mutated from A to F and the amino acid at position 138 is mutated from N to L or V; (e) the amino acid at position 136 is mutated from A to F or Y and the amino acid at position 208 is mutated from Y to F; (f) the amino acid at position 136 is mutated from A to Y, the amino acid at position 138 is mutated from N to L, and the amino acid at position 208 is mutated from Y to F; (g) the amino acid at position 136 is mutated from A to Y, the amino acid at position 208 is mutated from Y to F, and the amino acid at position 229 is mutated from V to Y; (h) the amino acid at position 136 is mutated from A to Y, the amino acid at position 138 is mutated from N to L, the amino acid at position 208 is mutated from Y to F, and the amino acid at position 229 is mutated from V to Y.

2. A composition characterized in that, The coenzyme regenerating enzyme and the ketoreductase mutant of claim 1.

3. The composition of claim 2, wherein The coenzyme regenerating enzyme is glucose dehydrogenase.

4. A biomaterial, characterized by, It comprises at least one of the following A)-D): A) a nucleic acid encoding the ketoreductase mutant of claim 1 or the composition of claim 2; B) a recombinant vector containing the nucleic acid of A); C) a host cell transformed or transfected with the recombinant vector of B); D) a mixture obtained by culturing the host cell of C).

5. Use of a ketoreductase mutant of claim 1 or a composition of claim 2 or a biological material of claim 4 in the reduction of 3-chloropropiophenone to prepare (3S)-3-chloro-l-phenyl-propanol. R )-3-chloro-l-phenyl-propanol.

6. A process for the preparation of (R)-3-chloro-1-phenyl-propan-1-ol, characterized in that R -3-chloro-1-phenyl-propan-1-ol, characterized in that including the reduction of 3-chloropropiophenone to prepare R )-3-chloro-1-phenyl-propanol using the ketoreductase mutant of claim 1 or the composition of claim 2 or the biological material of claim 4.

7. The production method according to claim 6, wherein The preparation process further comprises a co-substrate, a coenzyme, and a coenzyme regeneration enzyme; preferably, the co-substrate is glucose; preferably, the coenzyme is NADP + ; preferably, the coenzyme regeneration enzyme is glucose dehydrogenase.

8. The production method according to claim 6, wherein The concentration of the ketoreductase mutant of claim 1 or the composition of claim 2 or the biological material of claim 4 is 0.5-5 mg / mL.

9. The production method according to claim 7, wherein the concentration of the glucose is 1-10 mg / mL; and / or, the NADP + In the reaction system, 5% of NADP + an aqueous solution, and the amount is 0.5-5 mg / mL.

10. The production method according to claim 7, wherein The concentration of the coenzyme regenerating enzyme is 5-50 U / mL; and / or, the reaction temperature is 20-40℃; and / or, the buffer of the reaction is 20-200 mmol / L pH = 6.0-8.0 phosphate buffer; and / or, the 3-chloropropiophenone is dissolved in an organic solvent before the reaction, the concentration of 3-chloropropiophenone in the organic solvent is 1-15%, and the organic solvent is at least one selected from the group consisting of ethanol, diethyl ether, isopropyl alcohol, n-heptane, n-pentane, and tetrahydrofuran.

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