105 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 an alkene reductase mutant and an application of the alkene reductase mutant in synthesis of (R)-2-dimethyl methylsuccinate. According to the method, the olefin reductase is subjected to directed evolution to obtain the olefin reductase mutant with improved enzyme activity and substrate tolerance, the obtained mutant can be used for efficiently reducing dimethyl itaconate to prepare the high-optical-purity (R)-2-dimethyl methylsuccinate, and the reaction substrate concentration, the reaction yield and the optical purity of the product are high; the reaction process is simple to operate and low in energy consumption, and meets green chemical requirements. Therefore, the method disclosed by the invention can be applied to the biotransformation of the industrial production of the (R)-2-methyl dimethyl succinate.

The invention belongs to the technical field of biotechnology and biocatalysis, and particularly relates to a method for preparing D-tagatose by catalyzing isomerization of D-fructose. The invention provides a method for preparing D-tagatose by catalyzing isomerization of D-fructose, which comprises the following steps: by taking a wet cell obtained by inducible expression of a recombinant genetically engineered bacterium containing a D-tagatose-4-epimerase gene as a catalyst and D-fructose as a substrate, when the concentration of the substrate is higher than 300g / L, continuously supplementing the substrate and enzyme to prepare the D-tagatose by adopting a strategy of continuously supplementing the substrate and enzyme. The substrate inhibition is reduced, the conversion rate of the D-tagatose is increased, and the final conversion rate of the D-tagatose is increased from 19.5% to 24.2%.

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 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 invention belongs to the technical field of biochemical engineering, and particularly relates to a marine metagenome sourced meso-diaminopimelate dehydrogenase mutant and application thereof in D-tryptophan synthesis. According to the invention, a fast evolution method based on Rosetta guidance is adopted to modify meso-diaminopimelate dehydrogenase from a marine metagenome, and a mutant with high catalytic activity on 3-indolepyruvic acid is obtained through screening. The meso-diaminopimelate dehydrogenase mutant disclosed by the invention can be applied to direct catalysis of reductive amination of 3-indolepyruvic acid to generate chiral D-tryptophan, and can also be coupled with L-amino acid deaminase and coenzyme regeneration enzyme to realize configuration inversion from L-tryptophan to D-tryptophan, the substrate concentration reaches 0.1 M, and the product conversion rate is gt; the ee value is gt; therefore, the method has a relatively high practical application value.

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses a glutaminase mutant, immobilized glutaminase and application thereof. The glutaminase mutant is based on an amino acid sequence as shown in SEQ ID NO.1 and is subjected to amino acid mutation at the following sites: E113K, R136M and H223V, and the amino acid sequence of the glutaminase mutant is as shown in SEQ ID NO.3. The immobilized glutaminase is prepared by using the glutaminase mutant, the enzyme activity is increased by 5-10 times compared with that of a wild type, the substrate concentration in the L-theanine catalyzed and synthesized by the immobilized glutaminase reaches 80-150g / L, the conversion rate is greater than 99%, the L-theanine can be recycled through simple filtration, and the L-theanine can be repeatedly used for more than five times after being recycled. And the conversion rate during repeated use is basically the same as that during primary use, so that the method has relatively high industrial application potential.

The invention provides alkali-resistant glycosyl transferase and a coding gene, a biological material, a preparation method and application thereof. The glycosyl transferase has a specific amino acid sequence and can be efficiently expressed in escherichia coli through a genetic engineering means. The preparation method comprises the steps of codon optimization, recombinant plasmid cloning, expression strain transformation, induced expression, purification and the like. The obtained glycosyltransferase has significant application in preparation of gastrodin by enzymatic catalysis of p-hydroxybenzyl alcohol, and enzymatic hydrolysis conditions such as substrate concentration, enzyme concentration, UDP-glucose concentration and temperature, and buffer solution type and pH value are optimized. Particularly, when a glycine-sodium hydroxide buffer solution is used at the pH value of 9.5, the enzymolysis effect is remarkable. According to the alkali-resistant glycosyl transferase as well as the preparation method and the application thereof, a powerful tool is provided for biosynthesis of natural products such as gastrodin, and the alkali-resistant glycosyl transferase has an important industrial application value. Meanwhile, the biological material disclosed by the invention provides a basis for production and application of alkali-resistant glycosyl transferase.

The invention provides a method for improving tetrahydrofolic acid biosynthesis based on NADPH cofactor circulation, and aims to catalyze folic acid into tetrahydrofolic acid through an in-vitro enzyme catalysis technology. According to the invention, escherichia coli BL21 (DE3) is used as a host cell, and dihydrofolate reductase (SmDHFR) derived from serratia marcescens and formate dehydrogenase (pseudoFDH) derived from pseudomonas, which are obtained by screening, are expressed. A TB culture medium is used for fermentation, ultrasonic cell disruption and purification to obtain pure enzyme liquid of SmDHFR and pseFDH. SmDHFR is responsible for converting folic acid into tetrahydrofolic acid, and NADPH consumed in the process is converted into NADP < + >. And the pseudoFDH is used for reducing the generated NADP < + > into NADPH so as to realize the cyclic utilization of the NADPH cofactor. By exploring the optimal ratio of the addition concentrations of the two enzymes, the optimal reaction pH and the optimization of the substrate concentration, the final yield of tetrahydrofolic acid is 3661.14 mu M.

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).

The invention discloses an ethanol dehydrogenase mutant and an application of the ethanol dehydrogenase mutant in synthesis of symmetric chiral diol. The ethanol dehydrogenase mutant is obtained by carrying out single mutation or multiple mutations on amino acids at 74th, 187th, 210th, 211th and 352th sites of an ethanol dehydrogenase amino acid sequence as shown in SEQ ID NO.2 from Candida orbiculata Co. The invention further discloses a preparation method of the ethanol dehydrogenase mutant, and the preparation method comprises the following steps: carrying out single mutation or multiple mutations on the amino acids at the 74th, 187th, 210th, 211th and 352th sites of the ethanol dehydrogenase amino acid sequence as shown in SEQ ID NO.2. Alcohol dehydrogenase is utilized to catalyze prochiral symmetric diketone to synthesize symmetric chiral diol, the method is high in substrate concentration, high in stereoselectivity, mild in reaction, simple in operation process, environment-friendly and more suitable for industrial amplification production, and the problems that in the prior art, the substrate concentration is not high, the reaction condition is violent, the reaction process is complex and difficult to control, and the production cost is low are solved. And the method is not environment-friendly enough, and the like.

The invention discloses a recombinant D-amino acid oxidase mutant, a coding gene and application thereof, and belongs to the technical field of enzyme engineering. A D-amino acid oxidase mutant is constructed, the catalytic activity of enzyme is greatly improved, and the specific enzyme activity can be improved to 54.32 U / g. The constructed mutant is used for catalyzing D / L-glufosinate-ammonium, under the participation of glutamate dehydrogenase and ammonium formate dehydrogenase, a catalytic reaction can be carried out under the 1M substrate concentration, the catalytic conversion rate is 99%, the yield is 97%, and the e.e. Value is gt; and the effect is 99.9%.

The invention discloses a carbonyl reductase mutant, an immobilized carbonyl reductase and its application, and relates to the field of genetic engineering and enzyme engineering technology. A carbonyl reductase mutant, whose amino acid sequence is shown in SEQ ID NO.1; a carbonyl reductase mutant crude enzyme solution is mixed with chitosan microspheres, a glutaraldehyde solution is added dropwise, oscillation cross-linked, and washed with water to obtain an immobilized carbonyl reductase. The enzyme activity of the immobilized carbonyl reductase prepared in the present application is increased by 5-10 times compared with that of the wild type. When eslicarbazepine is prepared by asymmetric reduction catalyzed by immobilized carbonyl reductase, the substrate concentration reaches 80-150 g / L, the chiral purity value is greater than 99%, and the conversion rate is greater than 99%. The immobilized enzyme of the present invention can be recovered by simple filtration, can be repeatedly used more than 5 times after recovery, and the chiral purity and conversion rate of the reaction used for the first time are substantially the same when reused, and has a high potential for industrial application.

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 present invention relates to the field of biochemical engineering technology, and discloses a high-purity adenosylmethionine butane disulfonate preparation process, comprising the following steps: step 1, enzyme screening: selecting a catalytic enzyme that catalyzes the reaction of adenylic acid and butane disulfonic acid; step 2, enzyme expression and purification: expressing the catalytic enzyme by genetic engineering technology, and purifying by affinity chromatography to obtain an enzyme with a purity higher than 90%; step 3, reaction condition optimization: adjusting substrate concentration, pH value, temperature and additive conditions; step 4, enzyme-linked catalytic reaction. By optimizing the temperature and solvent system of the enzyme catalytic reaction, the reaction conversion rate is significantly improved, thereby increasing the yield of SAM. Under the optimized reaction conditions, the enzyme catalytic reaction shows the best reaction efficiency at 60°C, and can effectively convert the raw material into a high-yield SAM. In this way, the utilization rate of the raw material is improved, and at the same time, the reaction time can be reduced, the energy consumption can be reduced, and the production efficiency can be improved.

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%.