16 results about "Sucrose isomerase" patented technology

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

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 present invention relates to the field of biotechnology and discloses a sucrose isomerase mutant and its use in the preparation of isomaltulose. The present invention performs single-point mutations at positions 149, 222, and 297 of the amino acid sequence of a sucrose isomerase derived from Serratia symbiotica, or any combination of the above-mentioned multiple points, so that the catalytic efficiency of the obtained sucrose isomerase mutant in catalyzing the preparation of isomaltulose from sucrose is improved, which is specifically reflected in the improvement of isomaltulose selectivity and enzyme activity. When isomerizing sucrose to produce isomaltulose, the mutant significantly improves the selectivity of the product isomaltulose. At the same time, the catalytic activity of the sucrose isomerase mutant is also improved. Therefore, the catalytic preparation of isomaltulose by the sucrose isomerase mutant is highly efficient and has a short reaction time.

The invention discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the sucrose isomerase mutant, the thermal stability and the expression quantity are simultaneously improved, the mutant is used for preparing isomaltulose, the half-life period of the single mutant V447E at the temperature of 40 DEG C is prolonged by 1.38 times, the optimum temperature is increased to 35 DEG C from 30 DEG C, and the expression quantity is increased to 33.06 U * mL <-1 > from 18.89 U * mL <-1 >; other mutations are superposed on the basis of the mutant V447E, and the thermal stability of the constructed double mutant V447E / D232P is further improved. Compared with a wild type, the V447E / D232P has the advantages that the optimal temperature is increased by 5 DEG C, the half-life period at 40 DEG C is prolonged by 1.52 times, and the expression quantity is increased from 18.89 U.mL <-1 > to 30.73 U.mL <-1 >.

The invention provides a bacillus amyloliquefaciens strain BLBymyt-03, a production method of isomaltulose and application of the bacillus amyloliquefaciens strain BLBymyt-03, and the preservation number of the bacillus amyloliquefaciens strain BLBymyt-03 is CGMCC No.36184, and the preservation date is October 15, 2025. The strain can be used for preparing the sucrose isomerase through fermentation, so that the enzyme activity of the sucrose isomerase reaches 1252 U / mL. The strain can also be used for preparing isomaltulose and achieving conversion of high-concentration glycogen, the content of the converted isomaltulose is larger than 93%, the content of seaweed kelose is smaller than 5%, the substrate conversion rate reaches 92% or above, the primary crystallization purity is larger than 99%, and the yield is larger than 85%. Meanwhile, in crystals obtained through primary crystallization, the proportion of 30-80-mesh products reaches 80% or above, and the particles are uniform and good in fluidity.

The invention relates to the technical field of enzyme engineering, in particular to a mutant for improving the thermal stability of sucrose isomerase and a recombinant strain thereof. The amino acid of the sucrose isomerase mutant is as shown in SEQ ID NO. 1. The sucrose isomerase mutant is obtained by mutating the 304th amino acid from Leu to Pro on the basis of wild type sucrose isomerase from Pantoea dispersa, and the specific activity of the sucrose isomerase mutant is 1.21 times that of the wild type sucrose isomerase. The sucrose isomerase mutant provided by the invention is used for a catalytic experiment, and the purity of isomaltulose in a catalytic product can reach 98.87%. The invention lays a foundation for industrial application of sucrose isomerase.

The invention relates to the technical field of biology, and discloses a sucrose isomerase mutant and application thereof in catalytic synthesis of isomaltulose. According to the present invention, site-directed mutagenesis is performed on the sucrose isomerase gene derived from Raoultella terrigena, and the specific amino acid site is selected to modify so as to obtain the enzyme mutant capable of significantly improving the catalytic efficiency; in the process of catalyzing conversion of sucrose into isomaltulose, the mutant shows relatively high enzyme activity and can complete reaction within a relatively short time, so that the production efficiency of isomaltulose is remarkably improved. The enzymatic synthesis process is carried out under mild reaction conditions, the generation amount of byproducts is small, and the product purity is high. The application of the sucrose isomerase mutant not only improves the production efficiency of isomaltulose, but also has wide industrial application prospects, and is especially suitable for large-scale production in the industries of food, health care products, low-sugar drinks and the like.

Sucrose isomerase is used as a nutritional supplement, or can be mixed in with a powderous food / beverage formulation. When an animal, including a human, consumes sucrose, the sucrose isomerase will act on the sucrose present in the food, and will convert the sucrose to other sugars. This results in lowering of the glycemic index of the food without changing the formulation of the food.

The invention discloses a construction method of a bacillus subtilis chassis strain with improved sucrose isomerase expression capability, and belongs to the technical field of enzyme engineering and microbial modification. Based on a CRISPRi (clustered regularly interspaced short palindromic repeats i) system and an sgRNA (single guide ribonucleic acid) library of a targeted whole genome, key genes influencing PdSI expression are screened from bacillus subtilis and are combined, regulated and controlled, and a bacillus subtilis chassis strain with improved expression capability is obtained. And then, high-density fermentation is carried out in a 3-L tank, so that efficient recombinant expression of PdSI is realized, and the enzyme activity in the fermentation supernatant can reach 408.23 U.mL <-1 >.

The application discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application constructs a sucrose isomerase mutant with improved thermal stability and expression amount, and uses the mutant for preparation of isomaltulose, wherein a single mutant V447E has a half-life at 40 DEG C prolonged by 1.38 times, an optimum temperature increased from 30 DEG C to 35 DEG C, and an expression amount increased from 18.89 U.mL ‑1 -1 to 33.06 U.mL ‑1 -1; a double mutant V447E / D232P with other mutations superimposed on the basis of the mutant V447E has further improved thermal stability; compared with a wild type, the double mutant V447E / D232P has an optimum temperature increased by 5 DEG C, a half-life at 40 DEG C prolonged by 1.52 times, and an expression amount increased from 18.89 U.mL ‑1 -1 to 30.73 U.mL ‑1 -1.

The application discloses a mutant with improved sucrose isomerase enzyme activity and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering in molecular biology. The known sucrose isomerase parent is subjected to molecular modification by using a site-directed mutagenesis technology, and mutant enzymes T522S / K340D, D222H / K340D, G467N / K340D, T522S, K340D, D222H and G467N are obtained. The catalytic activity of the series of mutant enzymes provided by the application is increased by 10% to 25% compared with that of the original bacteria. The isomaltulose conversion rates of the combined mutant enzymes T522S / K340D, D222H / K340D and G467N / K340D are increased by 2.3%, 1.5% and 1.3% respectively compared with that of the original bacteria, and the combined mutant enzymes have good stability, can be used for biological preparation of isomaltulose, and have potential application prospect.

The invention belongs to the field of bioengineering and genetic engineering, and designs an expression strain of sucrose isomerase fused with a tag as well as a production method and application of the expression strain. The method comprises the following steps: by taking a GFP coding gene and a CBM2a-PalI coding gene as templates, respectively carrying out amplification through an overlap PCR (Polymerase Chain Reaction) method to obtain a GFP-CBM2a-PalI coding gene, then converting the GFP-CBM2a-PalI coding gene into a competent cell of expression host bacteria E. coli Rosetta (DE3) through heat shock, and finally obtaining an expression strain of the sucrose isomerase containing the fusion tag. Compared with a traditional immobilization method, the conversion rate is remarkably improved, the visual immobilization strategy provides innovative help for industrial production application, and the monitoring intuition of the production process is improved.

The present invention relates to the fields of genetic engineering and immobilized enzymes, and particularly to a sucrose isomerase recombinant and its construction method and application. The present invention performs molecular modification on the wild-type sucrose isomerase (Slase) derived from Pantoea dispersa, and after screening for affinity peptide ligands, performs fusion expression. The specific enzyme activity of the obtained sucrose isomerase recombinant is increased by 88% compared to that of the sucrose isomerase, significantly improving its enzyme activity level and improving its specific catalytic efficiency for sucrose substrates; it provides a reference for the molecular modification of other enzymes. Moreover, the sucrose isomerase recombinant has good thermal stability. Finally, research on optimizing the embedding system for one-step purification and immobilization of SIase is carried out to prepare an affinity-directed immobilized enzyme microsphere with relatively high hardness and strength, and which can effectively reduce the leakage or loss of enzyme molecules, significantly improving the reusability of the sucrose isomerase recombinant.

The application discloses a kind of preparation of carrier-free immobilized sucrose isomerase and its application in catalyzing sucrose to produce isomaltulose, and belongs to the field of bioengineering technology.The specific method of the application comprises the following steps: using polyethylene glycol as a precipitant, dextran aldehyde as a crosslinking agent, realizing the immobilization of sucrose isomerase by carrier-free immobilization-crosslinked enzyme aggregate method, adding bovine serum albumin as a stabilizer and amino donor in the preparation process, and then using the immobilized sucrose isomerase to convert sucrose raw materials to produce isomaltulose.The crosslinked sucrose isomerase aggregate prepared by the method has significantly improved temperature stability, pH tolerance and storage stability, and the enzyme activity recovery rate can be maintained at more than 90% after 10 times of continuous repeated use;after continuously preparing isomaltulose in batches, the substrate conversion rate of CLSIAs-BSA can be maintained at more than 85%.The application improves the efficiency of enzymatic preparation of isomaltulose, reduces production cost and separation and purification difficulty, is suitable for large-scale continuous production, and has great significance for improving the technical level of isomaltulose production in China.

The invention belongs to the technical field of biological fermentation and crystallization separation, discloses a method and a system for producing isomaltulose by coupling double-bacterium synergistic fermentation with gradient temperature control crystallization, and solves the problem that glucose generates feedback inhibition on sucrose isomerase in a fermentation stage. The method comprises a fermentation stage and a crystallization stage, a double-bacterium synergistic fermentation system of recombinant bacillus subtilis engineering bacteria and recombinant pichia pastoris engineering bacteria is adopted in the fermentation stage, and the inoculation ratio of the recombinant bacillus subtilis engineering bacteria to the recombinant pichia pastoris engineering bacteria is 3: 1-5: 1; in the crystallization stage, a gradient temperature control-ultrasonic-assisted coupling crystallization process is adopted, after the fermentation liquor is subjected to concentration pretreatment, the temperature is reduced to 25-28 DEG C at the speed of 0.5-0.8 DEG C / h, and heat preservation and ultrasonic treatment are performed; and cooling to 15-18 DEG C at the speed of 0.3-0.5 DEG C / h, preserving heat and carrying out ultrasonic treatment. The method provided by the invention realizes efficient production and accurate purification of isomaltulose.

The invention belongs to the technical field of bioengineering and enzyme engineering, and discloses a sucrose isomerase mutant with high catalytic activity, a method and application, the mutant is obtained by mutating lysine K into methionine M at the 160th amino acid residue of wild sucrose isomerase and mutating lysine K into tyrosine Y. The mutant is obtained by mutating lysine K into tyrosine Y at the 302nd amino acid residue of wild sucrose isomerase. According to the mutant, K160M and K302Y double mutation is introduced into 160th and 302th amino acid residues through a directed evolution technology, the obtained sucrose isomerase mutant with K160M and K302Y double-site mutation has the advantages that the catalytic efficiency is obviously improved (improved by nearly 3 times), the Km value is reduced (reduced by 61%), the thermal stability is improved, and the sucrose isomerase mutant can be used for preparing the sucrose isomerase mutant with K160M and K302Y double-site mutation. The catalyst has excellent application catalytic performance in industrial production of isomaltulose, and can shorten the reaction time, improve the conversion rate and reduce the production cost.