84 results about "Trehalose synthesis" patented technology

This invention relates to a new clone mycose synthesized zyme gene and its expression in the correlated host which zyme gene clone belong to thermobifida turning the malt sager to mycose under normal biochemical reaction, its zyme substrate conversion efficiency increases along with the decreasing of the reaction temperature, taking the malt sager or amylum as the raw material and turn it to alpha 1-mycose alpha-1 by cloned.

The invention relates to a mutant of trehalose synthetase from corynebacterium glutamicum. A method for obtaining the mutant performs site-directed saturation mutation on a phenylalanine residue at the 263rd site of an amino acid sequence of trehalose synthetase from corynebacterium glutamicum by using a site-directed mutation technology, and the method for obtaining the mutant also comprises the following step: the saturation mutation is performed on at least one amino acid residue in at least 80 percent of homologous trehalose synthetase sequences of an amino acid sequence including SEQ ID NO.1, SEQID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The reaction kinetics of the trehalose synthetase expressed by a mutated trehalose synthetase gene changes, various enzymology characteristics such as specific activity of enzyme, optimum reaction temperature, optimum pH value, and the like also change, and the enzyme can improve the transformation efficiency of the trehalose synthetase and further reduce the production cost of the trehalose.

The invention discloses trehalose synthase of streptomyces griseochromogenes and a coding gene and application of the trehalose synthase. The trehalose synthase gene is cloned from the streptomyces griseochromogenes. The trehalose synthase can convert maltose to generate trehalose, the proper reaction temperature of the trehalose synthase is 15-35 DEG C and is 20-25 DEG C preferably, and the proper pH (potential of hydrogen) of the trehalose synthase ranges from 6.0 to 8.0 and ranges from 7.0 to 7.5 preferably. The conversion efficiency of the trehalose synthase is high, the maltose is used as a substrate, conversion rate of the maltose is about 80% at the reaction temperature of 25 DEG C, and less than 5% of glucose is generated to be beneficial to increase of the conversion rate of the substrate. The trehalose synthase can be used for converting commercial ultrahigh malt syrup to prepare the trehalose, and a novel method using an enzymic method to produce the trehalose industrially can be provided.

The invention relates to a method for producing trehalose through utilizing immobilized trehalose synthase. The method comprises the following steps: (1) inoculating an enzyme production strain into a fermentation medium, and culturing for 30-80 hours so as to prepare a fermentation liquor; (2) carrying out thallus separating, thallus wall breaking and enzyme purifying on the fermentation liquor so as to prepare a trehalose synthase liquor; (3) carrying out a crosslinking embedding reaction so as to prepare the immobilized trehalose synthase; and (4) mixing the immobilized trehalose synthase with a maltose substrate solution, conversing and separating to obtain the trehalose. The immobilized trehalose synthase has high conversion rate, quick reaction speed, short action time, stable enzyme activity and high mechanical strength, so that the production efficiency of the trehalose is increased, the product cost is lowered, and the production period is shortened.

The invention relates to a streptomyces coelicolor trehalose synthase enzyme gene. The gene is cloned from streptomyces coelicolor (Streptomyces coelicolor, AS 4.1061). A trehalose synthase enzyme expressed by the gene can be used for converting maltose into trehalose under the normal biochemical reaction conditions and has the characteristics of high reaction speed, high conversion rate of a substrate and few byproducts. The content of trehalose in a product generated in the process of converting the maltose at a temperature of 25 DEG C can achieve over 75 percent and only little glucose is generated. The streptomyces coelicolor trehalose synthase enzyme gene is beneficial for improving the conversion rate of the substrate and reducing the production cost. The gene can be applied to production of the trehalose. The trehalose can be applied to the fields of food industry, beauty cosmetics, medical biological products and the like.

The invention relates to a Streptosporangium roseum trehalose synzyme gene and an application thereof. The gene is cloned from Streptosporangium roseum, and the expressed trehalose synzyme is capable of converting maltose into trehalose. The most important characteristic of the gene is as follows: the expressed trehalose synzyme has wide range of reaction temperature and pH, can maintain higher enzyme activity at temperature of 25-35 DEG C and pH of 6.5-7.5, and has high conversion efficiency and reaction rate; the content of trehalose in the reaction product produced by converting maltose into trehalose under an optimum reaction condition can reach 82 percent, and little glucose is produced, thereby the conversion efficiency of the substrate is improved, and the production cost is reduced. By application of the recombinant trehalose synzyme produced by the gene, trehalose can be prepared by taking commercial maltose syrup or maltose syrup prepared from starch as material, and the trehalose can be applied to fields of food industries, cosmetics, medicines, biological products and the like.

The invention discloses a thermomonospora curvata mycose synthetase gene and application thereof. The gene is cloned from thermomonospora curvata. Mycose synthetase expressed by the gene has two different transformation functions. The mycose synthetase can be used for transforming maltose into mycose and transforming cane sugar into kelp ketose under normal biochemical reaction conditions. The gene can be applied to the production of mycose by transforming maltose, and can be used for transforming cane sugar into kelp ketose. The mycose and the kelp ketose can be applied to the fields of food industry, beverages, functional foods, beautifying cosmetics, medical biological products and the like.

The invention discloses a method for producing trehalose synthase by taking integrated recombinant bacillus subtilis as a strain and producing trehalose from the trehalose synthase. The method specifically comprises the following steps of: integrating a trehalose synthase expression element in a bacillus subtilis chromosome to construct integrated recombinant bacillus subtilis, and fermenting in a nutrient culture medium to produce the trehalose synthase by taking the recombinant bacillus subtilis as a strain, wherein via simple separation, the trehalose synthase in the fermentation liquor can be directly used for manufacturing trehalose. The method disclosed by the invention has the advantages that the trehalose synthase is produced by virtue of a food safety expression system, the exogenous gene contained in the strain with integrated expression can be used for stable passage and expression, and the expressed trehalose synthase is secreted to the outside of cells, has no antimicrobial activity, and achieves the requirements of food applications for enzymic preparation, thus being beneficial to further manufacturing for trehalose.

The invention relates to a heatproof trehalose synthase fusion enzyme mutant gene and an application thereof. On the basis of optimized trehalose synthase of streptomyces roseus, an amino acid sequence capable of improving heat stability is linked to a C-end to construct to obtain a new fusion enzyme mutant; optimal reaction temperature of the trehalose synthase fusion enzyme mutant is increased from 20-25 DEG C to 50-55 DEG C, heat stability is also improved, yield of trehalose from catalytic maltose is 79% and higher than that, of 67%, of trehalose from Thermus thermophilus trehalose synthase, and the rehalose synthase fusion enzyme mutant is more beneficial for reducing production cost of the trehalose.

A mycose synthetase and its use are disclosed. It consists of (a) or (b) protein, (a) protein is made of amino-acid residue sequence in sequence 2; (b) protein is derived by (a) and substituted and/or lost and/or added by one or several amino-acid residue and has mycose synthetase activity. It can be used to produce mycose by catalyzed malt dust.

The invention discloses a trehalose synthetase as well as encoding genes and application thereof. The trehalose synthetase is a protein comprising one of the following amino acid residue sequences: 1) an SEQ ID No. 1 amino acid residue sequence in a sequence table; and 2) a protein which is derived from the SEQ ID No. 1, has trehalose synthetase related functions, and is obtained by replacing and/or deleting and/or adding the SEQ ID No. 1 amino acid residue sequence in the sequence table. Trehalose can be obtained by using maltose as a substrate and performing catalysis with the application of the protein of the trehalose synthetase; besides, the catalytic reaction has the advantages that the catalysis time is short, the concentration of the required substrate is low, a 90mM maltose is used as the substrate, the catalysis time is 8 hours, and the conversion rate reaches 69 percent.

The invention discloses a supervisory control method for producing trehalose by a double-enzyme method. The supervisory control method comprises the following steps of firstly mounting a near-infrared real-time monitoring and control system; collecting starch liquefied liquid, measuring a DE (Dextrose Equivalent) value by using a Fehling's reagent method, collecting catalysis liquid, measuring a pH (potential of Hydrogen) value by using a pH meter, measuring the content of the trehalose by using high performance liquid chromatography, associating with a correspondingly collected near-infrared spectrum, establishing a quantitative model of changes of the DE value, the content of the trehalose and the pH value in a production process, and correcting; afterwards, quickly detecting the DE value, the content of the trehalose and the pH value in the production process by utilizing the established model, and stabilizing the DE value to be 8 to 20 and stabilizing the pH value to be in the range, in which the activity of malto-oligosaccharide based trehalose synthase-MTSase and malto-oligosaccharide based trehalose hydrolase-MTHase is optimal, by utilizing a control system. The method can be used for monitoring the DE value of starch liquid and the content of the trehalose in reaction liquid in real time and can also used for automatically controlling the change of the pH value and the content of the trehalose and the result is accurate and reliable.

The invention provides a method for simultaneously preparing trehalose and glucolactone. The method comprises the steps that amylase is used for converting starch into maltodextrin; a branch of the maltodextrin is removed, and maltooligosyl trehalose synthase and malt oligosaccharyl trehalose hydrolase are used for obtaining trehalose; glucose oxidase and catalase are fed, and generated glucose is converted into sodium gluconate; saccharifying enzymes are added, after saccharifying is finished, glucose oxidase and catalase continue to be fed for reacting, and when the glucose content is lower than 1 g/L, reacting is finished; enzyme deactivation, decoloration, filtering and impurity removal are carried out. According to the method for simultaneously preparing trehalose and glucolactone, the conversion time of trehalose can be obviously shortened, the conversion rate is obviously increased to be larger than 78%, and meanwhile glucolactone can be produced.

This invention relates to a new mycose synthesized zyme gene clone and its expression in correlate host which zyme gene clone is from corynebacterium glutamicum CICC 10226 which can turn malt sager to mycose under normal biochemical reaction, its substrate conversion efficiency increases along with the reduction of the reaction temperature. It can take malt sager or amylum as the raw material to be turned to 1-mycose with the cloned zyme alpha alpha-1.

The invention relates to a trehalose synthase-trehalose hydrolase fusion enzyme, an expression gene thereof and application. Amino acid sequences of the maltooligosyl trehalose synthase-maltooligosyl trehalose tetrahydrolase fusion enzyme are shown as SEQ ID NO.2, and nucleotide sequences of the expression gene are shown as SEQ ID NO.1. The trehalose synthase-trehalose hydrolase fusion enzyme, the expression gene and the application have the advantages that the novel maltooligosyl trehalose synthase-maltooligosyl trehalose tetrahydrolase fusion enzyme is constructed by the aid of genetic engineering technologies and has the activity of maltooligosyl trehalose synthase and the activity of maltooligosyl trehalose tetrahydrolase, and accordingly production processes can be effectively simplified as compared with the traditional two-enzyme methods.

The invention discloses a method for producing trehalose through multienzyme coupling and application thereof, and belongs to the technical field of enzymes. According to the method and the application, rice starch is used as a substrate, the disproportionate activity of 4-alpha glycosyl transferase and cyclodextrin glycosyltransferase are used, and pullulanase, maltooligosyl trehalose synthase (MTSase), maltooligosyl trehalose hydrolase (MTHase), the 4-alpha glycosyl transferase (TaAM) and the cyclodextrin glycanotransferase (CGTase) are adopted for multienyme coupling to improve a substrateuse ratio, so that the yield of the trehalose is improved.

The invention relates to a method for converting maltose into trehalose by using genetically engineered bacteria. According to the technical scheme, the method comprises the following steps: demonstrating a Streptomycescoelicolor, AS4.1061 trehalose synthase gene on the surface of pichia pastoris to establish a genetically engineered bacterium with enzyme activity, performing multiplication culture on the genetically engineered bacterium to greatly increase the yield of the trehalose synthase, adding the genetically engineered bacterium with the demonstrated trehalose synthase into maltose or starch modified maltose, reacting for 1-8 hours at 25-45 DEG C so as to convert maltose into trehalose, wherein the content of trehalose in a maltose converted product can be 80%, and moreover the genetically engineered bacterium can be recycled and used as a solvent after being separated, washed and dehydrated. Therefore, the technical difficult that the trehalose synthase cannot be massively produced, is not easy to extract and is hard to recycle and repeatedly use in an immobilization manner are overcome.

The invention relates to starch induction type recombinant bacillus subtilis as well as a preparation method and application thereof. The starch induction type recombinant bacillus subtilis contains a recombinant vector, wherein the recombinant vector is characterized in that in front of a BamHI restriction enzyme cutting site of a PHT43 plasmid, a Pgrac promoter is replaced with an alpha-amylase promoter PamyQ by performing for three times a mode of overlapping PCR continuously, and then an MTSase-MTHase fusion enzyme gene is inserted behind the BamHI restriction enzyme cutting site. When the used alpha-amylase promoter and an amylase signal peptide are combined with the expression gene, i.e., the MTSase-MTHase fusion enzyme gene, the expression effect of the starch induction type is better than that of other induction types.

The invention relates to a method for producing trehalose by coupling fermentation on double enzyme fusion enzyme expressed by efficient secretion. The method is characterized in that a promoter P43,a signal peptide PhoD, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase) are inserted into bacillus subtilis for the first time; meanwhile, after lytic genes Xpf, SkfA, LytC and SdpC are knocked out, construction is carried out to obtain recombinant bacteria pHT01-P43-PhoD-MTSase-MTHase(LM1234), and the rigid connection of a special sequence is adoptedto realize the function expression of a target enzyme; after the trehalose is produced by practical fermentation, an amazing discovery shows that the reformed recombinant bacteria exoenzyme activitycan occupy 70% of total enzyme activity, exoenzyme production and conversion are synchronously carried out, so that a process that trehalose production is carried out after enzyme separation is carried out in traditional fermentation liquor is omitted, and the fermentation of the recombinant bacteria and the production of the trehalose are similar to coupling.

The invention relates to a Streptomyces griseus subsp.griseus trehalose synthetase gene and applications thereof. The gene is cloned from Streptomyces griseus subsp.griseus, and the synthetase gene expressed by the gene is capable of converting maltose into trehalose. The gene has the biggest characteristics of high conversion efficiency and high reaction speed. Under the reaction condition provided by the invention, the content of the trehalose in the reaction product of the trehalose generated by converting the maltose can reach 87 percent, and moreover, only 3.8 percent of glucose is produced, thereby being beneficial to increasing the conversion rate of a substrate and reducing the production cost. Recombinant trehalose synthetase produced by applying the gene can be used for preparing trehalose by using maltose syrup sold in the market or maltose syrup prepared from starch. The trehalose can be applied to the field of food industry, facial cosmetics, pharmaceutical and biological products and the like.

The invention relates to a method for constructing highly expressed trehalose synthase engineering bacteria by using a Pcry3Aa promoter. For a recombinant carrier, a Pcry3Aa-PhoD fragment by which a Pcry3Aa promoter fragment and a PhoD signal peptide fragment are connected by an overlap PCR (Polymerase Chain Reaction) is inserted at the upstream of a restriction enzyme cutting site BamHI of a shuttle plasmid PHT01, and a target protein trehalose synthase TreS fragment is inserted between two restriction enzyme cutting site, i.e., BamHI and AatII. The invention further relates to a method for constructing the highly expressed trehalose synthase engineering bacteria by using the recombinant carrier. According to the method disclosed by the invention, the Pcry3Aa promoter is adopted to naturally induce the synthesis of trehalose synthase; because the Pcry3Aa promoter contains a special STAB-SD structure, the stability of the Pcry3Aa promoter to transcribe mRNA is improved, the half-life period of mRNA is prolonged, the mRNA translation level of a downstream target gene is improved, and therefore the trehalose synthase is highly expressed.

The invention discloses thermal stability and trehalose yield improved MTSase mutant and belongs to the technical field of enzyme engineering and protein engineering. The maltooligosyl trehalose synthase mutant disclosed by the invention is prepared by mutating one amino acid site of maltooligosyl trehalose synthase. Compared with parent maltooligosyl trehalose synthase, the prepared maltooligosyltrehalose synthase has higher thermal stability, a lower enzyme adding amount and a higher trehalose producing conversion rate. Compared with a wild type, a half-life period of the maltooligosyl trehalose synthase mutant disclosed by the invention at 50 DEG C is increased by 12 hours, and the half-life period of the maltooligosyl trehalose synthase mutant disclosed by the invention is 1.3 times of the half-life period of the wild type.

The invention discloses thermal stability improved maltooligosyl trehalose synthase mutant and belongs to the technical field of enzyme engineering and protein engineering. The maltooligosyl trehalosesynthase mutant disclosed by the invention is prepared by mutating one amino acid site of maltooligosyl trehalose synthase. Compared with parent maltooligosyl trehalose synthase, the maltooligosyl trehalose synthase mutant has higher stability. Compared with a wild type, a half-life period of the maltooligosyl trehalose synthase mutant disclosed by the invention at 50 DEG C is increased by 41 hours; the half-life period of the maltooligosyl trehalose synthase mutant disclosed by the invention is 2 times of the half-life period of the wild type; namely, compared with the wild type, the stability of the maltooligosyl trehalose synthase mutant disclosed by the invention is improved by twice.

The invention relates to gene cloning of wholly new fucose synthetase and its expression in related host. The enzyme gene is cloned from Deinococcus radioduran, which can transform the malt to fucose under normal biochemical conditions. The substrate conversion rate rises while the reaction temperature reduces. The fucose can be used in food industry, sea product cold storage and pharmaceutical activity preserving.

The invention discloses a trehalose synthase and application thereof in trehalose production, and belongs to the technical field of enzyme engineering. The trehalose synthase of the invention has highenzyme activity, the specific enzyme activity of the trehalose synthase in a crude enzyme solution is as high as 35.2 U/mg when Escherichia coli carrying the trehalose synthase undergoes induction culture for 12 hours, and is as high as 33.5 U/mg when corynebacterium glutamate carrying the trehalose synthase undergoes induction culture for 12 hours. The specific enzyme activity and the trehaloseconversion rate of a trehalose synthase mutant of the present invention are significantly improved than those of wild-type trehalose synthase, wherein the specific enzyme activity of the trehalose synthase mutant K246A is improved by 1.43 times than that of the wild-type trehalose synthase, and the trehalose conversion rate of the trehalose synthase mutant K246A is improved by about 15% than thatof the wild-type trehalose synthase.