WO2018127143A1 - 一种高活性s-氰醇裂解酶及其应用 - Google Patents

一种高活性s-氰醇裂解酶及其应用 Download PDF

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WO2018127143A1
WO2018127143A1 PCT/CN2018/071619 CN2018071619W WO2018127143A1 WO 2018127143 A1 WO2018127143 A1 WO 2018127143A1 CN 2018071619 W CN2018071619 W CN 2018071619W WO 2018127143 A1 WO2018127143 A1 WO 2018127143A1
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mutated
amino acid
cyanohydrin
acid residue
lyase
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PCT/CN2018/071619
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English (en)
French (fr)
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田振华
瞿旭东
程占冰
孙传民
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上海弈柯莱生物医药科技有限公司
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Priority claimed from CN201710777767.9A external-priority patent/CN108277215A/zh
Application filed by 上海弈柯莱生物医药科技有限公司 filed Critical 上海弈柯莱生物医药科技有限公司
Priority to BR112019014116-2A priority Critical patent/BR112019014116A2/pt
Priority to EP18735996.3A priority patent/EP3567105A4/en
Priority to US16/476,349 priority patent/US11492607B2/en
Publication of WO2018127143A1 publication Critical patent/WO2018127143A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds

Definitions

  • the present invention is in the field of biotechnology, and in particular, the invention relates to a highly active S-cyanohydrin lyase and use thereof.
  • Cyanohydrin lyase is an industrial enzyme that is very useful in chemical production. Its natural activity is to catalyze the cleavage of cyanohydrin and release hydrocyanic acid.
  • the cyanohydrin lyase can catalyze the reverse reaction, that is, the addition of HCN to the aldehyde ketone, to obtain an optically active ?-cyanohydrin product.
  • S-type cyanohydrin (SCMB) of m-phenoxybenzaldehyde (m-PBAld) is a key intermediate for pyrethroid pesticides.
  • SCMB S-type cyanohydrin
  • m-PBAld m-phenoxybenzaldehyde
  • the traditional chemical method has the problem of low stereoselectivity, while the S-cyanohydrin lyase-catalyzed SCMB production process is selective.
  • Natural S-cyanohydrin lyase is present in a few plant tissues such as rubber, cassava and sorghum, with low abundance and difficulty in purification.
  • Wajant isolated the cassava cyanohydrin cleavage enzyme MeHNL from a cassava by five-step purification (Plant Sci., 1995, 108, 1);
  • White et al. used a three-step process to extract MeHNL from cassava leaves using salt.
  • the enzyme solution was obtained by means of precipitation and dialysis, but the stereoselectivity applied to chemical catalysis was not high (Plant Physiol 1998, 116, 1219).
  • MeHNL The cyanohydrin lyase derived from cassava (Manihotesculenta) is an S-cyanohydrin lyase. It has been reported in the literature that MeHNL is used to catalyze the chemical synthesis of S-type chiral cyanohydrin with an ee value >99%. It has important application value, but the enzyme activity is still not high enough to meet the requirements of practical applications.
  • a mutant S-cyanohydrin lyase which is mutated at one or more sites selected from the group consisting of: amino acid residue 103 a base, a 128th amino acid residue, a 2nd amino acid residue, an 81st amino acid residue, a 149th amino acid residue, a 94th amino acid residue, and a 176th amino acid residue, wherein the amino acid residue
  • the numbering uses the number shown in SEQ ID NO.
  • the catalytic activity of the mutated S-cyanohydrin lyase is increased by more than 30% compared to the catalytic activity of the wild-type S-cyanohydrin lyase; preferably by 50% or more; more preferably The land has increased by more than 80%.
  • the catalytic activity of the mutated S-cyanohydrin lyase is at least 2 times; preferably at least 5 times; more preferably at least 10 times the wild type S-cyanohydrin lyase.
  • amino acid sequence of the wild-type S-cyanohydrin lyase is as shown in SEQ ID NO.
  • the amino acid sequence of the mutated S-cyanohydrin lyase has at least 80% homology to SEQ ID NO. 1; more preferably, has at least 90% homology. Most preferably, having at least 95% homology; such as having at least 96%, 97%, 98%, 99% homology.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme comprises the amino acid residue at position 103; preferably, the amino acid residue at position 103 is mutated from H to L, I, V, C, S or M.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises the amino acid residue at position 128; preferably, the amino acid residue at position 128 is mutated from W to A, N, L, V More preferably, G or Y, the amino acid residue at position 128 is mutated from W to A.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises a second amino acid residue; preferably, the second amino acid residue is mutated from V to P, L, D, I , G, H, R, M, S, C, W, T, Q, or A.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises amino acid residue 81; preferably, the amino acid residue at position 81 is mutated from C to A, V or I.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises an amino acid residue at position 149; preferably, the amino acid residue at position 149 is mutated from L to I, C, A or P .
  • the mutation site of the mutated S-cyanohydrin cleavage enzyme further comprises an amino acid residue at position 94; preferably, the amino acid residue at position 94 is mutated from V to P, R, S, K. .
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises the amino acid residue at position 176; preferably, the amino acid residue at position 176 is mutated from K to P.
  • the mutated S-cyanohydrin lyase is further mutated at one or more sites selected from the group consisting of amino acid residue 209, amino acid residue 94, and 165th An amino acid residue, an amino acid residue at position 140, an amino acid residue at position 224, an amino acid residue at position 173, and an amino acid residue at position 36, wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO: .
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises amino acid residue at position 209; preferably, the amino acid residue at position 209 is mutated from K to R, A, S, C. , G, M, L, F, S, or C.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises an amino acid residue at position 94; preferably, the amino acid residue at position 94 is mutated from V to P, S, C, G , R, K, S, A, F, or T.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises amino acid residue 165; preferably, the amino acid residue at position 165 is mutated from G to P, D, S, or T.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises an amino acid residue at position 140; preferably, the amino acid residue at position 140 is mutated from T to H, G, K, I. , D, W, S, or R.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises amino acid residue 224; preferably, the amino acid residue at position 224 is mutated from K to P, E, V, S , I, H, D, N, A, or T.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises amino acid residue 173; preferably, the amino acid residue at position 173 is mutated from V to Q, L, S, A , C, I, or T.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme further comprises an amino acid residue at position 36; preferably, the amino acid residue at position 36 is mutated from L to A, F, I.
  • the mutated site of the mutated S-cyanohydrin cleavage enzyme comprises the amino acid residue at position 128, and the amino acid residue at position 103.
  • the mutation site of the mutated S-cyanohydrin cleavage enzyme includes the amino acid residue at position 128 and the amino acid residue at position 103; and the mutated S-cyanohydrin lyase is One or more sites selected from the group consist of a mutation: amino acid residue 2, amino acid residue 81, amino acid residue 149, amino acid residue 176, amino acid residue 209, 94 amino acid residue, amino acid residue 165, amino acid residue 140, amino acid residue 224, amino acid residue 173, and amino acid residue 36, wherein the amino acid residue number is SEQ. IDNO: The number shown in 1.
  • the number of mutation sites in the mutated S-cyanohydrin lyase is 1-5, preferably 2-4, such as 3.
  • the mutated S-cyanohydrin lyase is selected from each of the specific mutant enzymes in Table 2.
  • the mutated S-cyanohydrin lyase comprises a mutation site of each specific mutant enzyme in Table 2.
  • the mutated S-cyanohydrin lyase is selected from the mutant enzymes 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 in Table 2.
  • the mutated S-cyanohydrin lyase comprises a mutation site selected from the group consisting of:
  • Mutant enzyme number Mutation site 3 L36A, H103L, W128A 4 V94E, H103L, W128A 5 L36C, H103L, W128A 6 L36Y, H103L, W128A 9 V94L, H103L, W128A 10 L36Q, H103L, W128A 13 C81Y, H103L, W128A
  • a polynucleotide molecule encoding the mutated S-cyanohydrin lyase of the first aspect of the invention.
  • a vector comprising the nucleic acid molecule of the second aspect of the invention is provided.
  • a host cell comprising the vector or chromosome of the first aspect of the invention, comprising the nucleic acid molecule of the second aspect of the invention.
  • the host cell is a prokaryotic cell, or a eukaryotic cell.
  • the prokaryotic cell is Escherichia coli.
  • a method for the preparation of the mutated S-cyanohydrin lyase of the first aspect of the invention comprising the steps of:
  • the temperature at which the host cell is cultured in the step (i) is from 20 ° C to 40 ° C; preferably from 25 ° C to 37 ° C, such as 35 ° C.
  • an enzyme preparation comprising the mutated S-cyanohydrin lyase according to the first aspect of the invention.
  • a seventh aspect of the invention there is provided the use of the mutated S-cyanohydrin lyase according to the first aspect of the invention, the enzyme preparation of the sixth aspect of the invention, for preparing an optically active S-cyanohydrin product.
  • the use further comprises catalyzing an addition reaction of HCN with an aldehyde ketone.
  • the reaction substrate comprises m-phenoxybenzaldehyde, HCN (or sodium cyanide/potassium cyanide), and/or acetone cyanohydrin.
  • the temperature of the catalytic reaction is 0-20 °C.
  • Figure 1 shows the results of specific enzyme activity assays of wild type and some typical mutants of the present invention.
  • Figure 2 shows the results of catalytic reaction monitoring of wild type and some typical mutants of the invention.
  • mutation of the amino acid residue 103 of the wild-type S-cyanohydrin lyase can significantly increase the expression of the mutant enzyme in E. coli, and the expression is not required to be reduced.
  • Hydroxynitrile lyase is mainly derived from a few plant tissues such as rubber, cassava and sorghum. It mainly includes: cassava cyanohydrin lyase (MeHNL), laccase cyanohydrin lyase (HbHNL), and amylopectin lyase (PaHNL).
  • the cyanohydrin lyase is a tapioca cyanohydrin lyase.
  • the cassava cyanohydrin lyase wild type sequence is as follows:
  • the wild-type coding gene sequence is as follows:
  • the present invention has developed a specific high-throughput screening method based on the reported cassava-derived S-cyanohydrin cleavage enzyme MeHNL, and directed evolution has been carried out accordingly.
  • a further cyanohydrin lyase sequence with higher enzymatic activity was obtained by further screening.
  • the mutant enzymes were prepared by high-density fermentation of E. coli, and their catalytic performance and stereoselectivity were determined. It was found that these mutant enzymes have extremely high application value, and the highest mutant enzymes for m-PBAld
  • the enzyme activity is more than 10 times that of the wild type, and the ee value is as high as about 99%, which is higher than all the reported S-cyanohydrin lyases.
  • the enzyme catalytic reaction is shown in the following formula:
  • the catalytic reaction conditions are as follows:
  • Enzyme activity assay The enzyme activity of 1 U is defined as the amount of enzyme required to catalyze the production of 1 ⁇ mol of ether aldehyde per minute.
  • the enzyme activity assay was carried out by referring to the method reported by Selmar (Analytical Biochemistry 166 (1987), 208-211), m-phenoxybenzonitrile 10 mM, methanol 20 uL, 20 mM citrate buffer (pH 5.0), and an enzyme solution of 10 uL.
  • the above reaction solution was incubated at 25 degrees, and the change in absorbance at OD 310 nm was measured within 1-5 min.
  • the curve of time (min) and absorbance change was made.
  • the slope of the curve of the experimental group was set to ⁇ K, and the slope of the control group was zero. Under the same conditions, the change in absorbance at 310 nm wavelength was recorded at 25 ° C without adding an enzyme solution. As a control group, the control group should not have a change in absorbance.
  • the invention also provides a vector comprising the optimized cyanohydrin lyase gene of the invention, and a host cell comprising the vector.
  • the vector has the ability to be expressed in E. coli, more preferably in E. coli BL21 (DE3) strain.
  • the optimized cyanohydrin cleavage gene sequences of the invention can be obtained by conventional methods that can be used by one of ordinary skill in the art, such as full artificial synthesis or PCR synthesis.
  • a preferred method of synthesis is the asymmetric PCR method.
  • the asymmetric PCR method uses a pair of unequal primers to generate a large amount of single-stranded DNA (ssDNA) after PCR amplification.
  • the pair of primers are referred to as unrestricted primers and restriction primers, respectively, and the ratio is generally 50-100:1.
  • the amplified product is mainly double-stranded DNA, but when the restriction primer (low concentration primer) is consumed, the PCR guided by the non-limiting primer (high concentration primer) will Produces a large amount of single-stranded DNA.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the polynucleotide sequence of the present invention can express or produce a protein of interest by conventional recombinant DNA technology, including the steps:
  • transforming or transducing a suitable host cell preferably an E. coli cell
  • a polynucleotide (or variant) encoding a protein of the invention or with a recombinant expression vector containing the polynucleotide
  • expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals preferably the commercially available vector: pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the invention also provides a recombinant vector comprising the optimized MeHNL DNA sequence of the invention.
  • the promoter of the recombinant vector comprises a multiple cloning site or at least one cleavage site downstream.
  • the gene of interest is ligated into a suitable multiple cloning site or restriction site to operably link the gene of interest to the promoter.
  • the recombinant vector comprises, in the 5' to 3' direction: a promoter, a gene of interest, and a terminator.
  • the recombinant vector may also include the following elements: a protein purification tag; a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selectable marker (antibiotic resistance gene, fluorescent protein, etc.) ; enhancer; or operator.
  • the expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vector.
  • any plasmid and vector can be employed as long as it is capable of replication and stabilization in the host.
  • a person skilled in the art can construct a vector containing the promoter of the present invention and/or the gene of interest using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the expression vector of the present invention can be used to transform an appropriate host cell such that the host transcribes the RNA of interest or expresses the protein of interest.
  • the host cell may be a prokaryotic cell such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. . It will be apparent to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host When the host is a prokaryote (such as E. coli), it can be treated with the CaCl 2 method or by electroporation.
  • the host When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • plants For transformed plant cells, tissues or organs, plants can be regenerated by conventional methods to obtain transgenic plants.
  • operably linked means that a gene of interest intended for transcriptional expression is linked to its control sequence in a manner conventional in the art to be expressed.
  • the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell, and cultured under conditions suitable for growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the feed type should include carbon sources such as glycerin, methanol, glucose, etc., which can be fed separately or mixed;
  • the conventional induction concentration can be used in the present invention, usually the IPTG concentration is controlled at 0.1-1.5 mM;
  • (f) There is no particular limitation with respect to the induction time, and it is usually 2 to 20 hours, preferably 5 to 15 hours.
  • the cyanohydrin cleavage enzyme of the present invention is present in the cells of Escherichia coli cells, and the host cells are collected by a centrifuge, and then the host cells are disrupted by high pressure, machine power, enzymatic cell lysis or other cell disruption methods to release the recombinant protein, preferably High pressure method.
  • the host cell lysate can be purified by flocculation, salting out, ultrafiltration, etc., followed by chromatography, ultrafiltration, etc., or can be directly purified by chromatography.
  • Chromatography techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography and the like. Commonly used chromatographic methods include:
  • Anion exchange chromatography media include, but are not limited to, Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermented sample is high, affecting the binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography.
  • the sample can be replaced by dilution buffer, ultrafiltration, dialysis, gel filtration chromatography, etc. until it is similar to the corresponding ion exchange column equilibrium solution system, and then loaded for gradient elution of salt concentration or pH.
  • Hydrophobic chromatography media include, but are not limited to, Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose.
  • the sample is increased in salt concentration by adding NaCl, (NH 4 ) 2 SO 4 , etc., and then loaded, and eluted by reducing the salt concentration.
  • the hydrophobic proteins having a large difference in hydrophobicity are removed by hydrophobic chromatography.
  • Hydrophobic chromatography media include, but are not limited to, Sephacryl, Superdex, Sephadex.
  • the buffer system was replaced by gel filtration chromatography or further purified.
  • Affinity chromatography medium include (but are not limited to): HiTrap TM Heparin HP Columns.
  • the ultrafiltration medium includes an organic film such as a polysulfone membrane, an inorganic membrane such as a ceramic membrane, and a metal membrane. Purification and concentration can be achieved by membrane filtration.
  • the present invention also provides an enzyme preparation composition comprising the cyanohydrin cleavage enzyme of the present invention.
  • the enzyme preparation composition of the present invention may further comprise: citric acid, tartaric acid, and/or boric acid.
  • the invention also provides a preparation method of S-cyanohydrin, the method comprising the steps of:
  • the reaction substrate is m-phenoxybenzaldehyde, and acetone cyanohydrin (or, hydrogen cyanide (or sodium cyanide/potassium cyanide). .
  • the temperature of the catalytic reaction is 0-20 °C.
  • the mutated S-cyanool lyase according to the present invention has a catalytic activity which is significantly improved compared with the wild type, and the catalytic activity of some mutants is even more than 10 times that of the wild type;
  • the mutated S-cyanool lyase according to the present invention can be expressed in a large amount in engineered Escherichia coli, and thus the preparation cost is low.
  • the mutant S-cyanohydrin lyase according to the present invention can be expressed at a high temperature (about 25-37 ° C), greatly reducing the production cost and simplifying the fermentation process, while the wild type and some mutants are Expression at high temperature is inactive or extremely low in activity.
  • the construction steps of the mutant library are as follows:
  • H103-f 5'-GCAGCTGGCGTTTTCNNNAACTCCCTGCTGCCG-3' (SEQ ID NO. 3)
  • H103-r 5'-CGGCAGCAGGGAGTTNNNGAAAACGCCAGCTGC-3' (SEQ ID NO. 4)
  • the target band was amplified by PCR using the plasmid pET21a-meHNL as a template.
  • the procedure is as follows:
  • the PCR product was subjected to DpnI digestion at 37 ° C for 2 hr.
  • the reaction was completed by transforming competent cells E. coli BL21 (DE3), plated in LB medium containing 100 ug/mL ampicillin, and cultured overnight at 37 ° C to obtain a mutant library.
  • N in the sequence of the present application represents A, T, G or C.
  • Enzyme activity assay The enzyme activity of 1 U is defined as the amount of enzyme required to catalyze the production of 1 ⁇ mol of ether aldehyde per minute.
  • the enzyme activity assay was carried out by referring to the method reported by Selmar (Analytical Biochemistry 166 (1987), 208-211), m-phenoxybenzonitrile 10 mM, methanol 20 uL, 20 mM citrate buffer (pH 5.0), and an enzyme solution of 10 uL.
  • the above reaction solution was incubated at 25 degrees, and the change in absorbance at OD 310 nm was measured within 1-5 min.
  • the curve of time (min) and absorbance change was made.
  • the slope of the curve of the experimental group was set to ⁇ K, and the slope of the control group was zero. Under the same conditions, the change in absorbance at 310 nm wavelength was recorded at 25 ° C without adding an enzyme solution. As a control group, the control group should not have a change in absorbance.
  • a deoxyribonucleic acid sequence encoding a mutant enzyme was synthesized, and ligated into the NdeI and XhoI sites of the pET28a vector (purchased from Novagen) to obtain an E. coli plasmid pET28-MeHNL6 containing a T7 promoter.
  • the plasmid was transformed into Escherichia coli BL21 (DE3) (purchased from Invitrogene), and the corresponding strain was obtained on a Kana-resistant plate, inoculated into LB medium, cultured overnight at 37 ° C, and the strain was preserved with 20% glycerol.
  • the strain was inoculated into a 1 L shake flask containing 200 mL of LB medium, and cultured at 37 ° C, 180-220 rpm for 10-16 h.
  • the cultured seeds were inoculated in a 3 L upper tank fermentation medium (M9) at a ratio of 10% (v/v) (glucose 4 g/L, disodium hydrogen phosphate 12.8 g/L, potassium dihydrogen phosphate 3 g/L).
  • the fermentation method for wild type and partial mutants (such as mutant 2) is basically the same as above, except that the temperature is maintained at a low temperature (about 12-16 ° C) level during the fermentation.
  • the enzyme obtained by fermentation can be purified by a method conventional in the art.
  • the enzyme obtained by fermentation can also be purified by the following exemplary method, for example:
  • the wild-type sequence 1 L fermentation broth was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM sodium phosphate buffer (pH 5.5) in a ratio of 4 mL of buffer per g of the cells.
  • the mixture was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyacrylamide was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm.
  • the supernatant was concentrated 8 times (protein concentration 93 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 198 U/mL.
  • the 1st fermentation broth containing the mutant 9 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM potassium citrate buffer (pH 5.5) in a ratio of 4 mL of buffer per g of the cells. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyacrylamide was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 5 times (protein concentration 65 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 522 U/mL.
  • the 1st fermentation broth containing the mutant 27 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM potassium phosphate buffer (pH 5.5) in a ratio of 4 mL of buffer per g of the cells. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyacrylamide was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 5 times (protein concentration 69 mg/mL) with an ultrafiltration membrane, and the enzyme activity was 687 U/mL.
  • a 1 L fermentation broth containing the mutant 55 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 50 mM sodium citrate buffer (pH 5.5) in a ratio of 4 mL of buffer per gram of the cells. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyacrylamide was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 5 times (protein concentration 62 mg/mL) with an ultrafiltration membrane, and the enzyme activity was 958 U/mL.
  • a 1 L fermentation broth containing the mutant 72 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in a 20 mM sodium tartrate buffer (pH 5.0) in a ratio of 4 mL of buffer per g of the cells. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyethyleneimine was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 5 times (protein concentration 75 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 1530 U/mL.
  • the 1 column of the fermentation broth containing the mutant 113 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM sodium citrate-20 mM sodium phosphate buffer (pH 5.0) in a ratio of 4 mL per gram of the cells. Buffer. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyethyleneimine was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 3 times (protein concentration 64 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 1613 U/mL.
  • the 1 liter fermentation broth containing the mutant 135 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM sodium citrate-20 mM sodium phosphate buffer (pH 5.2) in a ratio of 4 mL per gram of the cells. Buffer. It was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyethyleneimine was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm. The supernatant was concentrated 3 times (protein concentration 55 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 1876 U/mL.
  • a 1 L fermentation broth containing the mutant 149 sequence was centrifuged (4000 rpm) to obtain 50 g of the cells; the cells were resuspended in 20 mM potassium phosphate buffer (pH 5.5) in a ratio of 4 mL of buffer per g of the cells.
  • the mixture was crushed by a high-pressure homogenizer (pressure: 800-1000 bar), polyacrylamide was added for flocculation (1-2 Torr), and the supernatant was collected by centrifugation at 4000 rpm.
  • the supernatant was concentrated 6 times (protein concentration: 56 mg/mL) with an ultrafiltration membrane, and the activity of the enzyme was 680 U/mL.
  • * represents a specific activity between 0-3.0 U / mg; ** represents a specific activity between 3.0-10.0 U / mg; *** represents a specific activity between 10.0-18.0 U / mg ;**** represents a specific activity between 18.0-26.0U / mg; ***** represents a specific activity between 26.0-34.0U / mg; ****** represents a specific activity > 34.0 Between U/mg.
  • the biocatalytic conversion of S-cyanohydrin is carried out by adding 20 mL of cyanohydrin lyase, 10 mL of aldehyde m-PBAld, 20 mL of methyl tert-butyl ether, 3 g of HCN, and stirring at 15 ° C in a 100 mL reaction flask.
  • the detection method is as follows:
  • the reaction was monitored by high performance liquid chromatography (HPLC): water and acetonitrile (45:55) as mobile phase, ODS-18 reversed phase column, Shimadzu LC-15C high performance liquid chromatography, UV absorption at 210 nm
  • HPLC high performance liquid chromatography
  • the reaction system was diluted with water and acetonitrile (45:55), centrifuged and filtered through a nylon membrane and then injected.
  • the progress of the reaction is detected by HPLC: after 1 hour of the reaction, the detection is 17.3 min for m-phenoxybenzaldehyde and 17.5 min for S-configuration cyanohydrin.
  • the product of the S-configuration prepared by the comparison of the present invention is identical to the target substance standard (purchased from Jiangxi Keyuan Biopharmaceutical Co., Ltd.).

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Abstract

提供了一种活性提高的S-氰醇裂解酶，是对野生型木薯S-氰醇裂解酶的103位氨基酸残基进行突变得到。该突变能显著提高突变酶在大肠杆菌中的表达，并且诱导表达时不需要降低温度。进一步对第128位等其它位点进行突变，获得了催化活性提高的突变体。

Description

一种高活性S-氰醇裂解酶及其应用 技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种高活性S-氰醇裂解酶及其应用。
背景技术
氰醇裂解酶是一种在化工生产中非常有用的工业用酶，其天然活性是催化氰醇的裂解并释放出氢氰酸。氰醇裂解酶可以催化逆反应，即HCN与醛酮的加成，得到具有光学活性的α-氰醇产物。间苯氧基苯甲醛(m-PBAld)的S-型氰醇(SCMB)是菊酯类农药的关键中间体。传统的化学法存在立体选择性不高的问题，而S氰醇裂解酶催化的SCMB生产工艺具有选择性。
天然的S-氰醇裂解酶存在于橡胶、木薯和高粱等少数几种植物组织中，丰度低，纯化难度大。1995年，Wajant采用五步纯化法从木薯中分离得到了木薯氰醇裂解酶MeHNL(Plant Sci.,1995,108,1)；White等人采用三步法从木薯叶中提取了MeHNL，采用盐析和透析的方式得到了酶液，但是应用于化学催化的立体选择性不高(Plant Physiol 1998,116,1219)。来源于木薯(Manihotesculenta)的氰醇裂解酶(MeHNL)是一种S-氰醇裂解酶，已有文献报道将MeHNL用于催化S-型手性氰醇的化学合成，ee值>99％，具有重要的应用价值，但是酶活仍然不够高，难以达到实际应用的要求。
因此，本领域技术人员致力于开发具有更高活性的S-氰醇裂解酶，以降低应用成本。
发明内容
本发明的目的在于提供一种高活性S-氰醇裂解酶及其应用。
本发明的第一方面，提供了一种突变的S-氰醇裂解酶，所述突变的S-氰醇裂解酶在选自下组的一个或多个位点发生突变：第103位氨基酸残基、第128位氨基酸残基、第2位氨基酸残基、第81位氨基酸残基、第149位氨基酸残基、第94位氨基酸残基、和第176位氨基酸残基，其中，氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述突变的S-氰醇裂解酶的催化活性与野生型S-氰醇裂解酶的催化活性相比提高了30％以上；优选地提高了50％以上；更优选地提高了80％以上。
在另一优选例中，所述突变的S-氰醇裂解酶的催化活性是野生型S-氰醇 裂解酶的至少2倍；优选地至少5倍；更优选地至少10倍。
在另一优选例中，所述野生型S-氰醇裂解酶的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述突变的S-氰醇裂解酶的氨基选序列与SEQ ID NO.1相比具有至少80％的同源性；更优选地，具有至少90％的同源性；最优选地，具有至少95％的同源性；如具有至少96％、97％、98％、99％的同源性。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点包括第103位氨基酸残基；优选地，第103位氨基酸残基由H突变为L、I、V、C、S或M。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第128位氨基酸残基；优选地，第128位氨基酸残基由W突变为A、N、L、V、G或Y，更优选地，第128位氨基酸残基由W突变为A。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第2位氨基酸残基；优选地，第2位氨基酸残基由V突变为P、L、D、I、G、H、R、M、S、C、W、T、Q、或A。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第81位氨基酸残基；优选地，第81位氨基酸残基由C突变为A、V或I。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第149位氨基酸残基；优选地，第149位氨基酸残基由L突变为I、C、A或P。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第94位氨基酸残基；优选地，第94位氨基酸残基由V突变为P、R、S、K。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第176位氨基酸残基；优选地，第176位氨基酸残基由K突变为P。
在另一优选例中，所述突变的S-氰醇裂解酶进一步地在选自下组的一个或多个位点发生突变：第209位氨基酸残基、第94位氨基酸残基、第165位氨基酸残基、第140位氨基酸残基、第224位氨基酸残基、第173位氨基酸残基、和第36位氨基酸残基，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第209位氨基酸残基；优选地，第209位氨基酸残基由K突变为R、A、S、C、G、M、L、F、S、或C。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第94位氨基酸残基；优选地，第94位氨基酸残基由V突变为P、S、C、G、R、K、S、A、F、或T。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第165位氨基酸残基；优选地，第165位氨基酸残基由G突变为P、D、S、或T。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第140位氨 基酸残基；优选地，第140位氨基酸残基由T突变为H、G、K、I、D、W、S、或R。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第224位氨基酸残基；优选地，第224位氨基酸残基由K突变为P、E、V、S、I、H、D、N、A、或T。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第173位氨基酸残基；优选地，第173位氨基酸残基由V突变为Q、L、S、A、C、I、或T。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点还包括第36位氨基酸残基；优选地，第36位氨基酸残基由L突变为A、F、I。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点包括第128位氨基酸残基、和第103位氨基酸残基。
在另一优选例中，所述突变的S-氰醇裂解酶的突变位点包括第128位氨基酸残基、和第103位氨基酸残基；并且，所述突变的S-氰醇裂解酶在选自下组的一个或多个位点发生突变：第2位氨基酸残基、第81位氨基酸残基、第149位氨基酸残基、第176位氨基酸残基、第209位氨基酸残基、第94位氨基酸残基、第165位氨基酸残基、第140位氨基酸残基、第224位氨基酸残基、第173位氨基酸残基、和第36位氨基酸残基，其中，氨基酸残基编号采用SEQ IDNO:1所示的编号。
在另一优选例中，所述突变的S-氰醇裂解酶中突变位点的数量为1-5个，优选为2-4个，如3个。
在另一优选例中，所述突变的S-氰醇裂解酶选自表2中的各具体突变体酶。
在另一优选例中，所述突变的S-氰醇裂解酶包括表2中的各具体突变体酶的突变位点。
在另一优选例中，所述突变的S-氰醇裂解酶选自表2中的突变体酶86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138或139。
在另一优选例中，所述突变的S-氰醇裂解酶包括选自下组的突变位点：
突变体酶编号 突变位点
3 L36A，H103L,W128A
4 V94E，H103L,W128A
5 L36C，H103L,W128A
6 L36Y，H103L,W128A
9 V94L，H103L,W128A
10 L36Q，H103L,W128A
13 C81Y，H103L,W128A
18 V94Q，H103L,W128A
20 V94H，H103L,W128A
21 H103L,W128A，V173T
22 C81Y，H103L，W128A
27 C81V，H103L，W128A
29 H103L,W128A，V173I
30 V94T，H103L,W128A
31 H103L,W128A，V173C
34 H103L,W128A，149A
35 V94F，H103L,W128A
36 H103L,W128A，V173A
37 L36I，H103L,W128A
38 H103L,W128A，V173S
39 L36F，H103L,W128A
40 H103S
41 C81I，H103L，W128A
42 V94A，H103L,W128A
43 V2P,H103L,W128A
44 V2W,H103L,W128A
45 V2T,H103L,W128A
46 V94S,H103L,W128A，K209R
47 H103L,W128A，V173L,K209C
48 V94R,H103L,W128A，K209C
49 H103V
50 H103L,W128A，G165T
51 H103L,W128A，V173L,K209S
52 V2H,H103L,W128A
53 H103L,W128A，K224T
54 V2D,H103L,W128A
55 V94G，H103L,W128A
56 V2P,C81A H103L,W128A,L149C
57 V2S,H103L,W128A
58 H103L,W128A，K224A
59 V2Q,H103L,W128A
60 H103L,W128A，K199P,K176P
61 V2R,H103L,W128A
62 V94R，H103L,W128A，V173L
63 H103I
64 H103L,W128A，K199P
65 H103L,W128A，K176P
66 V94C，H103L,W128A
67 H103L,W128A，K224N
68 H103L,W128A，K224D
69 V94S，H103L,W128A，V173L
70 H103L,W128A，K199P,K224P
71 V2C,H103L,W128A
72 H103L,W128A
73 H103L,W128A，K224P
74 H103L,W128A，V173L
75 H103L,W128A，K224H
76 H103L,W128A，K224I
77 H103L,W128A，K224S
78 H103L,W128A，K224V
79 H103L,W128A，G165S
80 H103L,W128A，K176P,K224P
81 H103C
82 H103L,W128A，V173Q
83 H103L,W128A，K224E
84 V94S,H103L,W128A，K209C
85 H103L,W128A，K224P
86 H103L,W128A，T140R
87 H103L
88 H103L,W128A，T140S
89 H103L,W128A，T140W
90 H103L,W128A，T140D
91 V94S,H103L,W128A，G165D
92 H103L,W128A，T140I
93 H103L,W128A，T140K
94 H103L,W128A，G165P
95 H103L,W128A，T140G
96 H103L,W128A，T140H
97 V94R，H103L,W128A
98 H103L,W128A，K209F
99 H103L,W128A，G165D
100 V94R,H103L,W128A，K209R
101 V94R,H103L,W128A，G165D
102 V94S，H103L,W128A
103 H103L,W128A，K209L
104 C81A，H103L,W128A
105 H103L,W128A，K209M
106 H103L,W128A，K209G
107 H103L,W128A，K209A
108 H103L,W128A，K209S
109 H103L,W128A，K209C
110 C81A,H103L,W128A，K224P
111 C81A，H103L，W128A
112 H103L,W128A，K209R
113 V2I，H103L，W128A
114 C81A,H103L,W128A，K176P
115 V2A,C81A,H103L,W128A,L149C
116 L36A，H103L,W128A
117 V2G,C81A,H103L,W128A
118 V2L,C81A,H103L,W128A
119 V2P,C81A,H103L,W128A
120 V2H,C81A,H103L,W128A
121 V2R,C81A,H103L,W128A
122 V2M,C81A,H103L,W128A
123 V2S,C81A,H103L,W128A
124 V2C,C81A,H103L,W128A
125 V2W,C81A,H103L,W128A
126 V2T,C81A,H103L,W128A
127 V2Q,C81A,H103L,W128A
128 V2A,C81A,H103L,W128A
129 C81A,H103L,W128A,L149P
130 C81A,H103L,W128A,L149I
131 C81A,H103L,W128A,L149C
132 C81A,V94P,H103L,W128A,K176P
133 C81A,94R,H103L,W128A,L149P
134 C81A,94K,H103L,W128A,L149P
135 V2P,C81A,H103L,W128A,L149C
136 H103I,W128A
137 H103V,W128A
138 H103C,W128A
139 H103S,W128A
140 H103I,W128Y
141 H103L,W128N
142 H103L,W128G
143 H103L,W128Y
144 H103I,W128N
145 H103I,W128G
146 H103C,W128V
147 H103C,W128G
148 H103C,W128Y；和
149 H103M,W128L。
本发明的第二方面，提供了一种多核苷酸分子，所述多核苷酸分子编码本发明第一方面所述的突变的S-氰醇裂解酶。
本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的核酸分子。
本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第一方面所述的载体或染色体整合有本发明第二方面所述的核酸分子。
在另一优选例中，所述宿主细胞为原核细胞、或真核细胞。
在另一优选例中，所述原核细胞为大肠杆菌。
本发明的第五方面，提供了一种制备本发明第一方面所述的突变的S-氰醇裂解酶的方法，包括步骤：
(i)在适合的条件下，培养本发明第四方面所述的宿主细胞，从而表达出所述的突变的氰醇裂解酶；和
(ii)分离所述的突变的氰醇裂解酶。
在另一优选例中，所述步骤(i)中培养所述宿主细胞的温度为20℃-40℃；优选为25℃-37℃，如35℃。
本发明的第六方面，提供了一种酶制剂，所述酶制剂包含本发明第一方面所述的突变的S-氰醇裂解酶。
本发明的第七方面，提供了本发明第一方面所述的突变的S-氰醇裂解酶、本发明第六方面所述的酶制剂的用途，用于制备具有光学活性的S-氰醇产物。
在另一优选例中，所述用途还包括催化HCN与醛酮的加成反应。
本发明的第八方面，提供了一种制备S-氰醇的方法，包括步骤：
(1)将本发明第一方面所述的突变的S-氰醇裂解酶与反应底物接触，进行催化反应，从而生成所述S-氰醇；
(2)分离并纯化所述S-氰醇产物。
在另一优选例中，所述步骤(1)中，所述反应底物包括间苯氧基苯甲醛、HCN(或氰化钠/氰化钾)，和/或丙酮氰醇。
在另一优选例中，所述步骤(1)中，催化反应的温度为0-20℃。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了野生型和本发明部分典型突变体的比酶活测定结果。
图2显示了野生型和本发明部分典型突变体的催化反应监测的结果。
具体实施方式
本发明人通过广泛而深入的研究，意外地发现，对野生型S-氰醇裂解酶的103位氨基酸残基进行突变能够显著提高突变酶在大肠杆菌中的表达，而且诱导表达时不需要降低温度，因而显著地降低了酶的制备成本；进一步地对第128位等其它位点进行突变获得了催化活性提高的S-氰醇裂解酶，实验结果表明，该类突变的S-氰醇裂解酶在催化间-苯氧基苯甲醛(m-PBAld)和HCN的加成反应的催化活性与野生型相比提高了30％以上，在此基础上完成了本发明。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
氰醇裂解酶
氰醇裂解酶(Hydroxynitrile lyase)主要来源于橡胶、木薯和高粱等少数几种植物组织。主要包括：木薯氰醇裂解酶(MeHNL)、漆树氰醇裂解酶(HbHNL)、杏仁氰醇裂解酶(PaHNL)。
在本发明优选的实施方式中，所述氰醇裂解酶为木薯氰醇裂解酶。
在本发明优选的实施方式中，优选地木薯氰醇裂解酶野生型序列如下：
Figure PCTCN2018071619-appb-000001
野生型编码基因序列如下：
Figure PCTCN2018071619-appb-000002
Figure PCTCN2018071619-appb-000003
突变的高活性氰醇裂解酶
本发明根据已报道的源于木薯的S-氰醇裂解酶MeHNL开发了特异性的高通量筛选方法，并据此进行了定向进化。通过进一步筛选得到了具有更高酶活的氰醇裂解酶序列。通过大肠杆菌的高密度发酵大量制备了突变体酶，并对其催化性能和立体选择性进行了测定，发现这些突变体酶具有极高的应用价值，其中最高的突变体酶对m-PBAld的比酶活达到野生型的10倍以上，同时ee值高达约99％，高于所有已报道的S氰醇裂解酶。酶催化反应如下式所示：
Figure PCTCN2018071619-appb-000004
优选地，催化反应条件如下：
酶活测定：1U的酶活定义为，每分钟催化生成1μmol醚醛所需的酶量。
酶活测定参考Selmar报道的方法(Analytical Biochemistry 166(1987)，208-211)，间苯氧基苯甲氰醇10mM，甲醇20uL，20mM柠檬酸缓冲液(pH5.0)，酶液10uL。上述反应液于25度温育，在1-5min内测定OD310nm的吸光度变化。做时间(min)与吸光度变化的曲线，实验组曲线斜率设为△K，对照组斜率为零。相同条件下，不加酶液的情况下，25℃条件，记录310nm波长吸光度的变化，作为对照组，对照组不应有吸光度的变化。
间氧基苯甲醛浓度标准曲线斜率为K。按照公式计算酶活：
Figure PCTCN2018071619-appb-000005
载体和宿主细胞
本发明还提供了一种包含本发明的优化的氰醇裂解酶基因的载体，以及含所述载体的宿主细胞。
在本发明的一个优选例中，所述载体具有在大肠杆菌(更佳地在大肠杆菌BL21(DE3)菌株)中表达的能力。
本领域的普通技术人员可以使用的常规方法获得本发明的优化的氰醇裂解酶基因序列，例如全人工合成或PCR法合成。一种优选的合成法为不对称PCR法。不对称PCR法是用不等量的一对引物，PCR扩增后产生大量的单链DNA(ssDNA)。这对引物分别称为非限制引物与限制性引物，其比例一般为50-100∶1。在PCR反应的最初10-15个循环中，其扩增产物主要是双链DNA，但当限制性引物(低浓度引物)消耗完后，非限制性引物(高浓度引物)引导的PCR就会产生大量的单链DNA。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明的多核苷酸序列可以通过常规的重组DNA技术，表达或生产目的蛋白，包括步骤：
(1)用编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞，较佳地大肠杆菌细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体，优选市售的载体：pET28。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
本发明还提供的重组载体，其包含本发明的经过优化的MeHNL DNA序列。在优选的实施方式中，所述重组载体的启动子下游包含多克隆位点或至少一个酶切位点。需要表达目的基因时，将目的基因连接入适合的多克隆位点或酶切位点，从而可操作地连接目的基因与启动子。
在另一优选的实施方式中，所述重组载体在5'到3'方向上包括：启动子，目的基因和终止子。如果需要，所述重组载体还可以包括以下元件：蛋白纯化标签；3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；选择标记(抗生素抗性基因、荧光蛋白等)；增强子；或操作子。
用于制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要其能够在宿主体内复制和稳定，任何质粒和载体都可以被采用。
本领域普通技术人员可以采用熟知的方法构建含有本发明启动子和/或目的 基因序列的载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明的表达载体，可以用于转化适当的宿主细胞，以使宿主转录目的RNA或表达目的蛋白质。宿主细胞可以是原核细胞，如大肠杆菌、谷氨酸棒杆菌、黄色短杆菌、链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
术语“可操作连接”是指将准备转录表达的目的基因以一种本领域的常规方式连接到它的控制序列以被表达。
工程菌的培养和目的蛋白发酵生产
在获得工程细胞后，便可在适合的条件下培养工程细胞，表达本发明的基因序列所编码的蛋白。根据宿主细胞的不同，培养中所用的培养基可选自各种常规培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在本发明中，可采用常规的发酵条件。代表性的条件包括(但并不限于)：
(a)就温度而言，氰醇裂解酶的发酵及诱导温度保持在25-37℃；
(b)就诱导期的pH值而言，诱导期pH控制在3-9；
(c)就溶氧(DO)而言，DO控制在10-90％，溶氧的维持可以用氧气/空气混合气体的通入来解决；
(d)就补料而言，补料种类宜包括甘油、甲醇、葡萄糖等碳源，可单独补料或混合补料；
(e)就诱导期IPTG浓度而言，常规诱导浓度都可用于本发明，通常IPTG浓度控制在0.1-1.5mM；
(f)就诱导时间而言，没有特别限制，通常为2-20小时，较佳地为5-15小时。
本发明的目的蛋白氰醇裂解酶存在大肠杆菌细胞胞内，通过离心机收集宿主细胞，然后通过高压、机器力、酶解细胞被或其他细胞破碎方法破碎宿主细胞，释放重组蛋白，优选的是高压法。宿主细胞裂解液可通过絮凝、盐析、超 滤等方法进行初步纯化后再进行层析、超滤等纯化，也可直接进行层析纯化。
层析技术包括阳离子交换层析、阴离子交换层析、凝胶过滤层析、疏水层析、亲和层析等技术。常用的层析方法包括：
1.阴离子交换层析：
阴离子交换层析介质包括(但不限于)：Q-Sepharose、DEAE-Sepharose。如果发酵样品的盐浓度较高，影响与离子交换介质的结合，则在进行离子交换层析前需降低盐浓度。样品可以用稀释、超滤、透析、凝胶过滤层析等手段进行平衡缓冲液的更换，直至与对应的离子交换柱平衡液系统相似，然后上样，进行盐浓度或pH的梯度洗脱。
2.疏水层析：
疏水层析介质包括(但不限于)：Phenyl-Sepharose、Butyl-Sepharose、Octyle-Sepharose。样品通过添加NaCl、(NH 4) 2SO 4等方式提高盐浓度，然后上样，通过降低盐浓度方法洗脱。通过疏水层析除去疏水性有较大差异的杂蛋白。
3.凝胶过滤层析
疏水层析介质包括(但不限于)：Sephacryl、Superdex、Sephadex类。通过凝胶过滤层析更换缓冲体系，或进一步精纯。
4.亲和层析
亲和层析介质包括(但不限于)：HiTrap TM Heparin HP Columns。
5.膜过滤
超滤介质包括：有机膜如聚砜膜、无机膜如陶瓷膜、金属膜类。通过膜过滤可以达到纯化和浓缩的目的。
制备酶制剂组合物
本发明还提供了一种酶制剂组合物，该酶制剂组合物中包含本发明的氰醇裂解酶。
本发明的酶制剂组合物还可以包含：柠檬酸、酒石酸、和/或硼酸。
S-氰醇的制备方法
本发明还提供了一种S-氰醇的制备方法，所述方法包括步骤：
(1)将本发明的突变的氰醇裂解酶与反应底物接触，进行催化反应，从而生成所述S-氰醇；
(2)分离并纯化所述S-氰醇产物。
在本发明优选地实施方式中，所述步骤(1)中，所述反应底物为间苯氧基苯甲醛、和丙酮氰醇(或，氰氢酸(或氰化钠/氰化钾)。
在本发明优选地实施方式中，所述步骤(1)中，催化反应的温度为0-20℃。
本发明的主要优点在于：
(1)根据本发明的突变的S氰醇裂解酶，其催化活性与野生型相比显著提高，某些突变体的催化活性甚至达到了野生型的10倍以上；
(2)根据本发明的突变的S氰醇裂解酶能够在工程化的大肠杆菌中大量表达，因而制备成本较低。
(3)根据本发明的突变的S氰醇裂解酶均能够在高温(约25-37℃)下表达，极大的降低了生产成本并简化了发酵工艺，而野生型及某些突变体在高温下表达无活性或活性极低。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1：突变体文库的构建
以H103位点突变为例，突变体文库的构建步骤如下：
定点饱和突变H103位点，设计引物
H103-f：5’-GCAGCTGGCGTTTTCNNNAACTCCCTGCTGCCG-3’(SEQ ID NO.3)
H103-r：5’-CGGCAGCAGGGAGTTNNNGAAAACGCCAGCTGC-3’(SEQ ID NO.4)
以质粒pET21a-meHNL为模板，PCR扩增目的条带，程序如下：
Figure PCTCN2018071619-appb-000006
对PCR产物进行DpnI消化，37℃，2hr。反应完成转化感受态细胞E.coliBL21(DE3)，涂布在含有100ug/mL氨苄青霉素的LB培养基，37℃培养过夜，得到突变体文库。
采用上述一样的方法，对其它位点进行突变体文库构建。针对其它位点突变体文库构建所设计的引物序列如下表所示： 表1
Figure PCTCN2018071619-appb-000007
Figure PCTCN2018071619-appb-000008
其中，本申请序列中N代表A、T、G或C。
实施例2：高通量筛选
按照如下实验步骤进行筛选：
1.选克隆子接种 96孔板1(每孔500μL TB培养基)，30℃过夜培养；
2.转新 96孔板2(每孔800μL TB培养基,0.15mM IPTG)，从 96孔板1接种100μL至 96孔板2，30℃过夜诱导培养。
3.收菌 96孔板2，加BugBuster Protein Extraction Reagent(Novagen)100μL，处理30min，离心取上清。
4.将酶液稀释至合理倍数。
5.制备酶标板，反应体系200μL，其中50mM柠檬酸缓冲液150μL(含15％甲醇)，底物SCMB(0.05g/mL溶于甲醇)5μL，酶液5μL反应2min，立即加入10μL溶液I(100mM N-氯代琥珀酰亚胺)反应2min，再加入30μL溶液II(65mM异烟酸，125mM巴比妥酸，溶于0.2M NaOH)，20min后在600nm波长处读数。
6.以野生型为参照系，选择600nm吸光度最强的最为阳性克隆子，分析其酶活和比酶活。
酶活测定：1U的酶活定义为，每分钟催化生成1μmol醚醛所需的酶量。
酶活测定参考Selmar报道的方法(Analytical Biochemistry 166(1987)，208-211)，间苯氧基苯甲氰醇10mM，甲醇20uL，20mM柠檬酸缓冲液(pH5.0)，酶液10uL。上述反应液于25度温育，在1-5min内测定OD310nm的吸光度变化。 做时间(min)与吸光度变化的曲线，实验组曲线斜率设为△K，对照组斜率为零。相同条件下，不加酶液的情况下，25℃条件，记录310nm波长吸光度的变化，作为对照组，对照组不应有吸光度的变化。
间氧基苯甲醛浓度标准曲线斜率为K。按照公式计算酶活：
Figure PCTCN2018071619-appb-000009
蛋白浓度的测定：按照Nanodrop2000的标准流程测定OD280的吸收，进而得出裂解液中蛋白的浓度c(mg/mL)；
比酶活的计算：比酶活(U/mg)＝酶活/蛋白浓度。
实施例3：高密度发酵
合成编码突变体酶的脱氧核糖核酸序列，连接到pET28a载体(购自Novagen公司)的NdeI和XhoI位点，得到含有T7启动子的大肠杆菌质粒pET28-MeHNL6。将质粒转化大肠杆菌BL21(DE3)(购自Invitrogene公司)，在Kana抗性平板上得到相应的菌种，接种到LB培养基中，37℃过夜培养，用20％甘油保存菌种。
将菌种接种于装有200mL LB培养基的1L摇瓶中，于37℃，180-220rpm培养10-16h。将上述培养好的种子按10％(v/v)的比例接种于3L上罐发酵培养基(M9)中(葡萄糖4g/L，磷酸氢二钠12.8g/L，磷酸二氢钾3g/L，氯化铵1g/L，硫酸钠0.5g/L，氯化钙0.0152g/L，六水氯化镁0.41g/L。)，在25-35℃，300-800rpm，空气流量2-6L/min的条件下培养。培养6-10h后，加IPTG诱导10-12h,同时以5-20mL/h的速率流加含60％甘油的补料培养基，持续至发酵结束。流加补料培养基数小时至OD 600达到80-100时，放罐，5000rpm离心收集菌体。菌体裂解后测定酶活。凝胶电泳检测与预期相符。
针对野生型及部分突变体的发酵制备
实验发现，在高温发酵(约25-37℃)条件下，工程菌表达的野生型(SEQ ID NO.1)和部分突变体(如突变体2)的活性极低，野生型基本没有活性。
因此，针对野生型和部分突变体(如突变体2)的发酵方法基本同上，不同在于：发酵过程中温度一直维持在低温(约12－16℃)水平。
实施例4：酶的纯化
可采用本领域常规的方法对发酵获得的酶进行纯化。也可以采用一下例举的方法对发酵获得的酶进行纯化，例如：
将含有野生型序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM磷酸钠缓冲液(pH5.5)重悬菌体，比例为每g菌体4mL缓冲液。用高 压均质机破碎(压力800-1000bar)，加入聚丙烯酰胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩8倍(蛋白浓度93mg/mL)，酶的活力为198U/mL。
将含有突变体9序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM柠檬酸钾缓冲液(pH5.5)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚丙烯酰胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩5倍(蛋白浓度65mg/mL)，酶的活力为522U/mL。
将含有突变体27序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM磷酸钾缓冲液(pH5.5)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚丙烯酰胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩5倍(蛋白浓度69mg/mL)，酶的活力为687U/mL。
将含有突变体55序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用50mM柠檬酸钠缓冲液(pH5.5)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚丙烯酰胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩5倍(蛋白浓度62mg/mL)，酶的活力为958U/mL。
将含有突变体72序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM酒石酸钠缓冲液(pH5.0)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚乙烯亚胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩5倍(蛋白浓度75mg/mL)，酶的活力为1530U/mL。
将含有突变体113序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM柠檬酸钠-20mM磷酸钠缓冲液(pH5.0)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚乙烯亚胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩3倍(蛋白浓度64mg/mL)，酶的活力为1613U/mL。
将含有突变体135序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM柠檬酸钠-20mM磷酸钠缓冲液(pH5.2)重悬菌体，比例为每g菌体4mL缓冲液。用高压均质机破碎(压力800-1000bar)，加入聚乙烯亚胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩3倍(蛋白浓度55mg/mL)，酶的活力为1876U/mL。
将含有突变体149序列1L发酵液离心收菌体(4000rpm)，得到50g菌体；用20mM磷酸钾缓冲液(pH5.5)重悬菌体，比例为每g菌体4mL缓冲液。用高 压均质机破碎(压力800-1000bar)，加入聚丙烯酰胺进行絮凝(1-2‰)，4000rpm离心收集上清液。将清液用超滤膜进行浓缩6倍(蛋白浓度56mg/mL)，酶的活力为680U/mL。
比酶活的结果如图1。
本发明涉及的野生型和筛选出的突变体酶的酶活检测结果如表2所示。
表2
突变体酶编号 突变位点 酶活
1 WT *
2 H103M，W128A *
3 L36A，H103L,W128A **
4 V94E，H103L,W128A **
5 L36C，H103L,W128A **
6 L36Y，H103L,W128A **
9 V94L，H103L,W128A **
10 L36Q，H103L,W128A **
13 C81Y，H103L,W128A **
18 V94Q，H103L,W128A **
20 V94H，H103L,W128A **
21 H103L,W128A，V173T **
22 C81Y，H103L，W128A **
27 C81V，H103L，W128A **
29 H103L,W128A，V173I **
30 V94T，H103L,W128A **
31 H103L,W128A，V173C **
34 H103L,W128A，149A **
35 V94F，H103L,W128A **
36 H103L,W128A，V173A **
37 L36I，H103L,W128A **
38 H103L,W128A，V173S **
39 L36F，H103L,W128A **
40 H103S **
41 C81I，H103L，W128A ***
42 V94A，H103L,W128A ***
43 V2P,H103L,W128A ***
44 V2W,H103L,W128A ***
45 V2T,H103L,W128A ***
46 V94S,H103L,W128A，K209R ***
47 H103L,W128A，V173L,K209C ***
48 V94R,H103L,W128A，K209C ***
49 H103V ***
50 H103L,W128A，G165T ***
51 H103L,W128A，V173L,K209S ***
52 V2H,H103L,W128A ***
53 H103L,W128A，K224T ***
54 V2D,H103L,W128A ***
55 V94G，H103L,W128A ***
56 V2P,C81A H103L,W128A,L149C ***
57 V2S,H103L,W128A ***
58 H103L,W128A，K224A ***
59 V2Q,H103L,W128A ***
60 H103L,W128A，K199P,K176P ***
61 V2R,H103L,W128A ***
62 V94R，H103L,W128A，V173L ***
63 H103I ***
64 H103L,W128A，K199P ***
65 H103L,W128A，K176P ***
66 V94C，H103L,W128A ***
67 H103L,W128A，K224N ***
68 H103L,W128A，K224D ***
69 V94S，H103L,W128A，V173L ****
70 H103L,W128A，K199P,K224P ****
71 V2C,H103L,W128A ****
72 H103L,W128A ****
73 H103L,W128A，K224P ****
74 H103L,W128A，V173L ****
75 H103L,W128A，K224H ****
76 H103L,W128A，K224I ****
77 H103L,W128A，K224S ****
78 H103L,W128A，K224V ****
79 H103L,W128A，G165S ****
80 H103L,W128A，K176P,K224P ****
81 H103C ****
82 H103L,W128A，V173Q ****
83 H103L,W128A，K224E ****
84 V94S,H103L,W128A，K209C ****
85 H103L,W128A，K224P ****
86 H103L,W128A，T140R *****
87 H103L *****
88 H103L,W128A，T140S *****
89 H103L,W128A，T140W *****
90 H103L,W128A，T140D *****
91 V94S,H103L,W128A，G165D *****
92 H103L,W128A，T140I *****
93 H103L,W128A，T140K *****
94 H103L,W128A，G165P *****
95 H103L,W128A，T140G *****
96 H103L,W128A，T140H *****
97 V94R，H103L,W128A *****
98 H103L,W128A，K209F *****
99 H103L,W128A，G165D *****
100 V94R,H103L,W128A，K209R *****
101 V94R,H103L,W128A，G165D *****
102 V94S，H103L,W128A *****
103 H103L,W128A，K209L *****
104 C81A，H103L,W128A *****
105 H103L,W128A，K209M *****
106 H103L,W128A，K209G *****
107 H103L,W128A，K209A *****
108 H103L,W128A，K209S *****
109 H103L,W128A，K209C *****
110 C81A,H103L,W128A，K224P *****
111 C81A，H103L，W128A *****
112 H103L,W128A，K209R *****
113 V2I，H103L，W128A *****
114 C81A,H103L,W128A，K176P *****
115 V2A,C81A,H103L,W128A,L149C *****
116 L36A，H103L,W128A *****
117 V2G,C81A,H103L,W128A ******
118 V2L,C81A,H103L,W128A ******
119 V2P,C81A,H103L,W128A ******
120 V2H,C81A,H103L,W128A ******
121 V2R,C81A,H103L,W128A ******
122 V2M,C81A,H103L,W128A ******
123 V2S,C81A,H103L,W128A ******
124 V2C,C81A,H103L,W128A ******
125 V2W,C81A,H103L,W128A ******
126 V2T,C81A,H103L,W128A ******
127 V2Q,C81A,H103L,W128A ******
128 V2A,C81A,H103L,W128A ******
129 C81A,H103L,W128A,L149P ******
130 C81A,H103L,W128A,L149I ******
131 C81A,H103L,W128A,L149C ******
132 C81A,V94P,H103L,W128A,K176P ******
133 C81A,94R,H103L,W128A,L149P ******
134 C81A,94K,H103L,W128A,L149P ******
135 V2P,C81A,H103L,W128A,L149C ******
136 H103I,W128A ****
137 H103V,W128A ****
138 H103C,W128A ****
139 H103S,W128A ****
140 H103I,W128Y **
141 H103L,W128N ***
142 H103L,W128G ***
143 H103L,W128Y ***
144 H103I,W128N ***
145 H103I,W128G ***
146 H103C,W128V ***
147 H103C,W128G ***
148 H103C,W128Y ***
149 H103M,W128L ***
注备：*代表比酶活在0-3.0U/mg之间；**代表比酶活在3.0-10.0U/mg之间；***代表比酶活在10.0-18.0U/mg之间；****代表比酶活在18.0-26.0U/mg之间；*****代表比酶活在26.0-34.0U/mg之间；******代表比酶活>34.0U/mg之间。
实施例5：生物催化转化S-氰醇及检测方法
生物催化转化S-氰醇的方法为，100mL反应瓶中，加入20mL氰醇裂解酶，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应。
检测方法如下：
采用高效液相色谱(HPLC)法监测反应：以水和乙腈(45:55)为流动相，色谱柱为ODS-18反相柱，岛津LC-15C高效液相色谱，210nm下检测紫外吸收；反应体系用水和乙腈(45:55)进行稀释，离心并用尼龙膜过滤后进样检测。在本发明优选地反应体系中，HPLC检测反应进程：反应1小时后，检测，17.3min为间苯氧基苯甲醛，17.5min为S-构型氰醇.
手性纯度采用Agilent 1260液相色谱进行分析，检测条件为：ChiralpakAD-H柱，正己烷:乙醇(0.1％DEA)＝90:10，0.8mL/min，检测波长为220nm。经过比对本发明制得的S-构型的产物与目标物质标准品(购自江西科苑生物药业有限公司)一致。
本发明中涉及的典型的催化反应及检测结果例举如下：
1.野生型
100mL反应瓶中，加入20mL氰醇裂解酶(50mg/mL，野生型SEQ ID NO.1)，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应2小时，每30 分钟取样检测反应进程,ee值95.9％。
2.突变体9
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL)，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应2小时，每30分钟取样检测反应进程,ee值99.5％。
3.突变体27
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL)，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应2小时，每30分钟取样检测反应进程,ee值97.7％。
4.突变体55
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL)，10mL醛m-PBAld，20mL甲基叔丁基醚，4.5g丙酮氰醇，15℃搅拌反应2小时，每30分钟取样检测反应进程,ee值98.1％。
5.突变体72
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL)，10mL醛m-PBAld，20mL甲基叔丁基醚，5g NaCN，1mL浓硫酸，15℃搅拌反应2小时，每30分钟取样检测反应进程,ee值97.8％。
6.突变体113
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL，突变体Seq IDNo.113)，10mL醛m-PBAld，20mL甲基叔丁基醚，5g KCN，1mL浓硫酸，15°C搅拌反应2小时，每30分钟取样检测反应进程，ee值99.1％。
7.突变体135
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL，突变体Seq IDNo.135)，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应2小时，每30分钟取样检测反应进程，ee值98.9％。
8.突变体149
100mL反应瓶中，加入20mL氰醇裂解酶浓缩液(50mg/mL)，10mL醛m-PBAld，20mL甲基叔丁基醚，3g HCN，15℃搅拌反应2小时，每30分钟取样检测反应进程,ee值99.1％。
催化反应监测的结果如图2所示。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种突变的S-氰醇裂解酶，其特征在于，所述突变的S-氰醇裂解酶在选自下组的一个或多个位点发生突变：第103位氨基酸残基、第128位氨基酸残基、第2位氨基酸残基、第81位氨基酸残基、第149位氨基酸残基、第94位氨基酸残基、和第176位氨基酸残基，其中，氨基酸残基编号采用SEQ ID NO.1所示的编号。
  2. 如权利要求1所述的突变的S-氰醇裂解酶，其特征在于，所述突变的S-氰醇裂解酶的突变位点包括第103位氨基酸残基；优选地，第103位氨基酸残基由H突变为L、I、V、C、S或M；更优选地，第103位氨基酸残基由H突变为L。
  3. 如权利要求1所述的突变的S-氰醇裂解酶，其特征在于，所述突变的S-氰醇裂解酶的突变位点还包括第128位氨基酸残基；优选地，第128位氨基酸残基由W突变为A、N、L、V、G或Y，更优选地，第128位氨基酸残基由W突变为A；和/或
    所述突变的S-氰醇裂解酶的突变位点还包括第2位氨基酸残基；优选地，第2位氨基酸残基由V突变为P、L、D、I、G、H、R、M、S、C、W、T、Q、或A；和/或
    所述突变的S-氰醇裂解酶的突变位点还包括第81位氨基酸残基；优选地，第81位氨基酸残基由C突变为A、V或I；和/或
    所述突变的S-氰醇裂解酶的突变位点还包括第149位氨基酸残基；优选地，第149位氨基酸残基由L突变为I、C、A或P；和/或
    所述突变的S-氰醇裂解酶的突变位点还包括第94位氨基酸残基；优选地，第94位氨基酸残基由V突变为P、R、S、K；和/或
    所述突变的S-氰醇裂解酶的突变位点还包括第176位氨基酸残基；优选地，第176位氨基酸残基由K突变为P。
  4. 一种多核苷酸分子，其特征在于，所述多核苷酸分子编码权利要求1所述的突变的S-氰醇裂解酶。
  5. 一种载体，其特征在于，所述载体含有权利要求4所述的核酸分子。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求5所述的载体或染色体整合有权利要求4所述的核酸分子。
  7. 一种制备权利要求1所述的突变的S-氰醇裂解酶的方法，其特征在于，包括步骤：
    (i)在适合的条件下，培养权利要求6所述的宿主细胞，从而表达出所述的突变的氰醇裂解酶；和
    (ii)分离所述的突变的氰醇裂解酶。
  8. 一种酶制剂，其特征在于，所述酶制剂包含权利要求1所述的突变的S-氰醇裂解酶。
  9. 权利要求1所述的突变的S-氰醇裂解酶、权利要求8所述的酶制剂的用途，用于制备具有光学活性的S-氰醇产物。
  10. 一种制备S-氰醇的方法，其特征在于，包括步骤：
    (1)将权利要求1所述的突变的S-氰醇裂解酶与反应底物接触，进行催化反应，从而生成所述S-氰醇；
    (2)分离并纯化所述S-氰醇产物。
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