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WO2016095223A1 - 双羰基还原酶突变体及其应用 - Google Patents

双羰基还原酶突变体及其应用 Download PDF

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WO2016095223A1
WO2016095223A1 PCT/CN2014/094422 CN2014094422W WO2016095223A1 WO 2016095223 A1 WO2016095223 A1 WO 2016095223A1 CN 2014094422 W CN2014094422 W CN 2014094422W WO 2016095223 A1 WO2016095223 A1 WO 2016095223A1
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Prior art keywords
pet
seq
amino acid
acid sequence
mutation
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French (fr)
Inventor
洪浩
詹姆斯盖吉
高峰
刘立辉
刘芳
于文燕
郭莉娜
崔瑜霞
唐芳荣
张娜
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Asymchem Laboratories Fuxin Co Ltd
Asymchem Laboratories Tianjin Co Ltd
Asymchem Laboratories Jilin Co Ltd
Asymchem Life Science Tianjin Co Ltd
Tianjin Asymchem Pharmaceutical Co Ltd
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Asymchem Laboratories Fuxin Co Ltd
Asymchem Laboratories Tianjin Co Ltd
Asymchem Laboratories Jilin Co Ltd
Asymchem Life Science Tianjin Co Ltd
Tianjin Asymchem Pharmaceutical Co Ltd
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Priority to PCT/CN2014/094422 priority Critical patent/WO2016095223A1/zh
Publication of WO2016095223A1 publication Critical patent/WO2016095223A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to the field of enzymes and enzyme engineering, and in particular to a biscarbonyl reductase mutant and uses thereof.
  • the enzyme As a biocatalyst, the enzyme can fully exert its high efficiency and high specificity in the living body.
  • Enzyme molecules must be engineered to suit different application requirements by means of protein engineering methods. Protein engineering methods can be summarized into three types: rational design, irrational design, and semi-rational design.
  • Rational design refers to the protein that produces new traits by changing the individual amino acids in the protein molecule by site-directed Mutagenesis or other methods based on understanding the spatial structure of the protein. This method is theoretically highly targeted and is mainly used to modify the catalytic activity, substrate specificity, stability, change of inhibitor type, and coenzyme specificity of natural enzyme proteins.
  • the fixed-point saturation mutation technique is an important technology in protein engineering. It belongs to the above semi-rational design but combines the advantages of rational design and irrational design to make up for their respective shortcomings. It transforms the gene encoding the target protein. A mutant in which the amino acid of the target site was replaced by the other 19 amino acids, respectively, was obtained in a short time. This technology is not only a powerful tool for protein orientation modification, but also an important means of protein structure-function relationship research. Studies have shown that multipoint mutations often yield more ideal evolutions than single point mutations. Multiple point mutations are not directly obtainable by site-directed mutagenesis. These problems that can not be solved by the fixed-point mutation technology are precisely the unique features of the fixed-point saturation mutation technology.
  • Carbonyl reductase is an oxidoreductase that plays an important role in many biotransformation processes in biological organisms. Based on its ability to catalyze the production of highly compatible chiral alcohols, carbonyl reductases are often used as a very important biocatalyst for the synthesis of chiral intermediates in the chemical and pharmaceutical industries.
  • the bis-carbonyl reductase can stereoselectively reduce the two carbonyl groups of the diketoester to the corresponding hydroxyl group, and can be used for the synthesis of key pharmaceutical intermediates, especially the chiral dihydroxyhexanoate chain for the synthesis of statins.
  • statins Such as the world's best-selling cholesterol-lowering drugs atorvastatin (Atorvastatin, Lipitor) and rosuvastatin (Rosuvastatin).
  • the currently known bis-carbonyl reductase can be used as a biocatalyst to reduce the diketone substrate in one step, and to obtain a key chiral intermediate 3R, 5S-dihydroxy-6-benzyloxy of a statin hypolipidemic drug with nearly single optical purity.
  • Tert-butyl hexanoate simplifies the synthesis step and reduces production pollution. But in the application of industrial production, there are still some questions. The problem needs to be further solved, such as lower enzyme catalytic activity, larger amount of enzyme solution, and an increase in the total volume of the reaction system, resulting in an increase in production batches and production costs.
  • the present invention aims to provide a biscarbonyl reductase mutant and use thereof to increase the enzyme stereoselectivity and catalytic activity of the dicarbonyl reductase (DKR).
  • a biscarbonyl reductase mutant having an amino acid sequence which is an amino acid sequence mutated by an amino acid sequence encoded by SEQ ID NO: 9 is provided,
  • the mutated amino acid sequence has at least two mutation sites: 94th, 151st, 231rd, 236th, and 251th, and the 94th I mutation is V, A, or G;
  • the V mutation at position is Q, N or S;
  • the F mutation at position 231 is W, Y or P;
  • the I mutation at position 236 is L, V or A;
  • the Q mutation at position 251 is H, R or K;
  • the amino acid sequence of the double carbonyl reductase mutant has a mutation site in the mutated amino acid sequence and an amino acid sequence having 90% or more homology with the mutated amino acid sequence.
  • amino acid sequence of the biscarbonylreductase mutant is the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5;
  • the amino acid sequence of the carbonyl reductase mutant has 95% or more homology with the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Amino acid sequence.
  • sequence of the DNA molecule is the sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or the sequence of the DNA molecule is SEQ ID NO :10.
  • SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 has a sequence having more than 95% homology.
  • a recombinant plasmid comprising any one of the above DNA molecules is provided.
  • the recombinant plasmids are pET-22b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-.
  • a host cell comprising any one of the above recombinant plasmids is provided.
  • the host cell comprises a prokaryotic cell, a yeast or a eukaryotic cell; preferably the prokaryotic cell is an E. coli BL21 cell or an E. coli DH5 ⁇ competent cell.
  • a process for producing a 3R,5S-dihydroxy compound comprising the step of catalytically reacting a diketone compound with a dicarbonyl reductase, wherein the biscarbonyl reductase is any of the above dicarbonyl groups. Reductase mutant.
  • diketone compound is a ketone compound of the formula I:
  • R1 is selected from the group consisting of an aryl group, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, an arylalkylheterocyclyl group, a cyclic heteroalkyl group or a cyclic heteroalkylated alkyl group; From an alkyl group, a cycloalkyl group, a haloalkyl group or a halocycloalkyl group; preferably the diketone compound is selected from the group consisting of 6-benzyloxy-3,5-dioxo-hexanoic acid tert-butyl ester, 6-benzyloxy- 3,5-dioxo-hexanoic acid neopentyl ester, 6-benzyloxy-3,5-dioxo-hexanoic acid methyl ester or 6-benzyloxy-3,
  • the above-mentioned biscarbonylreductase mutant of the present invention is further mutated by a site-directed saturation mutation based on the biscarbonylreductase F231W+I94V mutant encoded by SEQ ID NO: 9.
  • the double carbonyl reductase mutant having the above mutation site has the advantage of greatly improving the enzyme activity, and the enzyme activity is relatively
  • the bis-carbonyl reductase parent used in the present invention is increased by a factor of 2, or even 3 times, and the enzyme specificity is also correspondingly increased, thereby greatly reducing the cost in the industrial production of 3R, 5S-dihydroxy compounds.
  • Figure 1 shows the chemical reaction process of the synthesis of 3R,5S-dihydroxy compounds of the present invention
  • Figure 3 shows the chemical reaction equation for the synthesis of 3R,5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester according to the present invention
  • Figure 4 shows a three-dimensional structural simulation of a NAD-bound double carbonyl reductase
  • Figure 5 shows a three-dimensional structural simulation of a double-carbonyl reductase at a valid mutation site
  • Fig. 6 is a graph showing the results of protein electrophoresis detection of a biscarbonylreductase mutant in a preferred embodiment of the present invention.
  • the inventors of the present invention have based on the previously disclosed dicarbonyl reductase derived from Rhodococcus erythropolis SK121, since the prior art biscarbonylreductase has a defect of low catalytic activity and large amount of enzyme solution and is not suitable for industrial application. And its coding gene (CN201410188168), conducted a more in-depth study.
  • the bis-carbonyl reductase can be used as a biocatalyst to prepare a key chiral intermediate 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid of a statin hypolipidemic drug with nearly single optical purity by reducing the diketone substrate in one step.
  • Tert-butyl ester simplifies the synthesis step and reduces production contamination.
  • the inventors used a site-directed saturation mutation to modify the bis-carbonyl reductase (CN201410196920), and obtained a mutant F231W+I94V with greatly improved enzyme activity.
  • the mutant is in 3R, 5S-dihydroxy-
  • the amount of enzyme in the synthesis of 6-benzyloxy-hexanoic acid tert-butyl ester is reduced from 6wt to 2wt; but in the synthesis of 3R,5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester, the enzyme
  • the dosage is 9wt, and the amount of the enzyme solution is large, which increases the total volume of the reaction system, resulting in an increase in production batches and production costs.
  • the present invention is an improvement based on the dicarbonyl reductase mutant F231W+I94V to increase the stereoselectivity and catalytic activity of the dicarbonyl reductase (DKR) enzyme and to expand its application range.
  • the present invention mutates the gene of the mutant F231W+I94V (shown in SEQ ID NO: 9) of the dicarbonyl reductase (DKR) of Rhodococcus erythropolis SK121 strain as a starting gene, and directional screening The method obtains a series of dicarbonyl reductase mutants with increased enzymatic activity.
  • DKR dicarbonyl reductase
  • the mutated amino acid residue of the biscarbonylreductase mutant of the present invention is located at a substrate binding site or a region associated with substrate and NAD binding, associated with NAD proton transfer, for example, I94 is located in the NAD binding region, V151, F231, I236 And four amino acids of Q251 are in the vicinity of the substrate binding site, and these amino acid changes may increase the specificity of substrate binding, thereby increasing the activity of the enzyme.
  • the experimental results of the present invention indicate that the introduction of the Q251H mutation can greatly increase the activity of the dicarbonyl reductase based on the F231W mutation.
  • a single I236L mutation can significantly increase the catalytic specificity of the bis-carbonyl reductase, and a single Q251H mutation can significantly increase the catalytic activity of the bis-carbonyl reductase.
  • the introduction of the Q251H mutation also significantly increased the activity of its bis-carbonyl reductase based on the F231W and/or V151Q and/or I94V mutations.
  • the introduction of the I236L mutation into F231W and/or V151Q and/or I94V significantly increases the specificity of the dicarbonyl reductase product.
  • Combining F231W and/or I94V and/or I236L and/or Q251H is effective both to increase the activity of the dicarbonyl reductase and also to significantly increase the specificity of the dicarbonyl reductase product.
  • the biscarbonylreductase mutant obtained above can be overexpressed in E. coli by genetic engineering means linking its gene to pET-22b (+) and other expression vectors.
  • the expressed bis-carbonyl reductase mutant exhibits a molecular weight of about 30 KD on SDS-PAGE. Under 30 ° C and pH 6.0, the 3R, 5S-dihydroxy compound can be reduced in one step to obtain higher optical purity. 3R, 5S-dihydroxy compound.
  • the present invention provides a double carbonyl reductase mutant having an amino acid sequence which is a mutated amino acid sequence encoded by SEQ ID NO: 9 and which is mutated.
  • the amino acid sequence has at least two mutation sites: 94th, 151st, 231rd, 236th, and 251th, and the 94th I mutation is V, A or G; V mutation is Q, N or S; F mutation at position 231 is W, Y or P; I mutation at position 236 is L, V or A; Q mutation at position 251 is H, R or K;
  • the amino acid sequence of the carbonyl reductase mutant has a mutation site in the mutated amino acid sequence and an amino acid sequence having 90% or more homology with the mutated amino acid sequence.
  • the above-mentioned bis-carbonyl reductase mutant of the present invention is based on the bis-carbonyl reductase F231W+I94V mutant encoded by SEQ ID NO: 9, and is further mutated by a site-directed saturation mutation to change its amino acid sequence.
  • the structure and function of the protein are changed, and the bis-carbonyl reductase mutant of the present invention having the above-mentioned mutation site has the advantage of greatly improving the enzymatic activity, and the enzyme activity is relative to the dicarbonyl group used in the present invention.
  • the reductase parent is increased by a factor of 2, or even 3, and the enzyme specificity is also increased, thereby greatly reducing the cost of industrial production of 3R, 5S-dihydroxy compounds.
  • the amino acid sequence of the above double carbonyl reductase mutant is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Or It has 95% or more homology with the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
  • sequences defined by the present invention with varying degrees of homology must also have improved activity for dual carbonyl reductase activity.
  • amino acid sequence of the double carbonyl reductase mutant has 95% or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
  • SEQ ID NO: 1 amino acid sequence having improved biscarbonyl reductase activity.
  • the amino acid sequence of the above biscarbonylreductase is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
  • the mutation site of the amino acid sequence shown by SEQ ID NO: 1 is F231W+Q251H; the mutation site of the amino acid sequence shown by SEQ ID NO: 2 is F231W+I94V+I236L; the amino acid sequence of SEQ ID NO: 3
  • the mutation site is I94V+F231W+I236L+Q251H;
  • the mutation site of the amino acid sequence shown in SEQ ID NO: 4 is F231W+I94V+Q251H;
  • the mutation site of the amino acid sequence shown in SEQ ID NO: 5 is I94V+V151Q+ F231W+ Q251H.
  • the 3H,5S-dihydroxy compound was prepared from the biscarbonyl reductase mutant having the above amino acid sequence, and the obtained 3R, 5S-dihydroxy compound had an ee value of more than 99% and a de value of about 99%.
  • the above-mentioned bis-carbonyl reductase mutant of the present invention is a key pharmaceutical intermediate, and in particular, the synthesis of a chiral dihydroxyhexanoate chain of a statin provides a highly efficient catalyst for the industrial production cost of 3R, 5S-dihydroxy compound. It has been greatly reduced.
  • a DNA molecule encoding any of the above-described bis-carbonyl reductase mutants, which encodes a bis-carbonyl reductase having higher enzymatic activity, It is beneficial to reduce the cost in the industrial production of 3R, 5S-dihydroxy compounds.
  • the sequence of the above DNA molecule is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or the above DNA molecule
  • the sequence has 95% or more homology to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • a DNA molecule having the above sequence is capable of encoding a dicarbonyl reductase having further enhanced activity.
  • SEQ ID NO: 10 is the 691-693 bp TTC mutation in the biscarbonyl reductase gene sequence shown in SEQ ID NO: 9 to TGG, and the 751-753 bp CAA mutation is CAT or CAC.
  • SEQ ID NO: 11 is the 691-693 bp TTC mutation in the bis-carbonyl reductase gene sequence shown in SEQ ID NO: 9 is TGG, and the 280-282 bp ATT mutation is GTT, GTC, GTA or GTG, and the ATC mutation of the 706-708 bp is TTA, TTG, CTT, CTC, CTA or CTG.
  • the EQ ID NO: 12 is the 691-693 bp TTC mutation in the bis-carbonyl reductase gene sequence shown in SEQ ID NO: 9 is TGG, and the 280-282 bp ATT mutation is GTT, GTC, GTA. Or GTG, and the ATC mutation of the 706-708 bp is TTA, TTG, CTT, CTC, CTA or CTG, and the CAA mutation of the 751-753 bp is CAT or CAC.
  • SEQ ID NO: 13 is the 691-693 bp TTC mutation in the bis-carbonyl reductase gene sequence shown in SEQ ID NO: 9 is TGG, and the 280-282 bp ATT mutation is GTT, GTC, GTA or GTG, and the CAA mutation of the 751-753 bp is CAT or CAC.
  • SEQ ID NO: 14 is the 691-693 bp TTC mutation in the bis-carbonyl reductase gene sequence shown in SEQ ID NO: 9 is TGG, and the 280-282 bp ATT mutation is GTT, GTC, GTA or GTG, and the 451-453 bp GTC mutation is CAA or CAG, and the 751-753 bp CAA mutation is CAT or CAC.
  • the DNA molecule having the above sequence is capable of encoding a double-carbonyl reductase having a higher catalytic activity, and the enzyme activity is twice or even three times higher than that of the prior art biscarbonylreductase. Reduce the industrial production cost of 3R, 5S-dihydroxy compounds.
  • DNA molecules of the present invention may also exist in the form of "expression cassettes".
  • "Expression cassette” refers to a linear or circular nucleic acid molecule encompassing DNA and RNA sequences capable of directing expression of a particular nucleotide sequence in a suitable host cell.
  • a promoter operably linked to a target nucleotide is included, optionally operably linked to a termination signal and/or other regulatory elements.
  • the expression cassette may also include sequences required for proper translation of the nucleotide sequence.
  • the coding region typically encodes a protein of interest, but also encodes a functional RNA of interest, such as antisense RNA or untranslated RNA, in the sense or antisense orientation.
  • An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component thereof.
  • the expression cassette may also be naturally occurring, but obtained by efficient recombinant formation for heterologous expression.
  • a recombinant plasmid comprising a sequence of any one of the above DNA molecules.
  • the DNA molecule in the above recombinant plasmid is placed in the appropriate position of the recombinant plasmid such that the above DNA molecule can be correctly, smoothly replicated, transcribed or expressed.
  • plasmid includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule in a double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which may be a prokaryotic expression plasmid.
  • the recombinant plasmid is selected from the group consisting of pET-22b (+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b( +), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+ ), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+) , pET-32a(+), pET-35
  • a host cell comprising any of the above recombinant plasmids.
  • Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells, yeast or eukaryotic cells.
  • the prokaryotic cell is a eubacteria, such as a Gram-negative or Gram-positive bacterium. More preferably, the prokaryotic cells are E. coli BL21 cells or E. coli DH5 ⁇ competent cells.
  • a method for producing a 3R,5S-dihydroxy compound comprising the step of catalytically reacting a diketone compound with a dicarbonyl reductase, wherein the dicarbonyl reductase is Any of the above biscarbonyl reductase mutants. Since the above-mentioned biscarbonylreductase of the present invention has higher enzymatic activity, the 3R,5S-dihydroxy compound prepared by using the biscarbonylreductase mutant of the present invention can not only reduce the production cost, but also the 3R, 5S obtained.
  • the dihydroxy compound has an ee value of more than 99% and a de value of about 99%.
  • the raw material for preparing the 3R,5S-dihydroxy compound may be a commercially available raw material or a ketone compound which is easily prepared.
  • the diketone compound is represented by the general formula I.
  • R1 is selected from the group consisting of an aryl group, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, an arylalkylheterocyclyl group, a cyclic heteroalkyl group or a cyclic heteroalkylated alkyl group; From alkyl, cycloalkyl, haloalkyl or halocycloalkyl; more preferably the diketone is selected from the group consisting of 6-benzyloxy-3,5-dioxo-hexanoic acid tert-butyl ester, 6-benzyloxy -3,5-dioxo-hexanoic acid neopentyl ester, 6-benzyloxy-3,5-dioxo-hexanoic acid methyl ester or 6-benzyloxy-3,5-dioxo-hex
  • the biscarbonyl reductase has the advantages of increased catalytic activity, broadened substrate spectrum, increased thermal stability, and increased pH stability as compared with the prior art biscarbonylreductase, thereby catalyzing the above A more spectral substrate with higher catalytic activity.
  • the mutant of the present invention has a double carbonyl reductase mutant in a 3R, 5S-dihydroxy compound conversion reaction in an amount of only 50% of the amount of the dicarbonyl reductase encoded by the starting gene. %, and the de value of the product is increased to 99%, which is suitable for industrial applications.
  • DKR dicarbonyl reductase
  • DKR dicarbonyl reductase
  • the corresponding mutant primers were designed using Primer 5.0 (Table 1).
  • the pET22b(+) expression vector containing the biscarbonyl reductase gene purchased from Novagen, product number 69744 was used as a template, and a complete linear fragment was obtained by whole plasmid PCR, and the above PCR product was digested with DPnI to remove the female template, and then transformed.
  • Escherichia coli BL21 (DE3), it was applied to an LB culture dish containing 50 ⁇ g/ml ampicillin, and cultured at 37 ° C overnight.
  • Example 1 According to the content of Example 1, a single colony on the above solid medium was picked and inoculated into 96 deep-well plates, and 1 ml of LB liquid medium containing 50 ⁇ g/ml ampicillin was added in advance to each well, and cultured at 37 ° C, shaking at 220 rpm. After 3 h, the final concentration of IPTG was 0.1 mM, induced at 18 °C, 220 rpm for 16 h, and centrifuged at 4000 g for 15 min to collect the cells.
  • the cells were disrupted with a sonicator (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), 4 The supernatant was obtained by centrifugation at 12000 rpm for 5 min, that is, the mutant crude enzyme solution was used for the active screening of the microplate reader.
  • a sonicator JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.
  • the supernatant was obtained by centrifugation at 12000 rpm for 5 min, that is, the mutant crude enzyme solution was used for the active screening of the microplate reader.
  • enzyme activity (u/mL) ( ⁇ A ⁇ 60 ⁇ V1) / (6.22 ⁇ t ⁇ V2)
  • ⁇ A the amount of change in absorbance luminosity during the reaction
  • V1 the total volume of the reaction system
  • V2 The volume of the enzyme solution added.
  • a sonicator JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.
  • mutants with better catalytic activity than the female parent were selected for sequencing, the mutation sites were analyzed, and amplified, and the catalytic activity was determined to confirm the mutant F231W+Q251H (SEQ ID NO: 1), F231W+I94V+I236L (SEQ ID NO: 2), I94V + F231W + I236L + Q251H (SEQ ID NO: 3), F231W + I94V + Q251H (SEQ ID NO: 4) and I94V + V151Q + F231W + Q251H (SEQ ID NO: 5) catalytic activity is significantly greater than the parent of this program
  • the results of the re-screening are shown in Tables 2 and 3.
  • the three-dimensional structure of the dicarbonyl reductase was analyzed by software.
  • the I94 was located in the NAD binding region.
  • the four amino acids V151, F231, I236 and Q251 were in the vicinity of the substrate binding site. These amino acid changes may be improved on the one hand.
  • the affinity of the substrate binding, thereby increasing the activity of the enzyme, on the other hand may increase the directionality of substrate binding, thereby increasing the specificity of the enzyme (Fig. 5).
  • a in Tables 2 and 3 refers to the wet weight of each bis-carbonyl reductase mutant recombinant cell required to convert 1 g of substrate; 1 wt means that 1 g of dicarbonyl reductase mutant recombinant wet cells is required to convert 1 g of the main raw material.
  • NdeI and XhoI can be used to simultaneously digest the target gene and pET-22b(+) (other expression plasmids that can express proteins in E. coli), and digest the target gene and the larger fragment of the plasmid.
  • the ligation reaction was carried out with T4 DNA ligase, and the ligation product was transformed into competent cells of Escherichia coli DH5 ⁇ strain, and then the transformed competent cells were plated on an LB culture plate containing 50 ⁇ g/ml ampicillin, and cultured at 37 ° C overnight. .
  • the cloning vector was named pET22b(+)-RM and transformed into E. coli BL21 (DE3).
  • the transformed E. coli BL21 (DE3) was plated on LB culture plate containing 50 ⁇ g/ml ampicillin and cultured at 37 °C. overnight.
  • the bacterial solution was taken out, and the cells were collected by centrifugation at 6000 g for 15 min, and frozen at -20 ° C for use.
  • the cells were disrupted by a sonicator (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), and the supernatant and the precipitate were obtained by centrifugation at 10000 g for 20 min at 4 ° C.
  • the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • the expressed bis-carbonyl reductase mutant exhibited a molecular weight of about 30 KD on SDS-PAGE, as shown by the arrows in Figure 6.
  • Fig. 6 1 indicates mutant F231W+Q251H; 2 indicates mutant F231W+I94V+I236L; 3 indicates standard molecular weight protein marker: 97KDa, 66KDa, 43KDa, 31KDa, 14KDa from top to bottom, respectively; 4 indicates mutant I94V+F231W+I236L+Q251H;5 indicates mutant F231W+I94V+Q251H; 6 indicates mutant I94V+V151Q+F231W+Q251H; and 7 indicates female parent (F231W+I94V).
  • Examples 5 to 8 are mainly tert-butyl 6-benzyloxy-3,5-dioxo-hexanoate or 6-benzyloxy-3,5-dioxo-hexanoic acid neopentyl ester.
  • the bis-carbonyl reductase mutant provided by the present invention is used as an enzyme for catalytic reduction reaction.
  • the specific reaction equation is shown in FIG. 2 or as shown in FIG. 3, and the specific application process is as follows:
  • the diketone compound (Formula I) conforming to Formula I is selected as the starting material, wherein R1 is selected from the group consisting of an aryl group, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, and an aralkyl heterocyclic group. a cyclic heteroalkyl or cyclic heteroalkylated alkyl group; R2 is selected from the group consisting of alkyl, cycloalkyl, haloalkyl or halocycloalkyl.
  • the bishydroxy product is expressed by the following formula II: (Formula II) wherein R1 is selected from the group consisting of an aryl group, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, an aralkyl group. a cycloalkyl, cyclic heteroalkyl or cyclic heteroalkylated alkyl; R2 is selected from alkyl, cycloalkyl, haloalkyl or halocycloalkyl.
  • the nuclear magnetic data of the obtained product are as follows: 400 Hz, CDCl3: 7.29-7.35 (m, 5H), 4.53 (s, 2H), 4.21 (m, 1H), 4.05 (m, 1H), 3.43 to 3.39 (m, 4H), 2.40 (d, 2H), 1.65 (t, 2H), 1.42 (S, 9H).
  • the nuclear magnetic data of the obtained product were as follows: 400 Hz, CDCl3: 7.26 to 7.35 ppm (m, 5H), 4.58 ppm (s, 2H), 4.26 ppm (m, 1H), 4.09 ppm (m, 1H), 3.80 ppm (s, 1H), 3.47ppm (d, 2H), 3.33ppm (d, 1H), 2.46ppm (d, 2H), 1.81ppm (q, 2H), 1.62 to 1.67ppm (dd, 2H), 1.45ppm (s, 6H), 0.90 ppm (t, 3H).
  • the nuclear magnetic data of the obtained product are as follows: 400 Hz, CDCl3: ⁇ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
  • 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester in the system The ratio of (3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester) is 80 to 90%, the yield is 75 to 85%, the ee value is more than 99.3%, and the de value is 90 to 96%.
  • the nuclear magnetic data of the obtained product were as follows: 400 Hz, CDCl3: 7.26 to 7.35 ppm (m, 5H), 4.56 ppm (s, 2H), 4.24 ppm (m, 1H), 4.08 ppm (m, 1H), 3.79 ppm (s, 1H), 3.45 ppm (d, 2H), 3.30 ppm (d, 1H), 2.44 ppm (d, 2H), 1.79 ppm (q, 2H), 1.60 to 1.65 ppm (dd, 2H), 1.43 ppm (s, 6H), 0.88 ppm (t, 3H).
  • Example 7 Application of the biscarbonyl reductase mutant I94A+F231W+I236V shown in SEQ ID NO: 7 in the preparation of 3R,5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester
  • 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester in the system The ratio of (3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester) is 75 to 85%, the yield is 70 to 80%, the ee value is more than 99.5%, and the de value is 90 to 96%.
  • the nuclear magnetic data of the obtained product were as follows: 400 Hz, CDCl3: 7.26 to 7.35 ppm (m, 5H), 4.56 ppm (s, 2H), 4.24 ppm (m, 1H), 4.08 ppm (m, 1H), 3.79 ppm (s, 1H), 3.45 ppm (d, 2H), 3.30 ppm (d, 1H), 2.44 ppm (d, 2H), 1.79 ppm (q, 2H), 1.60 to 1.65 ppm (dd, 2H), 1.43 ppm (s, 6H), 0.88 ppm (t, 3H).
  • Example 8 The bis-carbonyl reductase having the sequence homology of 93.71% with the sequence shown in SEQ ID NO: 8 in Example 5 was prepared in the preparation of 3R,5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester.
  • 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester in the system The ratio of (3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid neopentyl ester) is 70 to 85%, the yield is 75 to 80%, the ee value is more than 99.5%, and the de value is 88 to 95%.
  • the nuclear magnetic data of the obtained product were as follows: 400 Hz, CDCl3: 7.26 to 7.35 ppm (m, 5H), 4.56 ppm (s, 2H), 4.24 ppm (m, 1H), 4.08 ppm (m, 1H), 3.79 ppm (s, 1H), 3.45 ppm (d, 2H), 3.30 ppm (d, 1H), 2.44 ppm (d, 2H), 1.79 ppm (q, 2H), 1.60 to 1.65 ppm (dd, 2H), 1.43 ppm (s, 6H), 0.88 ppm (t, 3H).
  • the above-described embodiments of the present invention achieve the following technical effects: by performing a fixed-point saturation mutation on the existing dimethyl-reductase mutant parent (I94V+F231W), and then by directional screening.
  • the biscarbonyl reductase mutant having the above-mentioned enzyme activity of the present invention is greatly improved, and the enzyme activity is relatively
  • the bis-carbonyl reductase parent used in the present invention is increased by a factor of 2, or even 3 times, and the enzyme specificity is also correspondingly increased, thereby greatly reducing the cost in the industrial production of 3R, 5S-dihydroxy compounds.

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Abstract

一种双羰基还原酶突变体及其应用。该双羰基还原酶突变体的氨基酸序列是SEQ ID NO：9所编码的氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列具有如下至少两个突变位点：第94位、第151位、第231位、第236位和第251位，且第94位的I突变为V、A或G；第151位的V突变为Q、N或S；第231位的F突变为W、Y或P；第236位的I突变为L、V或A；第251位的Q突变为H、R或K；或者双羰基还原酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有90％以上同源性的氨基酸序列。具有上述突变位点的双羰基还原酶突变体酶活性大幅度提高。

Description

双羰基还原酶突变体及其应用 技术领域
本发明涉及酶及酶工程领域，具体而言，涉及一种双羰基还原酶突变体及其应用。
背景技术
酶作为生物催化剂，在生物体内能充分发挥其高效和高特异性的特点。但是在工业应用中，却普遍存在无法适应工业生产条件和对非天然底物的催化能力低等问题。必须借助蛋白质工程方法对酶分子进行改造以适应不同的应用要求。蛋白质工程方法可以概括为三种：理性设计(rational design)、非理性设计(irrational design)和半理性设计(semi-rational design)。
理性设计是指在了解蛋白质的空间结构的基础上，通过定点突变(Site-directedMutagenesis)或其它方法改变蛋白质分子中的个别氨基酸，从而产生新性状的蛋白质。这种方法在理论上针对性较强，主要用于改造天然酶蛋白的催化活性、底物特异性、稳定性、改变抑制剂类型、辅酶特异性等方面。
定点饱和突变技术是蛋白质工程中的一门重要技术，属于上述的半理性设计但又结合了理性设计和非理性设计的优点，弥补了各自的不足，它通过对目的蛋白的编码基因进行改造，短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体。此技术不仅是蛋白质定向改造的强有力工具，而且是蛋白质结构－功能关系研究的重要手段。研究表明，多点突变往往能获得比单点突变更为理想的进化体。多点突变是定点突变不易直接获得的。而对于定点突变技术不能解决的这些问题，恰恰是定点饱和突变技术所擅长的独特之处。
羰基还原酶是一种氧化还原酶，在生物有机体的许多生物转化过程中发挥重要作用。基于其能催化产生高对应选择性的手性醇，羰基还原酶通常作为一种非常重要的生物催化剂应用于化学和制药工业中手性中间体的合成。而双羰基还原酶能够立体选择性地将二酮酸酯的两个羰基同时还原为相应羟基，可以用于合成关键的药物中间体，尤其是合成他汀类药物的手性二羟基己酸链，如世界上畅销的降胆固醇药物阿托伐他汀(Atorvastatin，立普妥)和瑞舒伐他汀(Rosuvastatin)。
目前已知的双羰基还原酶可以作为生物催化剂，一步还原二酮底物，制备得到近乎单一光学纯度的他汀类降血脂药物关键手性中间体3R，5S-二羟基-6-苄氧基-己酸叔丁酯，简化了合成步骤，降低了生产污染。但是在工业生产的应用中，依然有一些问 题需要进一步解决，如酶催化活性较低，使酶液的用量较大，且使反应体系总体积增加，导致了生产批次和生产成本的增加。
因此，仍需要对现有的双羰基还原酶进行改进，以提高双羰基还原酶(DKR)的酶立体选择性和催化活性。
发明内容
本发明旨在提供一种双羰基还原酶突变体及其应用，以提高双羰基还原酶(DKR)的酶立体选择性和催化活性。
为了实现上述目的，根据本发明的一个方面，提供了一种双羰基还原酶突变体，该双羰基还原酶突变体的氨基酸序列是SEQ ID NO：9所编码的氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列具有如下至少两个突变位点：第94位、第151位、第231位、第236位和第251位，且第94位的I突变为V、A或G；第151位的V突变为Q、N或S；第231位的F突变为W、Y或P；第236位的I突变为L、V或A；第251位的Q突变为H、R或K；或者双羰基还原酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有90％以上同源性的氨基酸序列。
进一步地，双羰基还原酶突变体的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5所示的氨基酸序列；或者双羰基还原酶突变体的氨基酸序列为与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQID NO：4或者SEQ ID NO：5所示的氨基酸序列具有95％以上同源性的氨基酸序列。
根据本发明的另一方面，提供了一种DNA分子，DNA分子编码上述任一种双羰基还原酶突变体。
进一步地，DNA分子的序列为SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14所示序列；或者DNA分子的序列为与SEQID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14具有95％以上同源性的序列。
根据本发明的又一方面，提供了一种重组质粒，重组质粒含有上述任一种DNA分子。
进一步地，重组质粒为pET-22b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、 pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。
根据本发明的再一方面，提供了一种宿主细胞，宿主细胞含有上述任一种重组质粒。
进一步地，宿主细胞包括原核细胞、酵母或真核细胞；优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。
根据本发明的另一方面，提供了一种生产3R，5S-二羟基化合物的方法，包括双羰基还原酶对二酮类化合物进行催化反应的步骤，双羰基还原酶为上述任一种双羰基还原酶突变体。
进一步地，二酮类化合物为通式I所示的酮类化合物：
Figure PCTCN2014094422-appb-000001
其中，R1选自芳香基、烷基、环烷基、烷基取代的芳香基、卤素取代的芳香基、芳烷杂环基、环状杂烷基或环状杂烷化烷基；R2选自烷基、环烷基、卤烷基或卤环烷基；优选二酮类化合物选自6-苄氧基-3，5-二氧代-己酸叔丁酯、6-苄氧基-3，5-二氧代-己酸新戊酯、6-苄氧基-3，5-二氧代-己酸甲酯或者6-苄氧基-3，5-二氧代-己酸乙酯。
应用本发明的技术方案，本发明的上述双羰基还原酶突变体是在SEQ ID NO：9所编码的双羰基还原酶F231W+I94V突变体的基础上，通过定点饱和突变的方法进行进一步突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的本发明的双羰基还原酶突变体具有酶活性大幅度提高的优势，其酶活相对于本发明所用的双羰基还原酶母本提高了2倍，甚至是3倍，并且酶特异性也有相应提高，从而大幅度降低了3R，5S-二羟基化合物工业生产中的成本。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明所涉及合成3R，5S-双羟基化合物的化学反应过程；
图2示出了本发明所涉及合成3R，5S-二羟基-6-苄氧基-己酸叔丁酯的化学反应方程式；
图3示出了本发明所涉及合成3R，5S-二羟基-6-苄氧基-己酸新戊酯的化学反应方程式；
图4示出了结合NAD的双羰基还原酶的三维结构模拟图；
图5示出了有效突变位点的双羰基还原酶的三维结构模拟图； 以及
图6示出了本发明优选实施例中双羰基还原酶突变体的蛋白电泳检测结果图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
由于现有技术中的双羰基还原酶存在催化活性低、酶液用量大而不适合工业化应用的缺陷，为了改善上述缺陷，本发明的发明人基于之前公开的来源于Rhodococcuserythropolis SK121的双羰基还原酶及其编码基因(CN201410188168)，进行了更深入的研究。该双羰基还原酶可以作为生物催化剂，通过一步还原二酮底物，制备得到近乎单一光学纯度的他汀类降血脂药物关键手性中间体3R，5S-二羟基-6-苄氧基-己酸叔丁酯，简化了合成步骤，降低了生产污染。在此基础上，发明人采用定点饱和突变的方法对该双羰基还原酶进行改造(CN201410196920)，得到了酶活性大幅度提高的突变体F231W+I94V，该突变体在3R，5S-二羟基-6-苄氧基-己酸叔丁酯的合成中酶的用量由6wt降低到2wt；但其在3R，5S-二羟基-6-苄氧基-己酸新戊酯的合成中，酶的用量为9wt，酶液的用量较大，使反应体系总体积增加，导致了生产批次和生产成本的增加。因此，本发明就是在双羰基还原酶突变体F231W+I94V的基础上进行的改进，以提高双羰基还原酶(DKR)的酶立体选择性和催化活性，扩大其应用范围。
本发明以红串红球菌(Rhodococcuserythropolis)SK121菌株的双羰基还原酶(DKR)的突变体F231W+I94V的基因(如SEQ ID NO：9所示)为起始基因进行基因突变，通过定向筛选的方法获得一系列酶活性提高的双羰基还原酶突变体。
通过采用软件对双羰基还原酶的三维结构进行计算机模拟分析，发现其中I94位于NAD结合区域(参见图4)，V151、F231、I236和Q251四个氨基酸均处于底物结合位点附近，这些氨基酸的改变一方面可能提高了底物结合的亲和性，从而使酶的活 性得到提高，另一方面可能提高了底物结合的方向性，从而使酶的特异性提高(参见图5)。
本发明的双羰基还原酶突变体的突变氨基酸残基位于底物结合位点或与底物和NAD结合相关、与NAD质子传递相关的的区域，例如I94位于NAD结合区域，V151、F231、I236和Q251四个氨基酸均处于底物结合位点附近，这些氨基酸的改变可能提高了底物结合的特异性，从而使酶的活性得到提高。本发明的实验结果表明，在F231W突变的基础上，引入Q251H突变可大量提高双羰基还原酶的活性。单一的I236L突变即可显著的提高双羰基还原酶的催化特异性，且单一的Q251H突变可显著的提高双羰基还原酶的催化活性。在F231W和/或V151Q和/或I94V突变的基础上，引入Q251H突变也可显著提高其双羰基还原酶的活性。I236L突变引入F231W和/或V151Q和/或I94V能显著提高双羰基还原酶产物的特异性。组合F231W和/或I94V和/或I236L和/或Q251H既能有效提高双羰基还原酶的活性，同时也能显著提高双羰基还原酶产物的特异性。
上述得到的双羰基还原酶突变体可以通过基因工程手段，将其基因连接到pET-22b(+)以及其它表达载体后在大肠杆菌中过量表达。经过量表达的双羰基还原酶突变体在SDS-PAGE上呈现的分子量约为30KD，在30℃，pH6.0条件下，可以一步还原3R，5S-双羟基化合物的制备原料得到光学纯度较高的3R，5S-双羟基化合物。
在上述研究结果的基础上，本发明提供了一种双羰基还原酶突变体，该双羰基还原酶突变体的氨基酸序列是SEQ ID NO：9所编码的氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列具有如下至少两个突变位点：第94位、第151位、第231位、第236位和第251位，且第94位的I突变为V、A或G；第151位的V突变为Q、N或S；第231位的F突变为W、Y或P；第236位的I突变为L、V或A；第251位的Q突变为H、R或K；或者双羰基还原酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有90％以上同源性的氨基酸序列。
本发明的上述双羰基还原酶突变体是在SEQ ID NO：9所编码的双羰基还原酶F231W+I94V突变体的基础上，通过定点饱和突变的方法进行进一步突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的本发明的双羰基还原酶突变体具有酶活性大幅度提高的优势，其酶活相对于本发明所用的双羰基还原酶母本提高了2倍，甚至是3倍，并且酶特异性也有相应提高，从而大幅度降低了3R，5S-二羟基化合物工业生产中的成本。
在本发明一种优选的实施例中，上述双羰基还原酶突变体的氨基酸序列为SEQ IDNO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5；或者 与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5所示的氨基酸序列具有95％以上的同源性。
本文使用的术语“同源性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同源性的规则、标准。本发明用不同程度同源性限定的序列还必须要同时具有改进的双羰基还原酶活性的活性。在上述实例中，优选双羰基还原酶突变体的氨基酸序列与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5具有95％以上的同源性并具有或编码具有改进的双羰基还原酶活性的氨基酸序列。本领域技术人员可以在本申请公开内容的教导下获得这样的变体序列。
而在本发明一种更优选的实施例中，上述双羰基还原酶的氨基酸序列为SEQ IDNO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5。SEQ IDNO：1所示的氨基酸序列的突变位点为F231W+Q251H；SEQ ID NO：2所示的氨基酸序列的突变位点为F231W+I94V+I236L；SEQ ID NO：3所示的氨基酸序列的突变位点为I94V+ F231W+I236L+Q251H；SEQ ID NO：4所示的氨基酸序列的突变位点为F231W+I94V+ Q251H； SEQ ID NO：5所示的氨基酸序列的突变位点为I94V+V151Q+F231W+ Q251H。具有上述氨基酸序列的双羰基还原酶突变体制备3R，5S-双羟基化合物，所获得的3R，5S-二羟基化合物的ee值大于99％，de值为99％左右。本发明的上述双羰基还原酶突变体为关键的药物中间体，尤其是他汀类药物的手性二羟基己酸链的合成提供了高效的催化剂，使3R，5S-二羟基化合物的工业生产成本得到大幅度降低。
在本发明另一种典型的实施方式中，提供了一种DNA分子，该DNA分子编码上述任一种双羰基还原酶突变体，其所编码的双羰基还原酶具有更高的酶催化活性，利于降低3R，5S-二羟基化合物工业生产中的成本。
在本发明一种优选的实施例中，上述DNA分子的序列为SEQ ID NO：10、SEQ IDNO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14；或者上述DNA分子的序列与SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14具有95％以上的同源性。具有上述序列的DNA分子能够编码活性进一步提高的双羰基还原酶。
在上述实施例中，SEQ ID NO：10为SEQ ID NO：9所示的双羰基还原酶基因序列中第691-693bp的TTC突变为TGG，且第751-753bp的CAA突变为CAT或CAC。上述优选实施例中，SEQ ID NO：11为SEQ ID NO：9所示的双羰基还原酶基因序列中第691-693bp的TTC突变为TGG，且第280-282bp的ATT突变为GTT、GTC、GTA或GTG，且第706-708bp的ATC突变为TTA、TTG、CTT、CTC、CTA或CTG。上 述优选实施例中，EQ ID NO：12为SEQ ID NO：9所示的双羰基还原酶基因序列中第691-693bp的TTC突变为TGG，第280-282bp的ATT突变为GTT、GTC、GTA或GTG，且第706-708bp的ATC突变为TTA、TTG、CTT、CTC、CTA或CTG，且第751-753bp的CAA突变为CAT或CAC。上述优选实施例中，SEQ ID NO：13为SEQ ID NO：9所示的双羰基还原酶基因序列中第691-693bp的TTC突变为TGG，且第280-282bp的ATT突变为GTT、GTC、GTA或GTG， 且第751-753bp的CAA突变为CAT或CAC。上述优选实施例中，SEQ ID NO：14为SEQ ID NO：9所示的双羰基还原酶基因序列中第691-693bp的TTC突变为TGG，且第280-282bp的ATT突变为GTT、GTC、GTA或GTG，且第451-453bp的GTC突变为CAA或CAG，且第751-753bp的CAA突变为CAT或CAC。
上述优选实施例中，具有上述序列的DNA分子能够编码酶催化活性更高的双羰基还原酶，其酶活性比现有技术中的双羰基还原酶的活性高2倍，甚至3倍，能够大大降低3R，5S-二羟基化合物工业生产成本。
本发明的上述DNA分子还可以以“表达盒”的形式存在。“表达盒”是指线性或环状的核酸分子,涵盖了能够指导特定核苷酸序列在恰当宿主细胞中表达的DNA和RNA序列。一般而言，包括与目标核苷酸有效连接的启动子，其任选的是与终止信号和/或其他调控元件有效连接的。表达盒还可以包括核苷酸序列正确翻译所需的序列。编码区通常编码目标蛋白，但在正义或反义方向也编码目标功能RNA，例如反义RNA或非翻译的RNA。包含目标多核苷酸序列的表达盒可以是嵌合的，意指至少一个其组分与其至少一个其他组分是异源的。表达盒还可以是天然存在的，但以用于异源表达的有效重组形成获得的。
在本发明又一种典型的实施方式中，还提供了一种重组质粒，重组质粒含有上述任一种DNA分子的序列。上述重组质粒中的DNA分子置于重组质粒的适当位置，使得上述DNA分子能够正确地、顺利地复制、转录或表达。
虽然本发明在限定上述DNA分子时所用限定语为“含有”，但其并不意味着可以在DNA序列的两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在DNA序列的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定将不能真实地覆盖这些情形。
本发明中所使用的术语“质粒”包括双链或单链线状或环状形式的任何质粒、粘粒、噬菌体或农杆菌二元核酸分子，优选为重组表达质粒，可以是原核表达质粒也可以是真核表达质粒，但优选原核表达质粒，在某些实施方案中，重组质粒选自pET-22b (+)，pET-3a(+)，pET-3d(+)，pET-11a(+)，pET-12a(+)，pET-14b(+)，pET-15b(+)，pET-16b(+)，pET-17b(+)，pET-19b(+)，pET-20b(+)，pET-21a(+)，pET-23a(+)，pET-23b(+)，pET-24a(+)，pET-25b(+)，pET-26b(+)，pET-27b(+)，pET-28a(+)，pET-29a(+)，pET-30a(+)，pET-31b(+)，pET-32a(+)，pET-35b(+)，pET-38b(+)，pET-39b(+)，pET-40b(+)，pET-41a(+)，pET-41b(+)，pET-42a(+)，pET-43a(+)，pET-43b(+)，pET-44a(+)，pET-49b(+)，pQE2，pQE9，pQE30，pQE31，pQE32，pQE40，pQE70，pQE80，pRSET-A，pRSET-B，pRSET-C，pGEX-5X-1，pGEX-6p-1，pGEX-6p-2，pBV220，pBV221，pBV222，pTrc99A，pTwin1，pEZZ18，pKK232-18，pUC-18或pUC-19。更优选，上述重组质粒是pET-22b(+)。
在本发明再一种典型的实施方式中，还提供了一种宿主细胞，该宿主细胞含有上述任一种重组质粒。适用于本发明的宿主细胞包括但不仅限于原核细胞、酵母或真核细胞。优选原核细胞为真细菌，例如革兰氏阴性菌或革兰氏阳性菌。更优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。
在本发明一种典型的实施方式中，还提供了一种生产3R，5S-二羟基化合物的方法，包括双羰基还原酶对二酮类化合物进行催化反应的步骤，其中，双羰基还原酶为上述任一种双羰基还原酶突变体。由于本发明的上述双羰基还原酶具有更高的酶催化活性，因而利用本发明的双羰基还原酶突变体制备的3R，5S-二羟基化合物不仅能够降低生产成本，而且所获得的3R，5S-二羟基化合物的ee值大于99％，de值为99％左右。
在上述方法中，3R，5S-双羟基化合物的制备原料，可以为已经在市场上商业化的原料或者容易制备的酮类化合物，在本发明中，优选二酮类化合物为通式I所示的酮类化合物：
Figure PCTCN2014094422-appb-000002
其中，R1选自芳香基、烷基、环烷基、烷基取代的芳香基、卤素取代的芳香基、芳烷杂环基、环状杂烷基或环状杂烷化烷基；R2选自烷基、环烷基、卤烷基或卤环烷基；更优选二酮类化合物选自6-苄氧基-3，5-二氧代-己酸叔丁酯、6-苄氧基-3，5-二氧代-己酸新戊酯、6-苄氧基-3，5-二氧代-己酸甲酯或者6-苄氧基-3，5-二氧代-己酸乙酯。本发明的双羰基还原酶突变体对通式I所示的二酮类化合物进行催化还原生成3R，5S-双羟基化合物的反应过程如图1所示。
本发明的上述方法中，双羰基还原酶与现有技术的双羰基还原酶相比，具有催化活性增加、底物谱变广、热稳定增加以及pH稳定性增加的优点，因而能够催化上述 更加光谱的底物，且催化活性较高。在本发明一个优选的实施例中，本发明的突变体在3R，5S-双羟基化合物转化反应中，其双羰基还原酶突变体的用量仅为起始基因编码的双羰基还原酶用量的50％，且产物的de值提高到99％，适用于工业应用。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。且下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本发明下述实施例中使用的各种抗体均来源于商业途径的标准抗体。
实施例1：
对来源于红串红球菌(Rhodococcus erythropolis)SK121菌株的双羰基还原酶(DKR)突变体(其氨基酸序列如SEQ ID NO：6所示)进行定点饱和突变。
将双羰基还原酶(DKR)的氨基酸序列在Swiss-model网站模拟蛋白质的三维结构，然后通过Docking进行底物与蛋白质的结合模拟，最后通过Pymol分析，选择有可能与底物和NAD结合相关、与NAD质子传递相关的氨基酸作为突变氨基酸(图4)。
根据突变氨基酸及其两侧的碱基序列(突变氨基酸请见表1.中的突变位点)，用Primmer5.0设计相应的突变引物(表1)。以含双羰基还原酶基因的pET22b(+)表达载体(购买于Novagen，产品编号69744)为模版，通过全质粒PCR获得完整的线性片段，将上述PCR产物经DPnⅠ消化除去母本模版后，转化到大肠杆菌BL21(DE3)中，涂布于含有50μg/ml氨苄青霉素的LB培养皿中，37℃培养过夜。
表1：点饱和突变引物序列
Figure PCTCN2014094422-appb-000003
Figure PCTCN2014094422-appb-000004
实施例2：双羰基还原酶突变体的初筛
根据实施例1所述内容，挑取上述固体培养基上的单菌落接种于96深孔板中，每孔预先加入1ml含有50μg/ml氨苄青霉素的LB液体培养基，于37℃，220rpm振荡培养3h后，加入IPTG终浓度为0.1mM，18℃，220rpm诱导培养16h，4000g离心15min收集菌体，菌体用超声破碎仪(JY92-2D，宁波新芝生物科技股份有限公司)破碎细胞，4℃，12000rpm离心5min获得上清液，即突变体粗酶液，用于酶标仪进行活性初筛。向96孔板中加入30μL DMSO，1.5μL主原料6-苄氧基-3，5-二氧代-己酸叔丁酯 (30mg/mL溶于DMSO)，2.5μL NADH(20mg/mL)，216μL磷酸盐缓冲液(100mM，pH＝6.0)，于340nm进行本底检测，然后分别向各孔中加入已经制备好的突变体酶液50μL，并立即于30℃检测340nm处吸光光度值的变化。
酶活计算公式：酶活(u/mL)＝(ΔA×60×V1)/(6.22×t×V2)
ΔA:反应过程中的吸光光度值变化量；
V1：反应体系的总体积；
6.22：消光系数；
t：ΔA的检测时间；
V2：加入的酶液体积。
实施例3：双羰基还原酶突变体的复筛
将实施例2中酶活高于母本的突变体接种于500ml含50μg/ml氨苄青霉素的LB液体培养基中，37℃振荡培养至OD600＝0.6时，加入IPTG至终浓度为0.1mM，在18℃下进行诱导表达。诱导16h后，6000g离心10min收集菌体。菌体用超声破碎仪(JY92-2D，宁波新芝生物科技股份有限公司)破碎细胞，4℃，10000g离心20min获得上清液，用于活性检测。向10ml反应瓶中加入0.05g主原料(6-苄氧基-3，5-二氧代-己酸叔丁酯或6-苄氧基-3，5-二氧代-己酸新戊酯)，0.5ml聚乙二醇PEG-400，原料溶解后，加入4.0ml磷酸盐缓冲液(100mM，pH＝6.0)，主原料均匀分散于缓冲液中；加入1.5mg NAD+，20.6mg甲酸铵，10mg辅酶甲酸脱氢酶和0.5ml双羰基还原酶，体系pH＝6.0，并于30±3℃保温16h后，薄层色谱(TLC)跟踪，选取转化产品点明显、主原料点不明显的体系进行乙酸乙酯萃取，静置分液，取有机相进行HPLC分析。
选取催化活性优于母本的突变体进行测序，分析突变位点，并进行放大培养，复测催化活性确定突变体F231W+Q251H(SEQ ID NO：1)、F231W+I94V+I236L(SEQID NO：2)、I94V+ F231W+I236L+Q251H(SEQ ID NO：3)、F231W+I94V+ Q251H(SEQID NO：4)和I94V+V151Q +F231W+ Q251H(SEQ ID NO：5)的催化活性比本方案母本显著提高，复筛结果如表2和表3所示。采用软件对双羰基还原酶的三维结构进行计算机模拟分析，其中I94位于NAD结合区域，V151、F231、I236和Q251四个氨基酸均处于底物结合位点附近，这些氨基酸的改变一方面可能提高了底物结合的亲和性，从而使酶的活性得到提高，另一方面可能提高了底物结合的方向性，从而使酶的特异性提高(图5)。
表2. 双羰基还原酶母本与突变体制备3R，5S-二羟基-6-苄氧基-己酸叔丁酯活性比较
SEQ ID NO: 位点 酶量a 转化 DE％ EE％
1 F231W+Q251H 1wt 80.14 90.08 100
2 F231W+I94V+I236L 3wt 74.42 98.98 100
3 I94V+F231W+I236L+Q251H 1wt 83.38 99 100
4 F231W+I94V+ Q251H 1wt 78.48 89.66 100
5 I94V+V151Q+F231W+Q251H 1wt 77.68 89.8 100
6 母本(F231W+I94V) 2wt 72.91 89.89 100
表3. 双羰基还原酶母本与突变体制备3R，5S-二羟基-6-苄氧基-己酸新戊酯活性比较
Figure PCTCN2014094422-appb-000005
注：表2和表3中的a指转化1g底物所需各双羰基还原酶突变体重组细胞的湿重；1wt指转化1g主原料需要1g双羰基还原酶突变体重组湿细胞。
实施例4：双羰基还原酶突变体的克隆与表达
为了便于双羰基还原酶突变体的表达以及鉴定，在其基因的5’和3’末端设计了兼容的限制性酶切位点。可以采用NdeⅠ和XhoⅠ分别将目的基因和pET-22b(+)(其他可在大肠杆菌中表达蛋白质的表达质粒也可使用)分别同时进行酶切，酶切后的目的基因和质粒的较大片段用T4DNA连接酶进行连接反应，将连接产物转化到大肠杆菌DH5α菌株的感受态细胞中，然后将转化后的感受态细胞涂布于含有50μg/ml氨苄青霉素的LB培养平板上，37℃培养过夜。
挑取上述培养皿上长出的单菌落接种于含有50μg/ml氨苄青霉素的LB液体培养基中，37℃振荡培养过夜，收集菌体进行质粒提取、PCR鉴定和双酶切鉴定后，将正 确的克隆载体命名为pET22b(+)- R-M并转化到大肠杆菌BL21(DE3)中，将转化的大肠杆菌BL21(DE3)涂布于含有50μg/ml氨苄青霉素的LB培养平板上，37℃培养过夜。挑取上述培养平板上长出的单菌落并接种于5ml含50μg/ml氨苄青霉素的LB液体培养基中，利用菌落PCR进行鉴定，将含有正确的表达载体的大肠杆菌进行后续的诱导表达。将上述菌液转接于500ml含50μg/ml氨苄青霉素的LB液体培养基中，37℃振荡培养至OD600＝0.6～0.7时，加入IPTG至终浓度分别为0.02～0.5mM，在18～25℃下进行诱导表达10～16h后，取出菌液，6000g离心15min收集菌体，于-20℃冻存备用。菌体用超声破碎仪(JY92-2D，宁波新芝生物科技股份有限公司)破碎细胞，4℃，10000g离心20min获得上清液和沉淀，上清液用垂直电泳仪进行SDS-PAGE检测。表达的双羰基还原酶突变体在SDS-PAGE上呈现的分子量约为30KD，具体见图6中箭头所指大小。
在图6中，1表示突变体F231W+Q251H；2表示突变体F231W+I94V+I236L；3表示标准分子量蛋白marker：由上到下分别为97KDa、66KDa、43KDa、31KDa、14KDa；4表示突变体I94V+F231W+I236L+Q251H；5表示突变体F231W+I94V+ Q251H；6表示突变体I94V+V151Q +F231W+Q251H；7表示母本(F231W+I94V)。
以下实施例5至8以6-苄氧基-3，5-二氧代-己酸叔丁酯或6-苄氧基-3，5-二氧代-己酸新戊酯为主原料，以本发明所提供的双羰基还原酶突变体作为酶进行催化还原反应，具体反应方程式如图2或如图3所示，具体应用过程如下：
实施例5：双羰基还原酶突变体I94V+F231W+I236L+Q251H在3R，5S-双羟基化合物制备中的应用
选用符合通式I的二酮化合物(通式I)为初始原料，其中R1选自芳香基、烷基、环烷基、烷基取代的芳香基、卤素取代的芳香基、芳烷杂环基、环状杂烷基或环状杂烷化烷基；R2选自烷基、环烷基、卤烷基或卤环烷基。所述的双羟基产物由以下化学通式II表达：(通式II)，其中R1选自芳香基、烷基、环烷基、烷基取代的芳香基、卤素取代的芳香基、芳烷杂环基、环状杂烷基或环状杂烷化烷基；R2选自烷基、环烷基、卤烷基或卤环烷基。
(1)双羰基还原酶突变体I94V+F231W+I236L+Q251H在3R，5S-二羟基-6-苄氧基-己酸叔丁酯制备中的应用
向100ml反应瓶中加入1g主原料6-苄氧基-3，5-二氧代-己酸叔丁酯：4ml聚乙二醇PEG-400，原料溶解后，加入35ml磷酸盐缓冲液(100mM，pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.03gNAD+，0.42g甲酸铵，0.1g辅酶甲酸脱氢酶和1wt双 羰基还原酶突变体I94V+F231W+I236L+Q251H的粗酶液，体系pH＝6.0，并于30±3℃保温17h；用40ml甲基叔丁基醚停止反应，用20g硅藻土过滤，40ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，产品3R，5S-二羟基-6-苄氧基-己酸叔丁酯：体系中
Figure PCTCN2014094422-appb-000006
(6-苄氧基-3，5-二氧代-己酸叔丁酯)的比例为81～83％，收率83～85％，ee值大于99.5％，de值99-99.3％。
所得产品的核磁数据如下：400Hz，CDCl3：7.29-7.35(m，5H)，4.53(s，2H)，4.21(m，1H)，4.05(m，1H)，3.43～3.39(m，4H)，2.40(d，2H)，1.65(t，2H)，1.42(S，9H)。
鉴于其他4个双羰基还原酶突变体对主原料6-苄氧基-3，5-二氧代-己酸叔丁酯的催化活性和反应方法类似，故在此不进行重复叙述。
(2)双羰基还原酶突变体I94V+F231W+I236L+Q251H在3R，5S-二羟基-6-苄氧基-己酸新戊酯制备中的应用
向100ml反应瓶中加入1g主原料6-苄氧基-3，5-二氧代-己酸新戊酯：4ml聚乙二醇PEG-400，原料溶解后，加入30ml磷酸盐缓冲液(100mM，pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.03g NAD+，0.42g甲酸铵，0.1g辅酶甲酸脱氢酶和2wt双羰基还原酶突变体I94V+F231W+I236L+Q251H粗酶液，体系pH＝6.0，并于30±3℃保温17h；用40ml乙酸乙酯停止反应，用20g硅藻土过滤，40ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，3R，5S-二羟基-6-苄氧基-己酸新戊酯：体系中
Figure PCTCN2014094422-appb-000007
(3R，5S-二羟基-6-苄氧基-己酸新戊酯)的比例为80～82％，收率81～83％，ee值大于99.5％，de值98.9～99.2％。
所得产品的核磁数据如下：400Hz，CDCl3：7.26～7.35ppm(m，5H)，4.58ppm(s，2H)，4.26ppm(m，1H)，4.09ppm(m，1H)，3.80ppm(s，1H)，3.47ppm(d，2H)，3.33ppm(d，1H)，2.46ppm(d，2H)，1.81ppm(q，2H)，1.62～1.67ppm(dd，2H)，1.45ppm(s，6H)，0.90ppm(t，3H)。
鉴于其他4个双羰基还原酶突变体对主原料6-苄氧基-3，5-二氧代-己酸新戊酯的催化活性和反应方法类似，故在此不进行重复叙述。
实施例6：双羰基还原酶母本(I94V+F231W)在3R，5S-双羟基化合物制备中的应用
(1)双羰基还原酶母本(I94V+F231W)在3R，5S-二羟基-6-苄氧基-己酸叔丁酯制备中的应用
向250ml反应瓶中加入5g主原料6-苄氧基-3,5-二氧代-己酸叔丁酯：20ml聚乙二醇PEG-400，原料溶解后，加入160ml磷酸盐缓冲液(100mM，pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.15g NAD+，20.6g甲酸铵，0.25g辅酶甲酸脱氢酶和2wt双羰基还原酶突变体I94V+F231W的粗酶液，体系pH＝6.0，并于30±3℃保温17h；用200ml乙酸乙酯停止反应，用125g硅藻土过滤，200ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，产品3R，5S-二羟基-6-苄氧基-己酸叔丁酯：体系中
Figure PCTCN2014094422-appb-000008
(6-苄氧基-3,5-二氧代-己酸叔丁酯)的比例为86～91％，收率80～86％，ee值大于99.5％，de值88～95％。
所得产品的核磁数据如下：400Hz，CDCl3：δ7.29 (m，5H)，4.54(s，2H)，4.22(m，1H)，4.07(m，1H)，3.45～3.40(m，4H)，2.41(d，2H)，1.65(t，2H)，1.43(S，9H)。
(2)双羰基还原酶突变体I94V+F231W在3R，5S-二羟基-6-苄氧基-己酸新戊酯制备中的应用
向500ml反应瓶中加入5g主原料6-苄氧基-3,5-二氧代-己酸新戊酯：10ml聚乙二醇PEG-400，原料溶解后，加入160ml磷酸盐缓冲液(100mM， pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.15g NAD+，20.6g甲酸铵，0.25g辅酶甲酸脱氢酶和9wt双羰基还原酶突变体I94V+F231W粗酶液，体系pH＝6.0，并于30±3℃保温17h；用200ml乙酸乙酯停止反应，用125g硅藻土过滤，200ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，3R，5S-二羟基-6-苄氧基-己酸新戊酯：体系中
Figure PCTCN2014094422-appb-000009
(3R，5S-二羟基-6-苄氧基-己酸新戊酯)的比例为80～90％，收率75～85％，ee值大于99.3％，de值90～96％。
所得产品的核磁数据如下：400Hz，CDCl3：7.26～7.35ppm(m，5H)，4.56ppm(s，2H)，4.24ppm(m，1H)，4.08ppm(m，1H)，3.79ppm(s，1H)，3.45ppm(d，2H),3.30ppm(d，1H)，2.44ppm(d，2H),1.79ppm(q，2H)，1.60～1.65ppm(dd，2H)，1.43ppm(s，6H)，0.88ppm(t，3H)。
实施例7：SEQ ID NO：7所示的双羰基还原酶突变体I94A+F231W+I236V在3R，5S-二羟基-6-苄氧基-己酸新戊酯制备中的应用
向500ml反应瓶中加入5g主原料6-苄氧基-3,5-二氧代-己酸新戊酯：10ml聚乙二醇PEG-400，原料溶解后，加入160ml磷酸盐缓冲液(100mM， pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.15g NAD+，20.6g甲酸铵，0.25g辅酶甲酸脱氢酶和4wt双羰基还原酶突变体I94V+F231W粗酶液，体系pH＝6.0，并于30±3℃保温17h；用200ml乙酸乙酯停止反应，用125g硅藻土过滤，200ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，3R，5S-二羟基-6-苄氧基-己酸新戊酯：体系中
Figure PCTCN2014094422-appb-000010
(3R，5S-二羟基-6-苄氧基-己酸新戊酯)的比例为75～85％，收率70～80％，ee值大于99.5％，de值90～96％。
所得产品的核磁数据如下：400Hz，CDCl3：7.26～7.35ppm(m，5H)，4.56ppm(s，2H)，4.24ppm(m，1H)，4.08ppm(m，1H)，3.79ppm(s，1H)，3.45ppm(d，2H),3.30ppm(d，1H)，2.44ppm(d，2H),1.79ppm(q，2H)，1.60～1.65ppm(dd，2H)，1.43ppm(s，6H)，0.88ppm(t，3H)。
实施例8：SEQ ID NO：8所示的与实施例5中序列同源性为93.71％的双羰基还原酶在3R，5S-二羟基-6-苄氧基-己酸新戊酯制备中的应用
向500ml反应瓶中加入5g主原料6-苄氧基-3,5-二氧代-己酸新戊酯：10ml聚乙二醇PEG-400，原料溶解后，加入160ml磷酸盐缓冲液(100mM， pH＝6.0)，将主原料均匀分散于缓冲液中；加入0.15g NAD+，20.6g甲酸铵，0.25g辅酶甲酸脱氢酶和5wt双羰基还原酶突变体I94V+F231W粗酶液，体系pH＝6.0，并于30±3℃保温17h；用200ml乙酸乙酯停止反应，用125g硅藻土过滤，200ml乙酸乙酯萃取两次，静置分液，有机相经干燥，过滤，浓缩得到粗品，3R，5S-二羟基-6-苄氧基-己酸新戊酯：体系中
Figure PCTCN2014094422-appb-000011
(3R，5S-二羟基-6-苄氧基-己酸新戊酯)的比例为70～85％，收率75～80％，ee值大于99.5％，de值88～95％。
所得产品的核磁数据如下：400Hz，CDCl3：7.26～7.35ppm(m，5H)，4.56ppm(s，2H)，4.24ppm(m，1H)，4.08ppm(m，1H)，3.79ppm(s，1H)，3.45ppm(d，2H),3.30ppm(d，1H)，2.44ppm(d，2H)，1.79ppm(q，2H)，1.60～1.65ppm(dd，2H)，1.43ppm(s，6H)，0.88ppm(t，3H)。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：通过对现有的双羰基还原酶突变体母本(I94V+F231W)进行定点饱和突变，再通过定向筛选的方法，得到本发明的上述酶活性大幅度提高的双羰基还原酶突变体，其酶活相对 于本发明所用的双羰基还原酶母本提高了2倍，甚至是3倍，并且酶特异性也有相应提高，从而大幅度降低了3R，5S-二羟基化合物工业生产中的成本。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种双羰基还原酶突变体，其特征在于，所述双羰基还原酶突变体的氨基酸序列是SEQ ID NO：9所编码的氨基酸序列发生突变的氨基酸序列，所述发生突变的氨基酸序列具有如下至少两个突变位点：第94位、第151位、第231位、第236位和第251位，且所述第94位的I突变为V、A或G；第151位的V突变为Q、N或S；第231位的F突变为W、Y或P；第236位的I突变为L、V或A；第251位的Q突变为H、R或K；或者所述双羰基还原酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与所述发生突变的氨基酸序列具有90％以上同源性的氨基酸序列。
  2. 根据权利要求1所述的双羰基还原酶突变体，其特征在于，所述双羰基还原酶突变体的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5所示的氨基酸序列；或者
    所述双羰基还原酶突变体的氨基酸序列为与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或者SEQ ID NO：5所示的氨基酸序列具有95％以上同源性的氨基酸序列。
  3. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1或2所述的双羰基还原酶突变体。
  4. 根据权利要求3所述的DNA分子，其特征在于，
    所述DNA分子的序列为SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14所示序列；或者
    所述DNA分子的序列为与SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或者SEQ ID NO：14具有95％以上同源性的序列。
  5. 一种重组质粒，其特征在于，所述重组质粒含有权利要求3或4所述的DNA分子。
  6. 根据权利要求5所述的重组质粒，其特征在于，所述重组质粒为pET-22b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b (+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求5或6所述的重组质粒。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述宿主细胞包括原核细胞、酵母或真核细胞；优选所述原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。
  9. 一种生产3R，5S-二羟基化合物的方法，包括双羰基还原酶对二酮类化合物进行催化反应的步骤，其特征在于，所述双羰基还原酶为权利要求1或2所述的双羰基还原酶突变体。
  10. 根据权利要求9所述的方法，其特征在于，所述二酮类化合物为通式I所示的酮类化合物：
    Figure PCTCN2014094422-appb-100001
    其中，R1选自芳香基、烷基、环烷基、烷基取代的芳香基、卤素取代的芳香基、芳烷杂环基、环状杂烷基或环状杂烷化烷基；R2选自烷基、环烷基、卤烷基或卤环烷基；优选所述二酮类化合物选自6-苄氧基-3，5-二氧代-己酸叔丁酯、6-苄氧基-3，5-二氧代-己酸新戊酯、6-苄氧基-3，5-二氧代-己酸甲酯或者6-苄氧基-3，5-二氧代-己酸乙酯。
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CN117126823B (zh) * 2023-09-01 2024-03-29 华南理工大学 一种酮还原酶突变体及其应用

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