4 results about "Sucrose isomerase" patented technology

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 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 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.