51 results about "Sucrose isomerase" patented technology

The invention discloses an Erwiniarhapontici NX-5 and a method of producing sucrose isomerase for the preparation of isomaltulose by the Erwiniarhapontici. The strain CGMCC No.2222 is inoculated in an aseptic culture medium containing carbon source, nitrogen source, inorganic salt and water for the aerobic culture. Cells containing the sucrose isomerase are obtained after the centrifugation or the ultrafiltration. Furthermore, dissociative cells containing the sucrose isomerase are directly adopted to be converted into sucrose to generate the isomaltulose or immobilized cells containing the sucrose isomerase which are then converted into the sucrose isomerase to generate the isomaltulose. With the strain adopted, the conversion rate of the sucrose reaches to 99.5 percent (w/w), the conversion rate of the isomaltulose reaches to 90 percent, and the concentration of the isomaltulose in the conversion solution reaches to 500g/L. no hydrolysis side effects happen. Almost no glucose and fructose are contained in the conversion solution. The product, the isomaltulose, can not be converted into other components with the reaction. The invention is beneficial to the industrial production of the isomaltulose.

The invention discloses a method for preparing isomaltulose by polyvinyl alcohol immobilized sucrose isomerase producing bacteria. The polyvinyl alcohol immobilized sucrose isomerase producing bacteria are obtained by adding 5 to 30 weight percent of sucrose isomerase producing wet bacteria into 4 to 50 percent of water solution pulp of an enzyme carrier which is polyvinyl alcohol or/and kieselguhr at 25 to 30 DEG C and uniformly stirring and hardening at -20 DEG C for later use, wherein the ratio of polyvinyl alcohol to kieselguhr is 2:(0.8-1.21). When used, the immobilized enzyme producing bacteria are taken out, naturally unfrozen at room temperature, cut into grains and added into sucrose solution to perform isomerization conversion; then produced sucrose isomerase is used to decompose sucrose remaining in conversion solution; the conversion solution is purified by decolorizing and ion exchange; and an isomaltulose product is obtained by concentration crystallization and centrifugal separation. The method has the advantages that: the operation is simple and efficiency; the material is cheap and readily available; the grains of immobilized bacteria have high strength and are insusceptible to breakage; the activity of sucrose isomerase is high; the service life of the sucrose isomerase is long; and the like. The isomaltulose has high quality, the purity of the isomaltulose is over 98 percent, the sucrose residue content is less than 0.1, and the isomaltulose is more suitable for preparing high-quality isomalt by hydrogenation.

The invention discloses gene engineering bacteria with a high yield of sucrose isomerase, and a method for efficiently producing the sucrose isomerase, and belongs to the technical field of gene engineering and fermentation engineering. According to the method, codon and signal peptide optimization is adopted to ensure that the expression quantity of a sucrose isomerase gene in bacillus licheniformis is improved by more than or equal to 3 times compared with the original expression quantity. Finally, a growth medium and a fermentation process suitable for synthesis and secretion of an enzyme, and recovery and preparation of a product are established, so that the content of isomaltulose in the product reaches 100%. Therefore, a firm foundation is laid for the application of the sucrose isomerase in food and other industries, and a technical support is provided for efficient preparation of other industrial enzyme preparations.

The invention provides a method for directly producing isomaltulose by condensation crystallization without a separating purification step. The method is characterized in that: in the process of preparing the isomaltulose, the complex processes of separating and purifying the isomaltulose by ion exchange resin and the like are omitted in industrialized production, and the vacuum condensation and crystallization of conversion solution of sucrose are directly performed to obtain the purified isomaltulose. In the method, cell debris containing activity of sucrose isomerase is firstly prepared into a biocatalyst, the sucrose is subjected to biological conversion by catalysis to generate a product of the isomaltulose not containing byproducts such as glucose, fructose and the like, and the conversion solution of sucrose is directly subjected to vacuum condensation, crystallization and drying to produce the crystallized product of the isomaltulose. The method has the advantages of greatly lowering production cost, saving a large amount of process water and simplifying the production process.

The invention discloses a sucrose isomerase mutant with improved thermal stability and catalytic efficiency and belongs to the technical fields of genetic engineering and enzyme engineering. According to the sucrose isomerase mutant with improved thermal stability and catalytic efficiency, site-specific mutagenes is performed on the amino acid with relatively high B factor in the sucrose isomerase crystal structure, namely mutating E at the 175 site of the sucrose isomerase into N; at the same time, E at the 175 site is further mutated into N on basis of the mutant K576D, so that two sucrose isomerase mutants with both improved thermal stabilities and catalytic efficiencies are obtained. Compared with the natural sucrose isomerase, the half life periods of the mutant E175N and the mutant E175N/K576D disclosed by the invention are respectively improved by 130% and 665% at 45 DEG C and the catalytic efficiencies Kcat/Km are respectively improved by 38% and 19%. Compared with natural enzymes, when catalysis is performed on sucrose by the sucrose isomerase mutant to produce isomaltulose, the maximum isomaltulose conversion rates of the mutants are respectively improved by 1.8% and 1.6%. Compared with natural sucrose isomerases, the two mutants obtained by the invention are more suitable for industrial applications.

The invention discloses a sucrose isomerase mutant with improved stability and a construction method thereof, and belongs to the field of gene engineering and enzyme engineering. On the basis of high-enzyme-activity dispersed pantoea-derived sucrose isomerase, two mutant strains V280L and S499F with improved single-point thermal stability and a combined mutant V280L/S499L are obtained through screening. The thermal stability of the three mutants is obviously improved, and the enzyme activity is not influenced. The mutants provided by the invention are more suitable for industrial production than natural sucrose isomerase, and have very huge application prospects and industrial values.

The invention discloses a sucrose isomerase mutant with high activity and high conversion rate and an application thereof, and belongs to the technical field of gene engineering. The mutants comprise mutants Y246L, H287R and H481P, and belong to the technical field of gene engineering. According to the sucrose isomerase mutant disclosed by the invention, a gene for coding the mutant is obtained by designing a site-directed mutagenesis primer and carrying out site-directed mutagenesis on the gene, and the gene is expressed in an escherichia coli expression system, so that the activity and the conversion rate of the obtained sucrose isomerase mutant are remarkably improved. The sucrose isomerase mutant prepared by the invention is a more efficient biocatalyst, and can improve the production efficiency and reduce the production cost when being applied to industrial production of isomaltulose.

The present invention provides a method of producing trehalulose, the method comprising: (a) contacting a sucrose solution with a sucrose isomerase enzyme to produce a sucrose-enzyme mixture; and (b) incubating the sucrose-enzyme mixture of (a) at a temperature of less than 10° C. for a period of time sufficient to convert the sucrose in the sucrose-enzyme mixture to a product comprising trehalulose.

The invention belongs to the technical field of enzyme engineering, and relates to a sucrose isomerase mutant and application thereof. The mutant carries out mutation on R434P and E273N in wild type sucrose isomerase with an amino acid sequence as shown in SEQ ID NO. 1. Compared with wild type sucrose isomerase, the mutant has the advantage that thermal stability and fermentation activity are obviously improved. The immobilized enzyme prepared from the mutant is higher in activity, has remarkably improved stability, an improved sucrose conversion rate and isomaltulose generation rate, has a lower impurity content and is more suitable for industrial application.

The invention discloses a method for producing isomaltulose by whole cell transformation, and belongs to the technical field of genetic engineering. The method includes utilizing whole cell transformation of bacillus subtilis engineering bacteria of a gene capable of expressing and encoding sucrose isomerase (nucleotide sequence as shown in SEQ ID NO. 1) to produce isomaltulose. Conversion of theisomaltulose by the method has the advantages of high conversion rate, production intensity and high yield. By means of conversion and production of the isomaltulose for 9h in the method, 500g/L sucrose in a reaction system can be converted into 443g/L isomaltulose, the conversion rate of the sucrose is as high as 94%, the yield of the isomaltulose is as high as 88.6%, and the space-time yield ofthe isomaltulose is as high as 49.2g/L h.

The invention is directed to novel enzymes that convert sucrose to isomaltulose. More particularly, the present invention discloses novel sucrose isomerases, polynucleotides encoding these sucrose isomerases, methods for isolating such polynucleotides and nucleic acid constructs that express these polynucleotides. Also disclosed are cells, including transformed bacterial or plant cells, and differentiated plants comprising cells, which contain these sucrose isomerase-encoding polynucleotides, as well as extracts thereof. Methods of producing isomaltulose are also disclosed which use the polypeptides, polynucleotides, cells, cell extracts and plants of the invention.

The invention discloses a preparation method of isomaltitol. The technological process comprises the following steps: (1) with a sucrose solution as a raw material, transforming the sucrose solution through sucrose isomerase to obtain the transformation liquid of isomaltulose and miscellaneous saccharides; (2) performing decoloration, low-temperature crystallization and re-crystallization on the obtained transformation liquid to obtain a high-purity isomaltulose crystal; and (3) performing catalytic hydrogenation with skeletal nickel to obtain isomaltitol. According to the method disclosed by the invention, in the first step, the yield of isomaltulose is increased by adding glucose in the reaction process, and the generation of other miscellaneous saccharides is reduced; and in the second step, the low-temperature crystallization and re-crystallization method is selected according to the solubility difference between isomaltulose and miscellaneous saccharides so as to remove the miscellaneous saccharides. The whole synthesis method is simple in process, the energy is remarkably saved, the production efficiency is improved, and the cost is reduced.

The invention relates to a preparation method of a cell wall fusion sucrose isomerase, and is characterized in that ultrasonic disruption technology is employed, and a cell wall fusion enzyme with sucrose isomerase activity is prepared and used as a catalyst to convert sucrose into isomaltulose. The method comprises the following steps: 1) performing bacterial activation of sucrose isomerase producing bacteria in a seed medium; 2) transferring the activated bacteria to a fermentation medium for culture so as to produce a sucrose isomerase; 3) performing ultrasonic disruption of the sucrose isomerase producing bacteria, collecting fragmented cells to obtain a cell wall fusion sucrose isomerase. When sucrose conversion is catalyzed by the sucrose isomerase prepared in the invention, the sucrose conversion rate is 99.8 wt%, the proportion of isomaltulose in the conversion product is 88.39%, and the proportion of trehalulose is 11.61%.

The invention provides polynucleotides encoding N-terminal truncated forms of sucrose isomerase polypeptides which are capable of conferring increased nematode resistance in a plant. The invention also provides methods of producing transgenic plants with increased nematode resistance, seeds of such transgenic plants, and expression vectors, all of which comprise the polynucleotides of the invention.

The invention discloses a functional food sweetening agent which comprises 2.1-6.0 percent by weight of 1,1-GPS(alpha-D-glucopyranosyl-1,1-D-sorbitol), 43.9-47.1 percent by weight of 1,6-GPS (alpha-D-glucopyranosyl-1,6-D-sorbitol) and 49.8-50.8 percent by weight of 1,1-GPM (alpha-D-Glucopyranosyl-1,1-D-mannitol). The preparation method comprises the following steps: (1), treating a saccharose water solution by using saccharose isomerase to obtain a saccharose conversion solution with a saccharose conversion rate above 95 percent; (2), treating the saccharose conversion solution by using active dry yeast; (3), treating a treated solution obtained in the step (2) by using a tubular centrifugal machine, treating through an ion exchange column after activated carbon adsorption to obtain a settled solution; and (4), carrying out hydrogenation reaction on the settled solution obtained in the step (3) by using raney nickel as a catalyst, then carrying out vacuum concentration until the baume degree reaches 50 DEG, spray-drying to obtain the powdery functional food sweetening agent. The functional food sweetening agent is pure in taste, and is capable of replacing saccharose as a food sweetening agent. The preparation method is simple in process, high in yield and low in cost.

The invention provides a prepared reagent for quickly detecting saccharose in honey and a storage method and a detection method of the prepared reagent. The prepared reagent is prepared from the following raw materials in parts by weight: 75-83 parts of a filler, 0.5-1.5 parts of a color developing agent, 15-20 parts of phosphate, 0.5-1.2 parts of sucrose isomerase, 0.6-1.2 parts of glucosyl oligosaccharide oxidase and 0.5-1.2 parts of horse radish peroxidase. The prepared reagent is convenient to carry, simple and efficient in operation process, high in security and strong in anti-jamming capability, large-scale instrument equipment is not needed, and detection results are visual and visible; the detection sensitivity is high, and by use of a spectrophotometer, the limit of detection canreach 1.0 mg/L; and the detection method is high in recovery rate and excellent in reproducibility.

The invention discloses a method for separating and preparing trehalose from sucrose isomerase enzymatic hydrolysate, and belongs to the field of functional sugar separation and preparation. The method comprises the following steps: decoloring enzymolysis by using resin;then removing macromolecular impurities and part of residual pigments in the sucrose isomerase enzymatic hydrolysate by using a solid-phase extraction technology, finally concentrating the treated enzymatic hydrolysate, and further separating and preparing on a self-made hydrophilic chromatographic column by using a hydrophilicchromatography to obtain the high-purity trehalose with the purity of more than 90%.

The invention relates to a preparation technology of isomaltulose, and specifically to a preparation technology for producing isomaltulose. The preparation technology comprises the following steps ofpreparing mother liquid, preparing immobilized enzyme-producing bacteria, preparing crude enzyme liquid and isomaltulose mother liquid, and preparing an isomaltulose finish product with the purity being greater 95%; the preparation technology for producing isomaltulose provided by the invention abandons the traditional technology of directly producing isomaltose through microbial fermentation (lowyield), produces sucrose isomerase through microbial immobilized fermentation, enables the sucrose isomerase to perform enzymolysis on sucrose in vitro, wherein the percent conversion reaches 95% orabove, the production cost is reduced, a product completely meets the newest international standards and Chinese national standards, and the production cycle is shortened.

The present invention is directed towards genetic modification of native gene encoding for sucrose isomerase and isomaltulose synthase to substantially increase the expression level of these enzymes and use of said enzymes in a process to produce rare disaccharides such as isomaltulose and trehalulose. Also disclosed in the present invention is expression constructs comprising the modified genes and a host cells to express the same.

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.