16 results about "Chitobiose" patented technology

The invention discloses a chitosanase mutant which is derived from bacillus subtilis and has the capacity of hydrolyzing chitosan to produce chitosan disaccharide per unit and belongs to a GH46 family, and application of the chitosanase mutant in hydrolyzing chitosan to produce chitosan oligosaccharide. Compared with wild type chitosanase capable of producing chitodisaccharide and chitotriose, the chitosanase mutant has the advantage that the chitodisaccharide can be produced per unit when the chitodisaccharide is hydrolyzed by the chitosanase mutant. The invention provides eight kinds of chitosanase mutants, and the mutants are obtained by carrying out site-specific mutagenesis on 21st-site glycine on the basis of wild type chitosanase and respectively mutating the 21st-site glycine into lysine, arginine, leucine, histidine, methionine, tyrosine, proline and glutamine. The mutant can hydrolyze chitosan under mild conditions to produce chitosan oligosaccharide with single polymerization degree, and can greatly relieve the difficulty in separation and purification in chitosan oligosaccharide production.

The invention relates to a method for producing 6 '-fucosylated chitosan disaccharide GlcNacbeta1-4 (Fucalpha1-6) GlcNAc through double-bacterium coupling fermentation, and belongs to the technical field of fermentation engineering. The method comprises the following steps: taking fucose and N-acetyl-D-glucosamine as substrates in a fermentation system; the 6 '-fucosylated chitosan disaccharide is produced through coupled fermentation of an engineering escherichia coli strain and yeast, and a GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc synthesis pathway including N-acetylhexosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucose kinase Fkp and N, N-diacetylchitosan phosphorylase Chbp is constructed in the engineering escherichia coli, so that the 6'-fucosylated chitosan disaccharide is obtained. According to the invention, the yeast is added to realize cyclic regeneration from GMP to GTP, so that not only is the problem of high oligosaccharide production cost caused by pure exogenous supplement of GTP effectively solved, but also efficient production of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is realized, and the yield of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is increased to 25.50 g / L by an optimized coupling fermentation production technology. The invention provides a novel synthesis technology of complex N-glycans, and has a wide application prospect.

The present application relates to a kind of chitosanase mutant WP-E80A, plasmid, recombinant bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnWP as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnWP, the 80th amino acid is subjected to site-directed mutagenesis.The present application also provides plasmid and recombinant engineering bacteria comprising the chitosanase mutant WP-E80A gene, and the chitosanase mutant WP-E80A can be prepared to generate chitotriose with chitobiose as substrate.

The invention relates to the technical field of gene engineering, and particularly discloses exo-chitinase Cq181 and application thereof in preparation of chitobiose. The amino acid sequence of the chitinase is as shown in SEQ ID NO: 1, and the nucleotide sequence of a Cq181 gene for coding the chitinase is as shown in SEQ ID NO: 2. According to determination, the molecular mass of the chitinase is 82.53 kDa, the chitinase shows high activity and specific production of chitobiose under the conditions that the temperature is 40 DEG C and the pH value is 7.0, and the chitinase is suitable for a mild and low-energy-consumption biological manufacturing process and particularly has good application potential in the aspect of preparing chitosan oligosaccharide with a specific polymerization degree (the polymerization degree is 2).

The invention relates to a method for high-yield production of chitobiose, and belongs to the technical field of fermentation. According to the method, N-acetylglucosamine serves as a substrate, ATP serves as a cofactor, recombinant bacteria over-expressing PPK, Nahk and Chbp are constructed for fermentation synthesis of the chitobiose, expression modes of the three enzymes are optimized, it is found that the yield of the chitobiose obtained through coupling fermentation of the recombinant bacteria over-expressing the three enzymes is the highest and is 1675 mg / L, and then the yield of the chitobiose obtained through coupling fermentation of the recombinant bacteria over-expressing the three enzymes is high through single-factor optimization. The highest yield of the obtained chitobiose is 5214 mg / L, and is improved by 310% compared with the yield before optimization. And the yield of the chitobiose obtained by fermenting the recombinant bacteria which simultaneously overexpresses the three enzymes is 1650mg / L. A reference method is provided for large-scale biological manufacturing of the chitobiose, cascade reaction of enzymes can be realized through a complete multienzyme system in cells, so that the defect that a cascade catalysis process is difficult to realize in enzymatic catalysis is overcome, the catalysis efficiency is improved, a tedious enzyme purification process is omitted, the preparation is simpler, and the production cost is lower.

The invention discloses pichia pastoris engineering bacteria capable of efficiently expressing chitinase and application of the pichia pastoris engineering bacteria in shrimp shell high-value processing, and belongs to the field of biological engineering. The pichia pastoris engineering bacterium takes pichia pastoris GS115 as a host, the high-purity CtChi70 enzyme preparation can be prepared by signal peptide optimization, high-copy strain screening and cofactor overexpression in combination with high-density fermentation in a fermentation tank, ultrafiltration concentration in a hollow fiber column and freeze-drying flow, and CtChi70 accounts for more than or equal to 90% of total protein in fermentation liquor. The lyophilized enzyme preparation is used for a chitin substrate extracted from shrimp shells after mild treatment, and a product mainly comprising chitobiose can be efficiently generated. According to the invention, high yield of CtChi70 and simple preparation of the preparation are realized, and an economical, environment-friendly and high-yield feasible path is provided for high-value utilization of waste shrimp shell resources.

The invention belongs to the technical field of green biosynthesis of multi-enzyme catalysis, and relates to a method for preparing chitobiose by a double-enzyme catalysis method, which comprises the following steps: 1) obtaining a substrate containing chitin; 2) preparing chitin hydrolase and chitin deacetylase in a recombinant expression manner; and (3) carrying out enzyme reaction on the chitin-containing substrate obtained in the step (1), the chitin hydrolase obtained in the step (2) and chitin deacetylase obtained in the step (2) in a buffer system to obtain chitobiose. The invention provides the method for preparing the chitobiose by the double-enzyme catalysis method, which can be used for remarkably improving the yield and the purity of the chitobiose and effectively reducing the environmental pollution.

The invention discloses an anti-aging beautifying oral liquid containing low-polymerization-degree chitosan oligosaccharide and a preparation method thereof, and belongs to the technical field of marine organisms. Chitosan oligosaccharide mainly comprising chitobiose and chitotriose is prepared by utilizing the synergistic fermentation effect of microorganisms, and the chitosan oligosaccharide is compounded with collagen, sodium hyaluronate, vitamin C and the like to prepare the oral liquid. The low-polymerization-degree chitosan oligosaccharide is an excellent in-vivo antioxidant, can remove excessive free radicals, inhibit lipid peroxidation and slow down damage of metabolites such as peroxided lipid to the body, and then the anti-aging effect is achieved; the anti-aging and beautifying oral liquid prepared by matching natural moisturizing and beautifying components such as collagen and sodium hyaluronate is good in taste and easy to absorb, and animal experiment results show that the anti-aging and beautifying oral liquid can have beautifying and anti-aging effects after being frequently taken.

The present invention discloses a chitosanase mutant and its use in the preparation of chitotetraose, belonging to the field of enzyme engineering. The invention screens for a mutant whose hydrolysis product has the potential to produce chitotetraose by performing site-directed saturation mutagenesis at position 149 of the amino acid sequence (SEQ ID NO: 1) of the Bacillus subtilis chitosanase BsCsn46A. Aspartic acid is mutated to glycine, labeled D149G. The hydrolysis product of the mutant produces chitotetraose in addition to chitobiose and chitotriose.

The application belongs to the technical field of enzyme engineering, and discloses a chitosanase mutant for producing chitobiose. A new 46 family glycoside hydrolase chitosanase (BsCsn46A) cloned from Bacillus subtilis is subjected to site-directed mutation, and the amino acid sequence is shown as SEQ ID NO:1. The chitosanase mutant is G21W, E235R and Y236K. Through hydrolysis reaction analysis, it is found that, different from the original enzyme hydrolysis chitosan product chitobiose (COS2) and chitotriose (COS3), the hydrolysis product of the mutant chitosanase is mainly COS2.

The invention relates to a chitosanase mutant WP-E80A, a plasmid, a recombinant bacterium and application of the chitosanase mutant WP-E80A, and belongs to the technical field of enzyme engineering, the amino acid sequence of the chitosanase mutant is shown as SEQ ID NO: 1, and the amino acid sequence of wild chitosanase CsnWP is shown as SEQ ID NO: 2. Compared with wild chitosanase CsnWP, the chitosanase CsnWP has the advantage that site-specific mutagenesis is carried out on the 80th amino acid. The invention also provides a plasmid and a recombinant engineering bacterium containing the gene of the chitosanase mutant WP-E80A, and the chitosanase mutant WP-E80A can be used for preparing and generating chitopentaose by taking chitobiose as a substrate.

The invention discloses application of combined application of chitobiose and bacillus in promoting tomato growth, preventing and controlling tomato soil-borne bacterial wilt and relieving tomato acid stress. The chitobiose and the bacillus amyloliquefaciens SQR9 are combined to promote the growth of the tomatoes, relieve the acid stress of the tomatoes and improve the tomato fruit quality. The chitobiose combined bacillus amyloliquefaciens SQR9 is applied to prevention and control of tomato soil-borne bacterial wilt and enhancement of tomato system resistance. Chitosan disaccharide can promote the growth of bacillus amyloliquefaciens, and also can significantly improve the inhibition ability of bacillus amyloliquefaciens to ralstonia solanacearum. In a greenhouse pot experiment, the chitobiose can improve the biomass of tomatoes and promote the growth and development of root systems. Chitosan disaccharide and bacillus amyloliquefaciens are simultaneously applied into soil by a root irrigation method, so that the disease index of tomato soil-borne bacterial wilt can be reduced, the activity of resistant enzyme of a plant system can be increased, the acid stress of tomatoes can be relieved, the biomass of the tomatoes can be increased, and the quality of tomato fruits can be improved.

The application discloses a chitinase mutant D615S with high thermal stability and a construction method and application thereof. A wild-type chitinase Chil is subjected to thermal stability modification through a semi-rational design method, and a point mutation of aspartic acid at the 615th position is changed into serine, so that the half-life of the obtained mutant at 40 DEG C is increased by 3.6 times, and the half-life at 45 DEG C is increased by 24 times. When 100 g / L powder chitin is degraded at 45 DEG C for 60 h, 48 g / L reducing sugar is obtained, and the degradation rate is 48%; when 100 g / L lobster shells with 20% chitin content are degraded for 60 h, 9 g / L reducing sugar is obtained, and the degradation rate can reach 45%; through identification of the degradation products, it is found that the products are N acetylglucosamine and chitobiose with a mass ratio of 4:1, and have a good market prospect.

The invention relates to the technical field of gene engineering, and particularly discloses three-function chitinase CmChi18B as well as an expression method and application thereof. The amino acid sequence of the CmChi18B is shown as SEQ ID No.1, the CmChi18B has the activity of chitobiose excision enzyme, the activity of chitin incision enzyme and the activity of beta-N-acetylglucosaminidase at the same time, and the enzyme activity reaches 7.04 U / mg. The enzyme can independently and efficiently convert chitin into GlcNAc in a low-temperature environment, colloid chitin can be efficiently converted into GlcNAc after the enzyme acts for 24 hours under the conditions that the temperature is 30 DEG C and the pH value is 5.5, the conversion rate reaches 91.15%, and the product purity exceeds 98%. The CmChi18B has low-temperature adaptability and a wide pH tolerance range. The invention provides an efficient and environment-friendly enzymolysis technology for high-value utilization of marine biomass resources, and is suitable for the fields of foods, medicines, cosmetics and the like.

The present invention belongs to the field of enzyme engineering technology and specifically relates to a thermostable chitinase mutant and its application. The specific technical solution includes: a chitinase mutant, based on a wild-type chitinase, undergoing any one or more of the following mutations: mutation of serine at position 67 to glycine, lysine at position 177 to arginine, alanine at position 220 to valine, asparagine at position 257 to tyrosine, and asparagine at position 271 to glutamate; the amino acid sequence of the wild-type chitinase is shown in SEQ ID NO: 1. Compared with the wild-type chitinase, the mutant exhibits no significant change in its chitin degradation pattern, with chitobiose still being the primary hydrolysis product; however, the mutant exhibits significantly higher optimal reaction and melting temperatures, and a significantly prolonged half-life at 60°C, suggesting significant application potential.

The invention discloses a chitosanase mutant capable of producing chitobiose and application of the chitosanase mutant. The chitosanase mutant is derived from glucoside hydrolase chitosanase of a bacillus subtilis GH46 family, and compared with wild type chitosanase producing chitodisaccharide and chitotriose, the chitosanase mutant has the advantage that the hydrolysate is mainly chitodisaccharide. According to the invention, two chitosanase mutants are obtained, and the mutants are obtained by site-specific mutagenesis of tyrosine at the 236th site into arginine and glycine on the basis of wild chitosanase. The mutant can hydrolyze chitosan under mild conditions to produce chitosan oligosaccharide with single polymerization degree, and provides great help for separation and purification work in chitosan oligosaccharide production.