59 results about "Chitobiose" patented technology

The invention relates to a chitinase gene chiC and encoded protein and application thereof, and belongs to the technical field of genetic engineering. According to the chitinase gene chiC and the encoded protein and the application thereof, pseudoalteromonas sp. DL-6 serves as a research object, the chitinase gene chiC with a nucleotide sequence showed in SEQ ID No 1 is cloned for the first time, and a high-activity chitinase is obtained. The preparation method of the high-activity chitinase is simple, the production cost is low, a product is easy to conserve, the appropriate hydrolysis temperature is 30 DEG C, The enzyme-decomposed PH is 9.0, high degradation activity of a colloidal chitin can reach to 1.569 +/- 0.017 U/ml, the chitinase chiC degrades an aipha chitin and a beta chitin to generate a chitobiose, and industrial production prospect is possessed.

The invention discloses a construction and application of an engineering bacteria secreting and expressing chitosan deacetylase, belonging to the technical field of fermentation engineering. The construction firstly constructs a recombinant Bacillus subtilis secreting and expressing chitosan deacetylase gene by a different source, and adds a signal peptide fragment yncM in the recombinant vector for a first time, wherein the signal peptide can secrete target protein chitosan deacetylase outside cells of the recombinant Bacillus subtilis, and obtain mutants in a non-translation region at 5' end, so that the expression of the target protein is significantly increased and steps of subsequent enzyme separation and purification are greatly simplified. The enzyme activity of obtained chitosan deacetylase is up to 1548.7 U/ mL and the yield of chitosan deacetylase is up to 620 mg/ L when fermentation medium is fermented and cultured for 50-60 hours. And meanwhile, the method has the advantages of low production cost, mild production conditions, simple purification process, safe operation and the like.

The invention relates to a preparation method and application of chitobiose. The preparation method comprises the following steps of dissolving chitobiose in glacial acetic acid solution; adding hydrogen peroxide; heating and stirring until the temperature is 40-90 DEG C and incubating; precipitating with ethanol after reaction is finished; filtering; and drying filter residues to obtain the chitobiose. The prepared chitobiose is used for preparing medicines for suppressing 10 strains of tumor cells of 5 kinds of humanized tumors. Biological compatibility and tumor atagonistics activity of the chitobiose are utilized effectively. By analyzing antitumor activity of the chitobiose, development of anti-tumor medicaments is promoted. The invention is used for the field of anti-tumor medicines.

The invention discloses chitosanase having an amino acid sequence as shown in SEQ ID NO.1. A gene encoding the chitosanase has a nucleotide sequence as shown in SEQ ID NO.2. The chitosanase is derivedfrom the GH5 family of glucoside hydrolase, can degrade chitosan to generate GlcN-(GlcN)4 and can be used for preparing an enzymatic COS mixture of a new component. The chitosanase has excellent biocatalytic activity, can hydrolyze chitosan to generate glucosamine, chitobiose, chitotriose and chitotetraose hydrochloride, and is relatively high in product purity; and when the pH is 10 and temperature is 50 DEG C, the chitosanase has relatively enzymatic activity of 898.6U/mg which is higher than those of most other known chitosanase, can effectively degrade chitosan, has mild reaction condition, easy control, high efficiency, environmental friendliness. The chitosanase has an industrial application value for producing novel chitosanase mixture.

The invention discloses a Beta-N-acetylglucosaminidase of Paenibacillus barungensis, an encode gene thereof and an application of beta-N-acetylglucosaminidase. The provided Beta-N-acetylglucosaminidase PbNag39 is excellent in the enzymatic property, and has a specific activity against chitosan is 28.3 U/mg, and the final products of hydrolysis of chitosan oligosaccharides are N-Acetyl glucosamine.The Beta-N-Acetylglucosaminidase and chitinase can hydrolyze chitin powder synergistically and obtain N-Acetyl glucosamine. The Beta-N-acetylglucosaminidase PbNag39 has the characteristics of high specific activity, good thermal stability and excellent hydrolysis characteristics. It can stabilize and play a catalytic role in a wide range of pH, and has important application value in chitin conversion.

Methods and compositions for treating myocardial dysfunction or inflammation are described. The methods of the invention involve administering an agent that can inhibit lysozyme to an animal in need thereof. Preferred lysozyme inhibitors include TAC and chitobiose.

The invention provides chitinase Chit46 as well as an expression and purification method and application thereof. The chitinase Chit46 is derived from trichoderma harzianum and an amino acid sequenceis shown as SEQ ID NO. 1; the fermentation solution enzyme activity of the chitinase is 31.4 U/mL and the specific enzyme activity is 34.5 U/mg, which is higher than that of like reported chitinase. The invention further provides the expression and purification method of the chitinase Chit46; the yield of the chitinase is greatly improved, the protein yield of a crude enzyme solution can reach 1.26 g/L and the yield of purified protein reaches 0.77 g/L. The chitinase provided by the invention can be used for hydrolyzing colloid chitin and has good hydrolysis activity; hydrolysis products are stable and the vast majority of hydrolysis products are chitobiose; a subsequent complicated purification and separation technology of each polymerization degree is omitted and the chitinase has a goodapplication prospect.

The invention discloses a detection method for a total content of chitosan oligosaccharides with a polymerization degree of 2 to 6. The detection method comprises the steps that firstly chitosan oligosaccharides standard substances (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose) and a chitosan oligosaccharides sample are matched to have an appropriate concentration, and injected into a high performance liquid chromatograph so as to be analyzed by high performance liquid chromatography; subsequently, the peak retention time and the peak area of the chitosan oligosaccharides standard substances are recorded, and the peak retention time and the peak area of the chitosan oligosaccharides sample are also recorded, thereby the chitosan oligosaccharides with the polymerization degree of 2 to 6 corresponding to each peak in the sample are determined by comparison with the peak retention time of the standard substances; finally, the concentration of the chitosan oligosaccharides with the polymerization degree of 2 to 6 in the sample is calculated from the ratio of the concentration to the peak area of the standard substances and the peak area of chitosan oligosaccharides with the polymerization degree of 2 to 6 in the sample, so that the total content of the chitosan oligosaccharides with the polymerization degree of 2 to 6 is calculated by the ratio of the concentration of the chitosan oligosaccharides with the polymerization degree of 2 to 6 to the sample preparation concentration. The detection method for the total content of the chitosan oligosaccharides with the polymerization degree of 2 to 6 fills the blank of content detection of chitosan oligosaccharides in the industry.

The invention relates to chitinase CmChi6 gene and cloning expression and application thereof. In the invention, exochitinase CmChi6 gene with the nucleotide sequence shown in SEQ ID No.1 is firstly cloned, and exochitinase with high activity is obtained. The preparation method is simple, the production cost is low, the product is easy to preserve, the suitable enzymolysis temperature is 45 DEG C,the optimum pH for enzymolysis is 7.4, the thermal stability is good at 25-50 DEG C, and the enzyme activity is maintained at 60% or above for 33 h; the optimum pH is 7.4, and the enzyme activity ismaintained at 75% or above for 33 h at pH of 7.0-10.0; Ba<2+> has a slight activation effect on the activity of CmChi6; CmChi6 has high degradation activity on colloid chitin, and can be used to prepare chitobiose, with high conversion rate, low energy consumption, environmental friendliness, and important economic value in application of industrial degrading of chitin.

The invention relates to a method for the large-scale preparation of chitobiose/chitotriose monomers, comprising the following steps: (1) dissolving chitooligosaccharide components in water to form a solution, decolorizing, filtering, and pumping into a strong acid cation exchange The column starts to load the sample, elutes with hydrochloric acid solution, and collects a tube at the same time; (2) each tube takes a sample to detect the sugar components in the collected solution, and merges the same sugar components; (3) the combined chitobiose and The two components of chitotriose are rotary evaporated to dryness, and hydrochloric acid is removed to obtain chitobiose monomer solid and chitotriose monomer solid respectively. Compared with the prior art, the process of the present invention is simple and convenient, and the yield is high, and high-purity chitobiose and chitotriose monomers and the like can be realized on a kilogram-level preparation scale.

The present invention discloses endochitinase and a coding gene and application thereof in production of chitobiose. The protein is as follows: (a) or (b) or (c), wherein (a) is a protein comprising 40th-site to 696th-site amino acid residues from N-terminal of a sequence 2; (b) is a protein comprising an amino acid sequence shown as the sequence 2 in a sequence table; and (c) is a chitinase-activity protein which is derived from a sequence 1 and obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of the (a) or (b). When the endochitinase is used for production of the chitobiose, conversion rate is high, energy consumption is less, no pollutant is produced, and the endochitinase has important economic value. The protein has a large potential for industrial application.

The invention discloses a preparation method of chitobiose derivative crosslinking fibers. The preparation method of the chitobiose derivative crosslinking fibers comprises the following steps of performing grafting modification on chitobiose through ring-opening reaction to obtain chitobiose derivative with polymerization reaction activity; adding photoinitiator to perform miscibility and then performing low-temperature freezing crystallization; performing irradiance crosslinking polymerization at low temperature; and removing a solvent by freeze drying to obtain a chitobiose derivative crosslinking fiber material. By the preparation method, the chitobiose derivative crosslinking fiber material with high mechanical property and mechanical strength is obtained. In a preparation process, a reagent for changing characteristics of chitobiose is not added, original excellent properties of the chitobiose serving as natural polymers are not changed, and the crosslinking chitobiose derivative fiber material can be widely used for various fields of medicine release systems, wound healing materials, sewage treatment, heavy metal recycling, membrane separation, daily-use chemical industry and the like.

The invention discloses a bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof. The molecular structure of the diacetylchitobiose deacetylase Dac and the RBS sequence of an expression vector pP43mut-N1-yncM-Dac are modified, so that the catalytic rate and translation expression rate of the diacetylchitobiose deacetylase Dac are increased, the enzyme activity is improved, and the bacillus subtilis capable of producing glucosamine GlcN at high yield is constructed. Compared with the prior art, the extracellular enzyme activity of the diacetylchitobiose deacetylase can reach 3769.5 U/mL. The conversion method is mild in condition, small in environmental pollution, high in substrate selectivity, specific in target product, high in reaction rate and high in product yield. The method is beneficial to solving the problems of serious pollution, tedious steps and the like in a chemical synthesis method, and is simple in process, easy to control and convenient to popularize and apply.

A chitin degradating bacterium is deformed by genetic engineering means to obtain the chitobiose deficient efficient genetically engineered chitin-degradating bacterium. It can effectively degradate the crystalline and colloidal chitin. After the chitin substrate is directly added to the fermented liquid containing them cultured in shake flask, the chitin oligose product without chitin monose can be directly obtained.

The invention relates to incision chitosanase CsnM with a thermal recovery characteristic and application thereof. The amino acid sequence of the chitosanase CsnM is shown as SEQ ID NO.1. The yield ofthe chitosanase disclosed by the invention can reach 680.2U/mL, the optimal reaction temperature is 40 DEG C, the chitosanase has the thermal recovery characteristic, and after the chitosanase is boiled by 10 minutes and incubated in ice water for 30 minutes, the activity can be restored by 89.1%. The enzyme action is in an incision manner, and main degradation products comprise chitobiose and chitotriose. The chitosanase disclosed by the invention is high in yield and novel in property, and has excellent industrial application potential.

The invention provides a marine streptomyces niveus chitosanase gene and application thereof. According to the preference of escherichia coli codon, a chitosanase coded gene(GenBank accession number:AQU65829) of the marine streptomyces niveus is optimized, the homology of an optimized nucleic acid sequence with an original nucleic acid sequence is 73.39%, the chitosanase SnCSN is successfully expressed in escherichia coli BL21 (DE3), and the obtained chitosanase SnCSN can efficiently degrade chitosan to obtain chitosan oligosaccharide with chitobiose as a main product; and the chitosanase SnCSN has higher hydrolytic activity on chitosan substrates with different degrees of deacetylation, and complex chitosan oligosaccharide with different degrees of deacetylation is obtained. The chitosanase can be used for large-scale preparation of chitosan oligosaccharide and chitin oligosaccharide, and the industrial application prospects are good.

The invention relates to a chitosan disaccharide deacetylase mutant, which is obtained by mutating glycine G at the 74th site of chitosan disaccharide deacetylase of extreme thermophilic archaea Pyrococcus horikoshii into alanine A. According to the invention, the enzyme activity, the expression quantity and the catalytic efficiency of the chitobiose deacetylase Dac are improved, and the problem that the efficiency of converting the chitobiose deacetylase Dac into glucosyl GlcN is not high is solved.

The invention discloses bacillus subtilis capable of stably expressing chitobiose deacetylase as well as a construction method and an application of the bacillus subtilis, and belongs to the technical field of genetic engineering. According to the invention, integration sites are screened, the integration copy number is increased, and a food safety level engineering strain capable of stably and efficiently expressing the chitobiose deacetylase Dac is constructed. Compared with the prior art, the extracellular enzyme activity of the chitobiose deacetylase is greatly improved, and the fermentation period is greatly shortened. The conversion method is simple and easy to implement, mild in condition, efficient, environmentally friendly and high in product yield. The method is beneficial to solving the problems of serious pollution, low yield and the like in the traditional synthesis method, and is easy to control and beneficial to popularization and application.

The invention discloses a method for determining contents of chitobiose and chitotriose in chitosan oligosaccharides. The ultraviolet response of the chitosan oligosaccharides can be enhanced by reaction of 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with amino groups in the chitobiose and the chitotriose for combination. Derivatized products are separated by high performance liquid chromatography and detected by a UV detector at a wavelength of 250-280 nm, and the contents of thethe chitobiose and the chitotriose can be calculated by an external standard method. The method has the characteristics of simple operation and sensitive detection, and is suitable for detecting the contents of the trace chitobiose and the chitotriose in the chitosan oligosaccharides.

The invention relates to a chitosan deacetylase combined mutant with optimal pH reduction. The chitosan deacetylase combined mutant is prepared by the following steps: analyzing and predicting chitosan deacetylase surface charge and enzyme activity related sites through a molecular model and a computer, constructing mutants of the related sites through a site-directed mutagenesis technology, screening out single mutants with optimal pH, enzyme activity and product GlcN stability during mutation of different sites, and constructing a combined mutant, thereby obtaining the combined mutant with the optimal pH reduced to 6.0 and the obviously improved enzyme activity.

The invention discloses use of chitobiose in preparation of bacteriostatic fishery drugs or bacteriostatic fish feeds, the inhibited bacteria are vibrio anguillarum, aeromonas hydrophila, aeromonas sobria, aeromonas punctata and flexibacrer marilimys. The chitobiose has obvious antibacterial activity, is a natural antibacterial substance free of toxic and side effects, and can significantly improve the disease resistance ability of cultured fishes by using as a fishery drug to add into basal feeds.

The invention belongs to the technical field of enzymes, and particularly relates to a chitosanase mutant and application thereof. The chitosanase mutant is characterized in that glycine at the 21 position of CsnMY002 wild type chitosanase is mutated into lysine. Compared with a wild strain, the chitosanase mutant has the advantages that chitosan oligosaccharide can be converted into chitobiose ina more targeted mode, separation is simpler, and the yield of a target product is higher.