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WO2016095223A1 - Double-carbonyle réductase mutante et application associée - Google Patents

Double-carbonyle réductase mutante et application associée Download PDF

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Publication number
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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Chinese (zh)
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/fr
Publication of WO2016095223A1 publication Critical patent/WO2016095223A1/fr
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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

La présente invention concerne une double-carbonyle réductase mutante et une application associée. Une séquence d'acides aminés de la double-carbonyle réductase mutante représente la séquence d'acides aminés mutante codée par SEQ ID No : 9, la séquence d'acides aminés mutante comporte au moins deux sites de mutation : No:94, No:151, No:231, No:236 et No:251, et la mutation I de No:94 est V, A ou G; la mutation V de No:151 est Q, N ou S; la mutation F de No:231 est W, Y ou P; la mutation I de No:236 est L, V ou A; la mutation Q de No:251 est H, R ou K; ou la séquence d'acides aminés de la double-carbonyle réductase mutante possède les sites de mutation dans la séquence d'acides aminés mutante, et possède plus de 90 % d'homologie avec la séquence d'acides aminés mutante. L'activité enzymatique de la double-carbonyle réductase mutante possédant les sites de mutation est sensiblement améliorée.
PCT/CN2014/094422 2014-12-19 2014-12-19 Double-carbonyle réductase mutante et application associée Ceased WO2016095223A1 (fr)

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CN113201511A (zh) * 2021-04-15 2021-08-03 华东理工大学 (R)-5-羰基癸酸(酯)还原酶突变体及其在制备(R)-γ/δ-内酯中的应用
CN113652407A (zh) * 2021-07-09 2021-11-16 浙江工业大学 羰基还原酶突变体及其不对称合成双手性化合物的应用
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Publication number Priority date Publication date Assignee Title
CN110004119A (zh) * 2019-04-18 2019-07-12 华东理工大学 ε-酮酯还原酶突变体及其催化合成(R)-α-硫辛酸前体的应用
CN113201511A (zh) * 2021-04-15 2021-08-03 华东理工大学 (R)-5-羰基癸酸(酯)还原酶突变体及其在制备(R)-γ/δ-内酯中的应用
CN113201511B (zh) * 2021-04-15 2022-08-30 华东理工大学 (R)-5-羰基癸酸(酯)还原酶突变体及其在制备(R)-γ/δ-内酯中的应用
CN113652407A (zh) * 2021-07-09 2021-11-16 浙江工业大学 羰基还原酶突变体及其不对称合成双手性化合物的应用
CN113652407B (zh) * 2021-07-09 2024-01-16 浙江工业大学 羰基还原酶突变体及其不对称合成双手性化合物的应用
CN117126823A (zh) * 2023-09-01 2023-11-28 华南理工大学 一种酮还原酶突变体及其应用
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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