482 results about "Enzyme method" patented technology

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M and a synthesis method of rebaudioside M, and belongs to the technical field of gene engineering. The glycosyl transferase mutant for synthesizing the rebaudioside M is a glycosyl transferase UGT76G4 mutant and / or a glycosyl transferase UGT91C1 mutant, and the glycosyl transferase mutant is a glycosyl transferase UGT76G4 mutant. The glycosyl transferase UGT76G4 mutant is obtained by mutation of an amino acid sequence as shown in SEQ ID NO: 1: 85th amino acid is mutated into R from L, and 90th amino acid is mutated into G from I; the glycosyltransferase UGT91C1 mutant is obtained by mutating an amino acid sequence shown in SEQ ID NO: 3 as follows: the 204th amino acid is mutated into A from L, the 208th amino acid is mutated into A from F, and the 380th amino acid is mutated into D from G. The mutant can realize efficient catalytic synthesis of rebaudioside M, and the yield of rebaudioside M synthesized by an enzymic method is improved.

The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 17th site, the 40th site and the 64th site of an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. Compared with a wild type enzyme, the carbonyl reductase mutant has high activity and high stereoselectivity, the enzyme activity can reach more than two times that of the wild type enzyme, the catalytic efficiency on a substrate precursor ketone is remarkably improved, the yield of a statin drug intermediate and chiral alcohol synthesized by an enzyme method of the carbonyl reductase mutant is remarkably improved, and the yield of a rosuvastatin intermediate (3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R) is remarkably improved. The highest yields of the (3R, 5R)-6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester and the atorvastatin intermediate (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester reach 99% and 97.6% respectively, e.e. Is larger than 99%, and the method has high industrial application value.

The invention discloses a Candida antarctica lipase B mutant with high transesterification activity and application thereof. The nucleotide sequence of the Candida antarctica lipase B mutant is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein of the Candida antarctica lipase B mutant is shown as SEQ ID NO.2. The invention further discloses a preparation method of the Candida antarctica lipase B mutant. Compared with an original enzyme, the mutant has higher fat hydrolysis activity and transesterification activity, the specific enzyme activity of the fat hydrolysis activity reaches 480.39 U / mg, the specific enzyme activity of the transesterification activity reaches 40.67 U / mg, the biodiesel conversion rate at the temperature of 30 DEG C can reach 92%, low-temperature high catalytic activity is shown, and the mutant can be used for enzymatic preparation of biodiesel and has potential industrial application value.

The invention belongs to the technical field of grease modification and lipid deep processing, and particularly relates to a method for enriching n-3 polyunsaturated fatty glyceride by an enzymic method. According to the method, alkyl-modified hollow mesoporous silica spheres are used as an immobilized enzyme carrier and an emulsifier, grease rich in n-3 polyunsaturated fatty acid is used as a raw material in a stable Pickering emulsion system, saturated fatty acid and monounsaturated fatty acid are selectively hydrolyzed by lipase, and polyunsaturated fatty glyceride is enriched. The method has the characteristics of fast reaction, high yield, wide substrate application range and reusability of the immobilized enzyme, and the content of polyunsaturated fatty acid in the polyunsaturated fatty glyceride reaches 58.05-90.53%.

The invention discloses a carbamoylase mutant SEQ ID NO: 3, which can efficiently catalyze the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine to D-p-hydroxyphenylglycine, has high thermal stability, and can be used for industrial dual-enzyme method to catalyze the generation of D-p-hydroxyphenylglycine.

The invention provides gamma-glutamyl peptide as well as a screening method and application thereof. Wherein the gamma-glutamyl peptide is selected from one or more of amino acid sequences as shown in SEQ ID NO: 92, SEQ ID NO: 91, SEQ ID NO: 83, SEQ ID NO: 130, SEQ ID NO: 150, SEQ ID NO: 60, SEQ ID NO: 82, SEQ ID NO: 90 or SEQ ID NO: 93. The nine kinds of gamma-glutamyl peptides to be protected have good taste, can be simply and conveniently synthesized through a biological enzyme method, and have important significance for improving the delicate flavor of food under the condition that the use amount of salt is reduced.

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a method for producing rebaudioside M by an enzyme method. The method comprises the following steps: S1, adding a mixed solution 1 of recombinant cell disruption liquid into a substrate, adding into a buffer solution to obtain a pre-reaction solution 1 (a mixture of recombinant cell disruption liquid of a recombinant strain UGT11 (S158F) and a recombinant strain AtSUS (P94N)), and collecting precipitate after reaction; and S2, after the precipitate is dissolved, adding a mixed solution 2 (a mixture of recombinant cell disruption solutions of a recombinant strain UGT76G1 (G83Q) and a recombinant strain AtSUS (P94N)) of recombinant cell disruption solutions, adding a buffer solution to obtain a pre-reaction solution 2, and collecting the reaction solution to obtain Reb M. The method provided by the invention can effectively solve the problems of low content and poor specificity of the extracted Reb M, and can realize the conversion rate of 93.33% from Reb A to Reb M.

The invention relates to the technical field of gene engineering and recombinase catalysis engineering, in particular to a recombinant strain for expressing phenolic acid decarboxylase as well as a construction method and application of the recombinant strain. According to the invention, a recombinant strain for expressing the phenolic acid decarboxylase PADCs is constructed by heterologous expression of a coding gene PADCs of the phenolic acid decarboxylase. The enzyme liquid of the recombinant strain for expressing the phenolic acid decarboxylase has remarkable catalytic performance, p-coumaric acid can be used as a substrate, the recombinant strain is used for inducing expression, the purified enzyme liquid is used for catalyzing decarboxylation reaction of p-coumaric acid, and p-hydroxystyrene is synthesized; meanwhile, the phenolic acid decarboxylase PADCs has a wide substrate spectrum, the 4-vinylphenol compound can be used as a substrate, bicarbonate is used as a carboxyl source, the freeze-dried whole cell of the recombinant strain is utilized to catalyze the 4-vinylphenol compound to carry out regioselective beta-carboxylation reaction, a series of phenolic acids with biological activity are synthesized by an enzymic method, and meanwhile CO2 is immobilized; and a huge application potential is provided for the utilization of CO2.

The invention relates to the field of bioengineering, in particular to an enzyme composition, a product and application of the enzyme composition in preparation of Ectoine. The invention provides an enzyme composition. The enzyme composition comprises a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydroperoxide hydrolase, a mutant of Ectoine synthetase, a mutant of pyruvic acid-CoA oxidase and lactobacillus helveticus lactic dehydrogenase. According to the method disclosed by the invention, a plurality of mutant enzymes are utilized for combined transformation for the first time, and cheap 2-hydroxy-4-aminobutyric acid is taken as a starting raw material to quickly and efficiently produce Ectoine; the enzyme method has the characteristics of easiness in control, short conversion time, few generated impurities and the like, so that the method has unique advantages in the aspects of low-cost and large-scale mass production of Ectoin.

The invention discloses an adenosine kinase mutant with high thermal stability and application thereof, and relates to the technical field of enzyme engineering, and the adenosine kinase mutant with high thermal stability is obtained by performing point mutation on the 253rd site or the 82nd site of adenosine kinase as shown in SEQ ID NO: 1. According to the invention, an adenosine kinase mutant is used as a biocatalyst in an enzyme catalysis manner; the method is used for enzymatic synthesis of 5 '-adenylic acid, adenosine and adenosine triphosphate react to prepare adenylic acid, generation of by-products and impurities in the reaction is reduced to a certain extent, production efficiency and purity of adenylic acid are improved, reaction conditions are mild in preparation in an enzyme catalysis mode, pollution to the environment is avoided, and the method is suitable for industrial production. And the high-thermal-stability adenosine kinase mutant is applied to adenylic acid preparation, so that the preparation efficiency and yield of adenylic acid can be improved.

The invention belongs to the field of gene engineering, and particularly relates to a PET hydrolase mutant with high catalytic activity and application thereof. According to the invention, amino acids at the 62nd site, the 65th site, the 66th site, the 71th site, the 154th site, the 156th site, the 177th site, the 183th site, the 184th site, the 209th site, the 213th site, the 214th site and the 215th site in the PES-H1L92F / Q94Y amino acid sequence of the PET hydrolase shown as SEQ ID NO.2 are mutated by utilizing a structural analysis and site-specific mutagenesis technology to obtain various mutants, and the mutants with improved catalytic activity are obtained. The PET hydrolase mutants promote the hydrolysis efficiency of ester bonds in PET so as to improve the degradation capacity of PET plastics, and can be applied to enzymatic depolymerization of PET plastics in life in the future.

The invention discloses a method for preparing plant-derived cholesterol through chemical catalysis, which is characterized in that plant-derived BA is used as a raw material, chemical catalytic reduction is carried out through an intermediate compound 3, compared with an enzyme method, a large number of proper enzymes do not need to be screened, the cost is low, the stability is good, in addition, the influence of reaction sites on the Grignard addition yield is avoided through hydroxyl protection, and the yield of the plant-derived cholesterol is improved. Finished product isomer impurities can be reduced, the product yield is improved, and purification treatment is facilitated. The whole preparation route does not need an expensive catalyst, the reaction condition is mild, the specificity is high, the high-purity cholesterol can be obtained only through simple aftertreatment in each step, the method is suitable for industrial production, and the quality can meet the standards of medical injection and pharmaceutic adjuvants.

The invention provides a high-thermal-stability deoxyadenosine kinase mutant and application thereof, and belongs to the field of enzyme engineering. Compared with an amino acid sequence as shown in SEQ ID NO.1, the high-thermal-stability deoxyadenosine kinase mutant provided by the invention has mutation of at least one amino acid site, and the mutation comprises V40G, D121T and S154L. In the high-thermal-stability deoxyadenosine kinase mutant provided by the invention, the half-life period of V40G at 50 DEG C is 1.02 h, the half-life period of D121T at 50 DEG C is 9.51 h, and the half-life period of S154L at 50 DEG C is 5.23 h, which are significantly higher than those of a wild type. The high-thermal-stability deoxyadenosine kinase provided by the invention can be used for enzymatic preparation of 2 '-deoxyadenosine monophosphate, deoxyadenosine and deoxyadenosine triphosphate react to obtain deoxyadenosine monophosphate by adopting a one-pot enzymatic method, and the high-thermal-stability deoxyadenosine kinase is simple to operate, can tolerate higher reaction temperature and has the advantage of industrial amplification.

The invention belongs to the technical field of dairy products and food biology, and particularly relates to a method for preparing hypoallergenic whey protein through combination of an enzyme method and glycosylation and application of the hypoallergenic whey protein. The method specifically comprises the following steps: carrying out enzymolysis on whey protein isolate by using alkaline protease to prepare a whey protein hydrolysate, adding allose, uniformly mixing, carrying out glycosylation reaction under a freeze-drying condition, and finally, carrying out ultrafiltration and dialysis to remove redundant sugar, so as to prepare a sample, namely the low-sensitization whey protein. According to the whey protein prepared by the method, the sensitization is effectively inhibited, the low sensitization after digestion is ensured, and the problem that the sensitization is increased in the digestion process is avoided.

The invention relates to the technical field of biological catalysis, and discloses an L204A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: L204A: leucine of the 204 amino acid sequence of UGT76G1 mutates into alanine; the high-efficiency in-vitro enzymatic preparation of the rebaudioside I (RI) is realized for the first time, the catalytic efficiency of the rebaudioside I (RI) is improved by more than 7 times (from about 7% to more than or equal to 50%) compared with that of an original enzyme, the high-efficiency performance can be stably maintained under different scales of 10mL to 5L, a solid foundation is laid for industrial application of the RI, and the preparation method is simple, controllable in process condition and suitable for popularization and application.

The invention discloses a method for synthesizing ceramide NP through two-step enzyme catalysis, which comprises the following steps of: firstly, adding lipase into vegetable oil to catalyze and combine fatty acid to decompose into free fatty acid, and centrifuging to remove the lipase; a small amount of phytosphingosine is added into the hydrolyzed vegetable oil, the raw material vegetable oil is used as a solvent or a squalane system solvent for incubation, and then immobilized lipase is added for catalytic synthesis of ceramide. The invention establishes a two-enzyme method for efficiently synthesizing the ceramide NP compound, and the process is natural, environment-friendly and non-toxic. The reaction conditions are optimized through response surface analysis, meanwhile, the immobilized lipase shows excellent cycle performance, and the method is easy to operate, environmentally friendly, green and economical and has wide application prospects in the fields of cosmetics, health care products, biological medicine and the like.

The invention discloses a method for preparing gamma-cyclodextrin by a precipitation technology and a separation enzyme method, which comprises the following steps: by taking gamma-cyclodextrin mother liquor prepared by a solvent method and an enzyme method as a sample solution, firstly dissociating a compound formed by gamma-cyclodextrin and a reaction aid under a high-temperature condition, and then separating the gamma-cyclodextrin by a precipitation separation technology to obtain the gamma-cyclodextrin. An inorganic salt precipitator is added to effectively separate out a reactant, so that the purification of a target product is realized, and an efficient and simple gamma-cyclodextrin separation and purification method is established. By adopting the method disclosed by the invention, the total conversion rate of the gamma-cyclodextrin can reach about 53.89%, the recovery rate of the separated gamma-cyclodextrin can reach 82.76%-90.84%, and the purity of the separated gamma-cyclodextrin is 92.56%-95.63%. The residual quantity of the reaction auxiliary agent is 0.02%.

The invention discloses a high-stability carbonyl reductase mutant and application of the high-stability carbonyl reductase mutant in chiral alcohol synthesis. An amino acid sequence as shown in SEQ ID NO.2 is mutated as follows: 20th leucine is replaced by any one of lysine, threonine, aspartic acid or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine. Compared with a wild type enzyme, the stability of the obtained mutant is remarkably improved, particularly, single-point mutants Mut-L20I and Mut-L55I and a combined mutant Mut-L20I-L55I show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, has the advantages of high catalytic efficiency, high thermal stability, strong substrate tolerance, easiness in fermentation production and the like, and is suitable for industrial application of enzymatic synthesis of chiral alcohols.

The invention belongs to the technical field of preparation of food additives, and particularly relates to a method for extracting sa-hao seed gum and oligosaccharide thereof by using a compound enzyme method. According to the method provided by the invention, complete artemisia desertorum seeds are taken as a raw material, enzymolysis is carried out by using a compound system consisting of xylanase, cellulase and pectinase, and after enzyme deactivation and alcohol precipitation, the artemisia desertorum gum or the artemisia desertorum gum oligosaccharide can be obtained. Compared with a traditional water extraction method and an acid-base extraction method, the extraction yield is remarkably increased through the enzymolysis synergistic effect, the process is green and safe, no chemical residue exists, the molecular weight of the product is controllable, and high purity and functional activity are both achieved. The method is easy and convenient to operate, low in cost and suitable for large-scale production, and has wide application prospects in the fields of food, medicine and functional oligosaccharide development.

The invention relates to a diacylglycerol acyltransferase capable of synthesizing triglyceride as well as a coding gene and application of the diacylglycerol acyltransferase, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the diacylglycerol acyltransferase is as shown in SEQ ID NO.1, and the amino acid sequence of the diacylglycerol acyltransferase is as shown in SEQ ID NO.2. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The invention discloses a method for preparing a functionalized polymer based on a macro-micro reactor chemical-enzyme method, which comprises the following steps: reacting a mixed solution of an acrylate monomer, an initiator, a ligand, a functional alcohol and a solvent in a micro reactor to obtain a reaction solution containing functionalized polyacrylate, and the micro reactor comprises the macro-micro reactor containing a copper internal member. In a macro-micro reactor, enzymatic ester exchange and Cu (0)-RDRP coupling reaction are carried out to obtain poly (methyl) acrylate with a series of chemical structures and copolymers of the poly (methyl) acrylate. The method is wide in applicable substrate range, and compared with a traditional synthesis method, the macro-micro reactor containing the internal component can remarkably shorten the polymerization time, secondary purification of the monomer is avoided, and the conversion rate is greatly increased. The characteristic scale of the macro-micro reactor reaches centimeter level, and high-flux synthesis can be realized. The invention provides a technical scheme for the preparation of the functionalized poly (methyl) acrylate polymer, and has an industrial conversion prospect.

The invention discloses diglyceride prepared by an enzymic method as well as a preparation method and application thereof, and belongs to the technical field of deep processing of grease. The method comprises the following steps: adding fatty acid into a choline chloride-glycerol-urea ternary eutectic solvent, uniformly mixing, and then adding candida antarctica lipase B for esterification reaction to obtain a reaction mixture; separating the reaction mixture, and recovering an upper-layer oil phase; and separating and purifying the upper-layer oil phase to obtain the diglyceride. According to the method, the reaction rate is high, the selectivity of the diglyceride in the esterification process is high, the yield of the diglyceride can reach 65% or above by reacting for 1 h, the selectivity of the diglyceride is 75% or above, and the purity of the separated and purified diglyceride is 90% or above; in addition, the method is simple to operate, and the reaction product and the eutectic solvent can be separated through simple centrifugation after the reaction is finished; in addition, the monoglyceride content in the product is low, and the subsequent separation and purification difficulty is greatly reduced.

The invention belongs to the technical field of food and chemical industry, and particularly relates to a gas-liquid-solid three-phase fluidized bed enzyme reactor and a synchronous preparation method of grease enzyme method deacidification and diglyceride by using the same. According to the method, the gas phase of the three-phase fluidized bed is high-purity dry inert gas, the liquid phase is a uniform solution formed by mixing reaction substrate high-acid-value oil and an acyl acceptor, and the solid phase is immobilized enzyme. The method comprises the following steps: mixing high-acid-value oil and an acyl acceptor to prepare a raw material solution, preheating a feeding tank to a reaction temperature, adding the raw material solution into a gas-liquid-solid three-phase fluidized bed reactor, and carrying out esterification and / or transesterification under the bubbling action of inert gas to realize the deacidification of the high-acid-value oil and the synchronous preparation of the functional lipid. The mixture of the deacidified grease and the functional lipid is obtained. The method has the characteristics of high catalytic efficiency, easiness in separation of the product from enzyme, high nutritional value of the product, no need of adding an organic solvent, difficulty in oxidation of grease and suitability for large-scale production.

The invention discloses application of double enzymes in one-step preparation of L-selenium methyl selenocysteine, and belongs to the technical field of biochemical engineering. In the method, racemic N-acetyl selenium methyl selenocysteine is used, and the L-selenium methyl selenocysteine with high optical purity is prepared in a green and efficient manner through co-catalysis of stereoselective L amino acid acylase and N-acetyl amino acid racemase. The catalyst is prepared through fermentation by adopting a one-bacterium two-enzyme method. Synchronous racemization and selective hydrolysis are realized by a one-step method, the theoretical conversion rate is 100%, the actual conversion rate is 95%, and the product is e.egt; the concentration of the substrate is 8%.

The invention discloses a marine-derived carboxylesterase and an application of the marine-derived carboxylesterase in degradation of PET (Polyethylene Terephthalate) plastics. The amino acid sequence of the enzyme is as shown in SEQ ID NO.1, experiments show that Tm of RbCE1 is 79.5 DEG C, 40 mM of depolymerization monomers can be generated after the enzyme treats low-crystallinity PET powder at 60 DEG C for 4 hours, the catalytic activity is equivalent to that of LCC, and good degradation activity is shown. Through escherichia coli heterologous expression, 0.4 g / L high expression quantity can be achieved in a shake flask, and the potential of remarkably reducing the enzyme production cost is achieved. Therefore, the novel carboxylesterase RbCE1 is suitable for degrading PET plastics and composite materials thereof, and is a potential enzyme preparation for efficient biological catalysis of a waste PET plastic waste enzymatic depolymerization recovery treatment process.

The invention relates to an alcohol oxidase mutant and application thereof. Specifically disclosed are a mutant derived from Gloeophyllum trabeum alcohol oxidase (GtrAOX-M2), a nucleic acid encoding the alcohol oxidase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a preparation method of a recombinant alcohol oxidase catalyst, and a preparation method of the recombinant alcohol oxidase catalyst. The invention also discloses a method for catalyzing methanol to be oxidized into formaldehyde by using the alcohol oxidase, the alcohol oxidase mutant or the recombinant alcohol oxidase catalyst. The activity of the alcohol oxidase mutant provided by the invention is obviously improved compared with that of a female parent, and the enzyme is high in activity and can be heterologously expressed in escherichia coli, so that the alcohol oxidase mutant can efficiently oxidize methanol into formaldehyde and oxidize the methanol into formaldehyde by a one-carbon unit biological utilization methanol enzyme method; wide application prospects are shown in the aspect of subsequent conversion and generation of polyhydric alcohols.

The invention relates to heparinase III with high activity on 6-sulfated heparin and application of the heparinase III. The amino acid sequence of the heparinase III (BeHepIII) is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention discovers and discloses a novel heparinase III (BeHepIII) for the first time, the heparinase III is derived from Bacteroides eggerthii, has a higher recombinant expression level in escherichia coli, has very high degradation activity on heparin containing a 6-site sulfated structure, and particularly has the enzyme activity which is 93.5 times of that of commercialized heparinase FhHepIII at present, and the heparinase III can be used for degrading heparin containing 6-site sulfated structure. Therefore, the heparinase III (BeHepIII) provided by the invention has a wide application potential in heparin degradation. In addition, by using heparinase III (BeHepIII), directional excision of a reducing end of heparin oligosaccharide synthesized by a chemical enzyme method is realized, and complete chemical enzyme method synthesis of a pentasaccharide structure with anticoagulant factor Xa activity is successfully realized.

The invention discloses a method for preparing butanediamine and spermidine by an enzyme method, and belongs to the field of bioengineering. The novel arginine decarboxylase mutant is successfully provided, the mutant shows excellent catalytic performance under the alkaline condition, and the specific enzyme activity of the mutant reaches 24.9 U / g and is improved by 8.3 times compared with that of wild type enzyme. The invention develops a two-step synthesis process based on the constructed arginine decarboxylase mutant, and the two-step synthesis process is used for efficiently preparing spermidine. Experimental results show that the process successfully realizes the remarkable increase of the yield of spermidine, and the final yield reaches 200.4 mg / L.

The invention provides a quality control method for synthesizing 3 '-sialic acid lactose by an enzymatic process, which comprises the following steps: A) dissolving and extracting a test sample by using a solvent to obtain a to-be-detected solution; the test sample is a reaction solution or a finished product of 3 '-sialic acid lactose synthesized by enzyme catalysis; b) determining the liquid to be detected by adopting a high performance liquid chromatography to obtain an HPLC (High Performance Liquid Chromatography) spectrum of the 3 '-sialic acid lactose; according to the method, an HPLC-UV-RI combined detection method is adopted, the specificity is high, the sensitivity is high, the linear correlation is good, and the pretreatment process of the sample is simple and rapid; according to the method, the content of 3 '-lactulose sialylate, 6'-lactose sialylate sodium salt, D-lactose, CMP, CMP-SA and SA in the 3 '-SL process development process can be accurately detected, control over the conversion rate and impurities in the process is facilitated, effective detection of a product 3'-SL related substance-isomer can be achieved, and control over the product quality is facilitated.

The invention discloses a nitrilase mutant and application of the nitrilase mutant in synthesis of a brivaracetam chiral intermediate, and provides an efficient (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing the nitrilase mutant with improved catalytic activity and stereoselectivity. The requirement of industrially synthesizing the brivaracetam key chiral intermediate is met, and a foundation is laid for industrial production of brivaracetam synthesized by a chemical-enzyme method.