70 results about "Substrate concentration" patented technology

The invention discloses a high-stability hydroxysteroid dehydrogenase mutant, a co-immobilized enzyme construction method and application. According to the invention, a site-directed mutant library is constructed based on computer-aided design, and mutants with significantly improved thermal stability and greatly improved catalytic efficiency compared with wild type mutants are obtained through screening; meanwhile, a co-immobilized enzyme construction method based on electrostatic adsorption and chemical crosslinking is established, the hydroxysteroid dehydrogenase mutant and lactic dehydrogenase are co-immobilized, the co-immobilized enzyme can still keep high activity under high substrate concentration, and the conversion rate is still maintained to be 90% or above after 30 batches of co-immobilized enzyme are repeatedly used. And an important foundation is laid for industrial application of the hydroxysteroid dehydrogenase.

The invention relates to the technical field of nourishment detection, and discloses a system and a method for detecting enzymolysis reaction components of brewing nourishment. The method comprises the following steps: collecting dynamic data such as an enzyme activity value, a substrate concentration value and a product generation rate value in an enzymolysis reaction process in real time through a multi-source sensor; performing multi-modal feature fusion processing on the dynamic data to generate an enzymolysis reaction feature vector; classifying enzymolysis reaction state categories based on the feature vectors, and dynamically adjusting detection parameter thresholds according to different state categories; and according to the deviation value between the real-time reaction state and the parameter threshold value, correcting an enzymolysis reaction process control instruction by adopting a nonlinear optimization algorithm. According to the method, the dynamic state of the enzymolysis reaction can be captured in real time, accurate detection of enzymolysis reaction components and reasonable regulation and control of the reaction process are achieved through multi-dimensional data fusion and dynamic adjustment, and the method is suitable for efficient detection of the enzymolysis reaction of the brewing nourishment.

The invention provides a transaminase mutant and application of the transaminase mutant in chiral amine synthesis. According to the transaminase derived from Mycobacterium sp. Provided by the invention, in a 1 mL reaction system for catalyzing N-Boc-3-piperidone to generate N-Boc-3-aminopiperidine, the 24-hour conversion rate is 95%, and the e.e. Value of the product is greater than 99%. On the basis, the invention further provides a series of mutants of the transaminase, the relative enzyme activity of the mutants reaches 153%-293%, and the catalytic activity of the transaminase is further improved. Wherein the 34-hour conversion rate of the optimal transaminase mutant is increased to 90% or above under the condition of 120g / L substrate concentration in a 10mL reaction system, and the e.e. Value of the product is greater than 99%. The production technology has the advantages of high catalytic activity, easiness in fermentation, environment friendliness and the like, and has good development and application values.

The application discloses a carbonyl reductase mutant and application thereof in preparation of (R)-8-chloro-6-hydroxyoctanoate compounds. The application relates to a mutant of carbonyl reductase with improved catalytic performance and stereoselectivity after rational molecular modification, a recombinant expression plasmid containing the mutant gene of the enzyme, a recombinant expression transformant, a preparation method of the recombinant enzyme, and application of the recombinant enzyme or the recombinant cell as a catalyst in asymmetric reduction of 8-chloro-6-carbonyloctanoate compounds (I) to prepare (R)-8-chloro-6-hydroxyoctanoate compounds (II), in particular, in an intermediate (R)-8-chloro-6-hydroxyoctanoate methyl ester of thioctic acid. The disclosed carbonyl reductase SsCR mutant has the advantages of high reaction efficiency and high catalytic activity, and exhibits the advantages of high substrate concentration, low catalyst consumption and short reaction time in the process of catalytic preparation of the compound (II).

This invention discloses a ketoisomerase mutant and its application in the preparation of D-chiral inositol, belonging to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, specifically by mutating amino acid position 62 and / or amino acid position 175 of the amino acid sequence shown in SEQ ID NO.1. After the above-mentioned mutation of the wild-type thermostable ketoisomerase, the resulting ketoisomerase mutant not only retains the advantages of the wild-type thermostable ketoisomerase—its heat resistance and susceptibility to temperature fluctuations—but also exhibits higher enzyme activity. When co-catalyzing the reaction of muscle inositol to D-chiral inositol with inositol dehydrogenase, it can further improve the conversion rate and yield of D-chiral inositol even at high substrate concentrations.

The invention discloses a nitrilase mutant and an application of the nitrilase mutant in preparation of (S)-3-methylheptanoic acid. The nitrilase mutant disclosed by the invention has high catalytic activity and stereoselectivity, and can overcome the defects of low substrate concentration, low stereoselectivity, high cost, environmental pollution and the like in the existing method, so that 3-methylheptonitrile can be quickly catalyzed to generate (S)-3-methylheptanoic acid under high substrate concentration. In addition, the method also has the effects of good enzyme catalysis effect, high product purity, high recovery rate, simple steps, greenness, environmental protection and the like.

A method for estimating an enzyme inhibition constant, according to one embodiment of the present disclosure, may comprise the steps of: measuring initial reaction rate data at a plurality of substrate concentrations and one inhibitor concentration; setting the one inhibitor concentration to be greater than or equal to a predetermined half-maximal inhibitory concentration; and using the measured initial reaction rate data and the predetermined half-maximal inhibitory concentration value so as to estimate an inhibition constant by fitting to an enzyme inhibition model and applying a normalization term that includes the relationship between the half-maximal inhibitory concentration and at least one candidate inhibition constant.

The application discloses a kind of Baeyer-Villiger monooxygenase and its mutant in synthesis optical pure 3-substituted chiral butyrolactone Application, the application with the Baeyer-Villiger monooxygenase ArBVMO of Acinetobacter radioresistens (CGMCC No.25186) source of resistance to radiation Acinetobacter (Acinetobacter radioresistens) and its mutant as biocatalyst, can catalyze the asymmetric oxidation of series latent chiral 3-substituted cyclobutanone, preparation corresponding optical pure chiral butyrolactone.Compared with prior art, the application has the advantages of high enzyme reaction substrate concentration, mild reaction condition, environment-friendly, high space-time yield, high product optical purity, and has good application prospect.

The application discloses a carbonyl reductase mutant, a combined enzyme and application of the combined enzyme in synthesis of S-configuration boselike, and belongs to the technical field of biological catalysis and pharmacy. The amino acid sequence of the wild-type carbonyl reductase mutant provided by the application is shown as SEQ ID NO:1, and the mutant is obtained by mutation of the wild-type carbonyl reductase, and the mutation mode is that F at the 147th position is mutated into L, V at the 163rd position is mutated into Y, L at the 176th position is mutated into E, and P at the 200th position is mutated into R. By using the mutant, a catalytic system is formed by combining the mutant with formic acid dehydrogenase (FDH), and optical pure S-configuration boselike can be efficiently and highly selectively prepared under high substrate concentration of 250-320 g / L, separation and purification problems caused by generation of a large amount of soluble by-products in catalysis of other combined enzymes are avoided, a post-treatment process is obviously simplified, and production cost and environmental burden are reduced.

The invention provides a bionic targeted motion research method of a biological enzyme driven nano-robot. The method comprises the following steps: preparing an enzyme driven nano-robot; preparing a micro-fluidic chip, wherein the micro-fluidic chip is provided with an H-shaped micro-fluidic channel structure which is diluted layer by layer; determining motion characteristics and enzyme properties of the enzyme-driven robot in composite field environments such as a gradient substrate concentration field, an enzyme chemotactic agent field and a gradient viscosity field; a chemotactic factor solution, a solution containing the enzyme-driven nano-robot and a urea solution are injected into a middle channel of the micro-fluidic chip through an injection pump, and the chemotactic behavior and targeted movement of the robot in a fluid composite field are observed by controlling the fluid concentration and flow velocity and representing a chemotactic factor concentration gradient field in a stable flow state. According to the technical scheme provided by the invention, the chemotactic sensitivity of the bio-enzyme driven nano-robot to different types of chemotactic factors can be explored, and the chemotactic movement of ultralow-concentration chemical signal molecules is realized.

The invention relates to a mycobacterium hydrolase and application thereof in preparation of flavors and fragrances, and belongs to the fields of enzyme engineering and mining and application of new enzyme resources. The hydrolase comes from mycobacteria, and the amino acid sequence of the hydrolase is shown as SEQ ID No: 2; the hydrolase MdHDL disclosed by the invention has relatively high catalytic activity and substrate concentration tolerance, and can efficiently utilize benzyl alcohol or phenethyl alcohol to react with p-nitrophenyl methyl acetate pNPA, p-nitrophenyl methyl butyrate pNPB or p-nitrophenyl methyl caprylate pNPO to synthesize essence and perfume substances with different flavors; the method is suitable for preparing flower fragrance and fruit fragrance type flavors and fragrances such as jasmine flower fragrance, rose fragrance, pineapple fragrance, banana fragrance and apricot fragrance by an enzyme method, has obvious industrial production potential, and provides technical basis and method guidance for preparing the flower fragrance and fruit fragrance type flavors and fragrances by the enzyme method.

The present application relates to aldose reductase activity analysis technical field, specifically relates to a kind of aldose reductase activity and its inhibitory effect quantitative analysis method.Local substrate concentration regulation is constructed by dividing multiple detection segments, combining bypass sampling channel, electrochemical detection cavity and compensation injection point, and the substrate environment of each detection segment is maintained consistent;Enzyme-free reference channel parallel to microfluidic reaction channel is set, and the total absorbance change amount background is separated, to obtain the real enzymatic absorbance change amount;Based on sliding time window, extract local dynamic enzyme reaction rate, combined with wall adsorption correction to eliminate the influence of enzyme activity attenuation along the way, identify stable reaction interval through local fluctuation coefficient of variation, determine local half response time and local onset time;Local onset time is used as the integration starting point, to calculate comprehensive inhibition intensity, realize inhibitor onset order and inhibition strength spatial quantitative analysis, improve the accuracy, stability and spatial comparability of detection result.

The invention discloses recombinant escherichia coli for producing lysine decarboxylase as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of biology. The invention constructs lysine decarboxylase, and the amino acid sequence of the lysine decarboxylase is as shown in SEQ ID NO.7. According to the invention, a gene engineering technology is utilized to carry out heterologous expression on a strain (Klebsiella quaipneumonae HFUT7) screened out in a laboratory, a recombinant escherichia coli CadA-KW01 for expressing the lysine decarboxylase is constructed, the lysine decarboxylase expressed by the recombinant strain is utilized to carry out catalytic reaction, and the maximum enzyme activity of the enzyme can reach 260 U / mL. The conversion rate is 91.3% when stirring is performed for 90 min at the substrate concentration of 1 mol / L and the temperature of 45 DEG C, and efficient production of 1, 5-pentanediamine can be achieved. The method disclosed by the invention has the advantages of environmental friendliness (normal temperature and normal pressure and no toxic solvent), high efficiency (one-step reaction), high conversion rate, simplicity and convenience in purification and the like, the production cost is remarkably reduced, and the method has a good application prospect in the production of 1, 5-pentanediamine.

The invention relates to the technical field of genetic engineering, in particular to recombinant acidic pectinase GsPG3 and application thereof. The amino acid sequence of the recombinant acidic pectinase GsPG3 is as shown in SEQ ID NO: 4, the optimum pH of the recombinant acidic pectinase GsPG3 is 3.5, the optimum temperature of the recombinant acidic pectinase GsPG3 is 65 DEG C, and the recombinant acidic pectinase GsPG3 has excellent stability under an acidic condition (pH is 3-5) and can be activated by Ca < 2 + >. The enzyme can be efficiently expressed in pichia pastoris, the shake flask enzyme activity reaches 7235.4 U / ml, and the fermentation activity in a 30L fermentation tank reaches 91,150.3 U / ml. According to the method, the GsPG3 enzyme is specifically applied to beet pulp enzymolysis for the first time, the optimal process conditions are obtained through system optimization: the pH is 3.0, the temperature is 50-55 DEG C, the enzyme concentration is 2,000 U / ml, the substrate concentration is 0.25 g / ml, the time is 3h, and the pectin degradation rate of the beet pulp can reach 45.4% under the conditions.

The invention discloses an ethanol dehydrogenase mutant, engineering bacteria and application in synthesis of a sinobamate intermediate, the ethanol dehydrogenase mutant is utilized to catalyze prochiral ketone (B) to synthesize chiral alcohol (A), the method is high in substrate concentration, simple and convenient in post-treatment step and more suitable for industrial large-scale production, and the problems that in the prior art, the substrate concentration is not high, and the yield is high are solved. Post-treatment is complicated, and the like.

This invention discloses an enzyme preparation and a method for preparing D-chiral inositol, belonging to the field of genetic engineering technology. The enzyme preparation includes inositol dehydrogenase and 2-keto-inositol isomerase. The amino acid sequence of inositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of 2-keto-inositol isomerase is shown in SEQ ID NO. 8. Compared with the prior art, the enzyme activities of the inositol dehydrogenase and 2-keto-inositol isomerase with specific amino acid sequences of this invention are further improved. When catalyzing the reaction of muscle inositol to D-chiral inositol, both can further improve the conversion rate and the yield of D-chiral inositol. Furthermore, this inositol dehydrogenase has high tolerance to muscle inositol, and can still efficiently catalyze the reaction to produce D-chiral inositol even at high substrate concentrations, alleviating substrate inhibition.

The invention provides a 5-hydroxymethylfurfural oxidase mutant and application thereof, and belongs to the technical field of biology. The sequence of the 5-hydroxymethylfurfural oxidase mutant is as shown in SEQ ID NO: 4. The invention also provides a gene for coding the 5-hydroxymethylfurfural oxidase mutant, a recombinant vector and a recombinant cell containing the gene, and applications of the 5-hydroxymethylfurfural oxidase mutant, the recombinant vector and the recombinant cell in catalyzing 5-hydroxymethylfurfural to generate 2, 5-furandicarboxylic acid. According to the 5-hydroxymethylfurfural oxidase mutant provided by the invention, the catalytic reaction effect is obviously superior to that of a wild type, the specific activity is improved to 21.4 times, and the FDCA yield is improved to 18.0 times. The mutant can tolerate high substrate concentration, the reaction product is single and high in purity, the FDCA yield is larger than 95%, and the mutant has high industrial value and application prospects.

This invention belongs to the fields of genetic engineering and enzyme engineering technology, and discloses a glutaminase mutant, immobilized glutaminase, and their applications. The glutaminase mutant is based on the amino acid sequence shown in SEQ ID NO.1, with amino acid mutations at the following sites: E113K, R136M, and H223V, and its amino acid sequence is shown in SEQ ID NO.3. Immobilized glutaminase prepared using the glutaminase mutant exhibits 5-10 times increased enzyme activity compared to the wild type. The substrate concentration in the synthesis of L-theanine catalyzed by the immobilized glutaminase reaches 80-150 g / L, with a conversion rate greater than 99%. It can be recovered through simple filtration and reused more than 5 times, with the conversion rate remaining essentially the same during repeated use, demonstrating high potential for industrial application.

This invention discloses a sucrose isomerase site mutant, a genetically engineered bacterium, and a method for catalyzing the production of isomaltulose. This invention utilizes PROSS online analysis combined with sequence alignment analysis to screen key amino acid sites related to catalytic activity in sucrose isomerases. Through site-directed mutagenesis, the mutant WT-Gro7-Q474T was obtained, with a maximum specific enzyme activity of 683 U / mg. k cat (s ‑1 The concentration reached 718. By optimizing protein expression conditions, including temperature, IPTG concentration, bacterial concentration, and time, and combining this with the method of co-expression of molecular chaperone proteins, a highly efficient heterologous expression system for sucrose isomerase was established. This invention established an optimal isomaltulose-catalyzed reaction system: in a 2 L reactor, with 1 L of reaction solution and a substrate concentration of 800 g / L sucrose, after 6 hours of reaction, all sucrose was converted, yielding isomaltulose 792.50 g / L, with a conversion rate >99.5%.

The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-resistant lactase mutant and application thereof in galactooligosaccharide synthesis. In order to overcome the defects that lactase for GOS synthesis in the prior art is insufficient in heat resistance and cannot efficiently catalyze under the conditions of high temperature and high substrate concentration, the invention provides a lactase mutant BglSS obtained through rational design, and the amino acid sequence is as shown in SEQ ID NO: 3. The mutant keeps high catalytic activity, the thermal stability is remarkably improved, GOS synthesis can be stably catalyzed for a long time under the conditions of 65-70 DEG C and 600-800 g / L high-concentration lactose, the reaction time is remarkably shortened, the production efficiency is improved, and the energy consumption and the production cost are reduced.

This invention belongs to the field of genetic engineering technology and discloses a high-efficiency expression method for recombinant collagenase and its application in the preparation of bovine bone collagen peptides. The collagenase gene derived from Bacillus sp. HUB-I-004 strain (accession number J9CTX3) was optimized using E. coli-preferred codons. A recombinant expression vector was constructed and transformed into E. coli BL21(DE3). After optimization of induction conditions and Ni-NTA affinity purification, a highly active recombinant collagenase with a specific activity of 231.05 U / mg was obtained. Bovine bone collagen was extracted using a high-temperature, high-pressure method. Bovine bone collagen peptides were prepared under optimal conditions of a substrate concentration of 10 mg / mL, an enzyme addition of 20%, and enzymatic hydrolysis at 37℃ for 8 h, significantly improving peptide yield. The obtained product is mainly composed of small molecule peptides and exhibits significant osteoblast proliferation-promoting activity, making it widely applicable in the preparation of health foods and pharmaceuticals.

This invention provides a phase separation system comprising H-Laf1, P-Laf1, and T-Laf1 fusion proteins. The H-Laf1 fusion protein is obtained by fusing the H protein of the GCS system with the IDR region of the Laf1 protein; the P-Laf1 fusion protein is obtained by fusing the P protein of the GCS system with the IDR region of the Laf1 protein; and the T-Laf1 fusion protein is obtained by fusing the T protein of the GCS system with the IDR region of the Laf1 protein. This invention also provides a method for CO2 fixation and amino acid production via phase separation. By constructing a phase separation system, this invention forms a dynamic membrane-free microreactor in vitro, achieving substrate concentration and mass transfer optimization, significantly increasing glycine yield. Simultaneously, based on the temperature response characteristics of the IDR tag, it enables rapid enzyme separation, truly realizing enzyme recycling.

The invention discloses difunctional glutathione synthetase based on ancestor sequence reconstruction and application of the difunctional glutathione synthetase, and belongs to the technical field of enzyme engineering and bioengineering. A difunctional glutathione synthetase Anc427 with an amino acid sequence shown as SEQ ID NO: 1 is obtained through an ancestral sequence reconstruction technology, and compared with St-GshF, the obtained difunctional glutathione synthetase Anc427 has the advantages that the melting temperature is increased to 56.2 DEG C from 45.4 DEG C through a stability test and an amplification reaction; at the temperature of 40 DEG C, the half-life period is prolonged from 173.29 min to 3465.74 min; the half-life period at 50 DEG C is 266.6 min, and the thermal stability is remarkably improved. Meanwhile, the activity of the bifunctional glutathione synthetase Anc427 is improved by 17% compared with that of St-GshF, and the conversion rate under the substrate concentration of 100 mM is improved by 13.6%. The industrial application value is extremely high.

Disclosed are a self-assembly enzyme system supplying α-ketoglutarate (α-KG) and application thereof in catalytic synthesis of 4-hydroxyisoleucine. In the present disclosure, glutamate oxidase catalyzes glutamate to generate α-KG, and catalase-peroxidase decomposes a byproduct H2O2. An interaction between RIAD and RIDD and a covalently linked combined state can mediate higher-order structures of various self-assembly enzymes. The LGOX / KatG self-assembly system is constructed through the affinity of short peptides in vitro to eliminate H2O2 in situ, thereby eliminating the inhibitory effect of H2O2 on Fe(II) / α-KG DOs, and facilitating efficient and high-yield production of 4-HIL in a one-pot cascade reaction with IDO, with a highest yield up to 95% at a substrate concentration of 100 mM.

This invention discloses a mulberry leaf antihypertensive active peptide, its preparation method, and its application. Using mulberry leaves as raw material, this invention prepares mulberry leaf albumin. Enzymatic hydrolysis parameters, including hydrolysis time, pH, enzyme-to-substrate ratio, and substrate concentration, were optimized through single-factor and orthogonal experiments, providing optimal hydrolysis conditions for preparing crude ACE-inhibiting peptides. Further purification, peptide sequencing, and virtual screening identified highly active ACE-inhibiting peptides with strong solubility and good ACE-inhibiting activity. The two peptides with the highest activity, VPSCFDLTGK and RLPDFHGL, showed the highest IC50 values. 50 The values ​​were 8.23 ​​μmol / L (8.765 μg / mL) and 23.01 μmol / L (23.58 μg / mL), respectively. These bioactive peptides can act as angiotensin-converting enzyme inhibitory peptides, thereby lowering blood pressure by inhibiting ACE activity.

The invention relates to the technical field of biology, in particular to a zymomonas mobilis recombinant strain for producing ethanol by taking cellobiose as a raw material and application of the zymomonas mobilis recombinant strain. On the basis of recombinant zymomonas mobilis expressing beta-glucosidase, an efficient cellobiose conversion system is constructed through the synergistic effect of promoter and signal peptide optimization and gene multi-copy regulation, the activity of extracellular beta-glucosidase is remarkably improved, cellobiose is accelerated to be hydrolyzed into glucose, and the activity of beta-glucosidase is improved. The yield of ethanol is promoted to be obviously improved. Based on the fact that no linear positive correlation exists between the activity of beta-glucosidase and the yield of ethanol, the bottleneck of cellulose degradation under high substrate concentration is solved through multi-element coordinated regulation and dynamic matching of enzyme activity and substrate metabolism, and the energy efficiency and industrial application value of biomass energy conversion are remarkably improved.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a D-psicose 3-epimerase mutant derived from rumen clostridium and application of the D-psicose 3-epimerase mutant. The mutant disclosed by the invention is obtained through specific single-point mutation on the basis of a wild type amino acid sequence of D-psicose 3-epimerase, and specifically, the mutant is obtained through mutation of the following five sites: L159V, Q164E, N238T, E288G and C289I; wherein mutants containing all five single point mutations are named as XT mutants. The mutants significantly improve the thermal stability of the enzyme and the catalytic conversion rate under high substrate concentration, especially under the high substrate concentration of 300-700 g / L, the conversion rate of D-psicose of the XT mutant is improved by 2-3% compared with that of a wild type, and the thermal stability of the XT mutant is improved by 28 times compared with that of the wild type. The mutant is suitable for a high-temperature and high-substrate-concentration industrial conversion system, the production cost of D-psicose can be remarkably reduced, and the mutant has important industrial application value.

The present application relates to the field of temperature measurement technology, and specifically to a method and system for real-time monitoring of the ambient temperature of a bio-fertilizer fermentation tank. The method comprises: performing anomaly detection on temperature data acquired by each temperature sensor disposed in the bio-fertilizer fermentation tank; obtaining an anomaly factor of the temperature data acquired at the current moment based on the similarity between the temperature data acquired by each temperature sensor and the remaining temperature sensors; performing anomaly detection on the discreteness of the temperature data to determine a first temperature anomaly degree; obtaining a substrate consumption rate at each moment; analyzing the similarity between the substrate consumption rate and the change in temperature data to determine a correction factor at the current moment; performing a comparative analysis on the relationship between the temperature data and the substrate concentration to determine a second temperature anomaly degree; and determining a final anomaly result of the temperature data acquired at the current moment to monitor the ambient temperature of the bio-fertilizer fermentation tank. The present application aims to reduce the error in temperature data anomaly detection and improve the accuracy of temperature monitoring.

The invention discloses a D-tagatose-3-epimerase mutant and application thereof, the D-tagatose-3-epimerase mutant has a single-point mutation or a multi-point mutation in the 45th mutation, the 48th mutation, the 50th mutation, the 80th mutation, the 93th mutation, the 98th mutation and the 144th mutation relative to an amino acid sequence SEQ ID NO.1 of a wild type D-tagatose-3-epimerase, and the D-tagatose-3-epimerase mutant has a single-point mutation or a multi-point mutation in the 45th mutation, the 48th mutation, the 50th mutation, the 80th mutation, the 93th mutation, the 98th mutation and the 144th mutation. The invention also provides a corresponding coding gene, a recombinant vector, a genetically engineered bacterium, a biocatalyst and application in preparation of rare sugars. The invention develops the D-tagatose-3-epimerase mutant which has stronger adaptability with glucose isomerase and is more stable in a cascade reaction, is beneficial to improving the efficiency of reaction for preparing rare sugar by using xylose, can simultaneously consider higher substrate concentration and D-xylose conversion rate, and reduces the production cost of the product.

The application belongs to the technical field of enzyme engineering and genetic engineering, and discloses a ketoreductase mutant, method and application. The mutant comprises the following point mutations: the isoleucine at the 91th position of the amino acid sequence shown in SEQ ID No. 1 is mutated into glycine (I91G), and the alanine at the 139th position is mutated into methionine (A139M). The ketoreductase mutant obtained by the application can produce N-BOC-cis-3-hydroxy-L-proline methyl ester in high selectivity and high yield. Under the conditions of OD 600 =10, 300mM glucose, 2mM NADP + / NAD + , 50mg / ml ls GDH, and a substrate concentration of 200mM, the yield is 96.5% and de>99% after 18h of reaction.