136 results about "Trehalose synthase" patented technology

The invention relates to a separation and purification method of trehalose, which comprises the following steps: (1) carrying out enzymolysis on a trehalase reaction solution prepared by a trehalose synthase conversion process, and filtering to obtain a trehalose crude solution; (2) decolorizing the trehalose crude solution through activated carbon, and filtering to obtain a mixed solution; (3) passing the mixed solution through an ion exchange resin to obtain a glucose-trehalose sugar solution; and (4) concentrating the glucose-trehalose sugar solution, and separating by continuous chromatography with a simulated moving bed to obtain a trehalose solution and a glucose solution. The trehalase reaction solution generated by converting maltose by trehalose synthase is used as the raw material, the maltose which is isomeric with the trehalose is converted by an enzyme process, and impurity glucose, remaining maltose and impurities, such as proteins, pigments, metallic ions and the like, are removed to obtain the high-purity trehalose.

The invention provides a method for preparing trehalose. The method comprises the following step: with starch as a substrate and producing trehalose by using malt oligosaccharyl trehalose synthase and malt oligosaccharyl trehalose hydrolase. According to the method, the trehalose production efficiency is improved by improving processes of liquifying, saccharifying and the like.

The invention relates to a production method of trehalose, in particular to a method for producing trehalose by utilizing pseudomonas putida, belonging to the technical field of fermentation engineering and microorganism. The method for producing trehalose by utilizing pseudomonas putida comprises the following steps of: culturing the pseudomonas putida, treating beta-mercaptoethanol and fermenting. In the method, maltose is used as a substrate, and then the maltose is transformed to the trehalose in one step through preparing the permeable cells of pseudomonas putida by utilizing trehalose synthase in the cells of the pseudomonas putida. Through determination, about 3-5% of trehalose is generated in the permeable cell solution of the pseudomonas putida treated by using the beta-mercaptoethanol.

The invention relates to a method of synthesizing trehalose by virtue of whole cell catalysis. The method comprises the following steps that: recombinant Escherichia coli cells used for massively producing trehalose synthase are cultivated in a fermenting manner, permeable treatment is carried out on the recombinant cells by using an ecological biosurfactant, and the trehalose is synthesized by the treated cells in a phosphate buffering system by using maltose as a substrate. According to the method disclosed by the invention, the Escherichia coli after the permeable treatment is used for synthesizing the trehalose by using the 25-3 percent of maltose as the substrate, the conversion rate of the substrate can reach 55-60 percent after reaction for 16-20 hours at the temperature of 20-25 DEG C; in addition, compositions of a reaction solution are very simple, thus greatly simplifying an extraction and purification technology of the permeable treatment. By utilizing the method provided by the invention, the trehalose with high quality can be efficiently produced in a large scale with low cost.

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 maltose inducible trehalose synthase synthesis engineering bacterium, a method for preparing the same and application. The maltose inducible trehalose synthase synthesis engineering bacterium is characterized in that maltose inducible promoters are inserted in the fronts of BamHI cleavage sites of PHT01 plasmids of recombinant plasmid vectors instead of Pgrac promoters on the PHT01 plasmids, expression genes of Tat type signal peptides are inserted in the fronts of the BamHI cleavage sites, and expression genes of trehalose synthase are inserted in the rears of the BamHI cleavage sites. The maltose inducible trehalose synthase synthesis engineering bacterium, the method and the application have the advantage that expression effects realized after the maltose inducible promoters and the trehalose synthase are fused with one another are obviously superior to other inducible expression effects.

The invention relates to a method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces. The method is characterized in that Cot series spore capsid protein is used as the molecular vehicle for displaying heterologous protein on spore surfaces, series primers of the Cot molecular vehicle are designed, the molecular vehicle can be replaced and target heterologous protein can be changed through double enzyme digestion, the gene technology is used to display the trehalose synthase on bacillus subtilis spore surfaces, and the spore capsid protein which can efficiently display the trehalose synthase is screened. The method has the advantages that the trehalose synthase efficiently displayed on the spore surfaces has no antibacterial activity, meets the requirements of enzyme preparation applied to food and facilitates next-step production of trehalose.

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 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 method of industrial production for trehalose by way of microbial fermentation comprises the steps of fermentation of trehalose hydrolase and trehalose synthase, starch liquefaction, catalytic reaction, glycosylation, decolorization and filtration, ion exchange, concentration, crystallization, centrifugation, drying, and membrane separation. The method of industrial production for trehalose by way of microbial fermentation has the following advantages: 1, starch is used as the raw material in the method to produce the trehalose through enzymatic catalysis, allowing mass and cheap industrial production of the trehalose; 2, high activity and low dosage of enzymes cause reduction of the production cost; 3, a centrifuged trehalose mother liquor is subjected to membrane separation such that the content of the trehalose is increased, and blending crystallization may be implemented to further increase the yield of the trehalose; 4, an innovative trehalose crystallization process causes the content of the trehalose to be above 50%, the content of glucose to be below 20%, and the content of the one-time crystallization product of the trehalose to be above 98.5%; 5, compared with a process of transforming the trehalose by using maltose, the method has great advantages, such as higher yield and lower production cost.

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 discloses a TPS gene which is derived from rice and related with stress tolerance, which encodes following proteins: firstly, an amino acid sequence with SEQ ID NO: 1 in the table or the amino acid sequence whose N-end is lack of 1-130 amino acid residues and secondly the amino acid sequence in the first step encodes derivative proteins with the function of regulating plant stress tolerance after substitution or deletion or addition of 1-10 amino acid residues. Experiments show that the tolerance of the rice to adversity stress can be increased through transforming the gene of the invention to the rice and the normal growth and the economy deseription of the rice are not affected by the gene clearly. The proteins and encoding genes has great theoretical and practical significance for researching plant stress tolerance mechanism, increasing plant stress tolerance, and improving relative deseription and will play an important role in improving plant (especially cereal crops) stress tolerance gene engineering with broad application prospects.

The invention relates to a trehalose synthase and a coding gene and application thereof. A nucleotide sequence of an expression gene of the trehalose synthase is shown in SEQ ID No. 1; the amino acid sequence of the trehalose synthase is shown in SEQ ID No. 2. According to the trehalose synthase and the coding gene and application thereof, the trehalose synthase is obtained from pseudomonas stutzeri for the first time, and a preparation method of the trehalose synthase is simple and convenient and is high in yield and high in purity; proved by experiments, the thermal stability of the trehalose synthase is good, the trehalose conversion ratio can reach 70% in 1-hour reaction, and the reaction time is greatly shortened compared with that of the existing trehalose synthase, so that the production cost of trehalose can be reduced, and a foundation is laid for the industrial production of trehalose.

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 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 provides a nanofiber biological membrane immobilized bi-enzyme system and a trehalose catalytic synthesis method thereof. A starchiness nanofiber biological membrane is arranged on the surface of an escherichia coli cell generating high-temperature-resistant trehalose synthase through fermentation, polypeptide tag SpyTag-SpyCatcher capable of gene coding is used for specificity covalent binding and efficient immobilization recombination of beta-amylase, a trehalose synthase-beta amylase bi-enzyme catalytic system is constructed autonomously, and finally extracellular and intracellular two-step catalysis is conducted continuously. The immobilization efficiency of beta-amylase can reach 50-62%, maltose is generated by 20-25wt% of soluble starch under catalysis of beta-amylase on the extracellular biological membrane, enters the cell and reacts with trehalose synthase to generate trehalose, and the conversion rate of starch can reach 45-55% after the immobilized cell is reused for 10-16 times.

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 a maltooligosyl trehalose synthase and coding genes and application thereof. The maltooligosyl trehalose synthase is a protein which has one of following amino acid residue sequences: 1) an amino acid residue sequence with the No.1 SEQ ID in a sequence list; and 2) SEQ ID No.1 amino acid residue sequence derivative proteins which are obtained through the substitution and / or deletion and / or addition of one or a plurality of the amino acid residues from or to the SEQ ID No.1 amino acid residue sequence in the sequence list and are additionally provided with the relevant functions of the maltooligosyl trehalose synthase. The maltooligosyl trehalose synthase protein has the activity of the maltooligosyl trehalose synthase. The maltooligosyl trehalose synthase has molecular weight of 90 kDa, an enzyme activity pH range of 7.0 to 9.0, an optimal pH value of 8.0, an enzyme activity temperature range of 20 to 40 DEG C, and an optimal reaction temperature of 35 DEG C.

A method for culturing the new stress-resistant variety of sugar cane includes such steps as transferring the mycose synthetase gene into the enbryonic cells of sugar cane by agrobacterium mediation method and culturing the transgenic plant of sugar cane. Its advantages are short period, high efficiency and high stress-resistant effect.

The invention provides a Streptomyces trehalose synthase gene and application thereof. The gene is cloned from Streptomyces sp GXT6; trehalose synthase expressed by the gene can transform maltose into trehalose. The biggest characteristic of the gene is that a substrate has high transformation efficiency and a fast reaction speed; under optimal reaction conditions, a reaction product obtained in transformation of maltose into trehalose contains 86.68% of trehalose, only 3.56% of glucose is produced, so a transformation rate of the substrate is increased, and production cost is reduced. The trehalose synthase can be used in production of trehalose, and the trehalose can be used in fields like the food industry, cosmetics and pharmaceutical and biological products.

The invention discloses a method for improving expression level of a trehalose synthase gene by molecular chaperone co-expression. According to the method, a pET-22b-tttreS plasmid containing the trehalose synthase gene and plasmids comprising sigma32, GroeL, GroeS, DnaK and DnaJ independently or randomly combined are converted in recombinant escherichia coli expressing thermophilic bacteria trehalose synthase by applying a molecular biological technique, so that the trehalose synthase and molecular chaperone are co-expressed. Under the action of joint use of three molecular chaperones, the soluble expression quantity of a foreign protein is increased. According to the method disclosed by the invention, the trehalose synthase and molecular chaperone are co-expressed, so that the expression level of the trehalose synthase is improved.

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 relates to mutant trehalose synthase as well as an expression gene and application thereof. The nucleotide sequence of the expression gene of the mutant trehalose synthase is as shown in SEQ ID NO. 1. The amino acid sequence of the mutant trehalose synthase is as shown in SEQ ID NO. 2. On the basis of a three-dimensional structure predicted by pseudomonas stutzeri Qlu3, the invention conducts key amino acid site-specific mutagenesis on the active center for the first time, so that the mutant protein of the trehalose synthase is obtained; the mutant trehalose synthase is simple and convenient in preparation method, high in yield, high in purity and good in thermal stability; and compared with wild trehalose synthase, the trehalose conversion rate of the mutant is improved by 4.5% and the generation amount of glucose, as a byproduct, is reduced by 69.4%.

The invention discloses maltooligosyl trehalose synthase, a gene of the synthase, a recombinant expression vector containing the gene, and a recombinant bacterium, and preparation of the synthase. The synthetase has an amino acid sequence shown in SEQ ID NO:1, and can be obtained from the gene including the amino acid sequence shown in SEQ ID NO:2 or a mutant of the gene or a complementary sequence thereof by expression. The synthetase MTsase disclosed by the invention is high in optimal reaction temperature and thermal stability and low in optimal pH, reduces the contamination risk, improves the production stability, obviously improves the efficiency for producing trehalose from reducing starch hydrolysate by joint action with pullulanase, and obviously improves the production efficiency of the trehalose. The gene of expressing the synthase is obtained by the method; the MTSase is produced by a gene recombination technology; the enzyme expression quantity is high; the preparation efficiency is obviously improved; the cost is obviously reduced; and the production cost of the trehalose is also reduced.