255 results about "Chitosanase" patented technology

The invention discloses a bacillus subtilis chitosanase as well as a preparation method and application thereof. The invention optimizes an encoding gene of bacillus subtilis chitosanase according to the preference of pichia pastoris codon. The optimized nucleotide sequence is shown as SEQ ID NO.2. A pichia pastoris expression system is further utilized to perform efficient secretory expression on the optimized chitosanase encoding gene, so as to obtain the bacillus subtilis chitosanase with the amino acid sequence as SEQ ID NO.1. The bacillus subtilis chitosanase obtained according to the invention has higher hydrolytic activity to chitosan substrates at different degrees of deacetylation; the crude enzyme generated through shake-flask fermentation has the hydrolysis capacity of degrading 5g of chitosan by 1mL crude enzyme (0.3mg of protein), about 150mg of non-specific commercial enzyme is required for degrading the same amount of chitosan, and the efficiency is theoretically increased by 500 times; and the bacillus subtilis chitosanase has excellent industrial application prospects.

The invention belongs to the technical field of biological engineering, and provides a chitosanase high-yield producing strain, a liquid and solid fermentation method for preparing chitosanase with high activity by using the strain, and chitosan oligosaccharide production method by using the chitosanase. The chitosanase producing strain in the invention is identified as a strain of Bacilluscereus with a strain code of JBSH-003 by 16SrDNA identification, and has a preservation number of CGMCCNo.6129 in China General microbiological culture collection center. The strain can be applied to the production of chitosan to obtain chitosan oligosaccharide with high content, molecular weight range of 1500Dalton-2500Dalton, and relatively concentrated molecular weight range.

The invention provides a compound enzyme for producing reducing sugar by degrading maize straws. The compound enzyme is prepared from pectase, cellulose, hemicellulase, ligninolytic enzyme, amylase, prolease, lipase and chitosanase. Compared with the prior art, 1) the yield of the reducing sugar by degrading the maize straws has high yield, and the gross amount of the reducing sugar of straws per gram reaches over 480 mg, so the compound enzyme has wide application prospect in the fuel alcohol industry; 2) compared with sugar preparation by acid process, severe acid pollution is prevented to facilitate subsequent fermentation; and 3) compared with monomer cellulose or simple compound enzyme, the pretreatment of steam explosion with high energy consumption is not required, and the amount of the reducing sugar is increased by over 1 time; meanwhile, glucose and xylose in a degradation product have high content, side products are few so as to contribute to subsequent fermentation, the industrial application value of the maize straws is improved, and the compound enzyme can be applied to production of single cell protein and fuel alcohol.

The present invention is production process of UV and NTG mutagenizing Bacillus subtilis TQS6 to culture high efficiency expressing chitosanase strain TQK and fermentation producing high activity chitosanase. The chitosanase strain is first slant cultured and then fermentation cultured while introducing oxygen-rich air to increase dissolved oxygen to 30-80 %; and after culturing for 18-24 hr, average enzyme activity may reach 718 mu/ml. High activity chitosanase may be obtained through fermentation in optimized enzyme producing condition. The process of the present invention can obtain chitosanase with high yield, high stability and high activity, and the produced chitosanase is applied in converting chitosan into chitin oligose effectively and economically.

A composition for treating cancer is provided. The composition is a hexosaminidase covalently attached to a cancer cell targeting ligand and improves selectively of the hexosaminidase for tumor cells. In certain embodiments, the hexosaminidase is alternately chitinase (N-acetyl-glucosaminohydrolase), chitosanase, or N-acetyl-hexosaminidase and the targeting ligand is either a monoclonal antibody, an antibody fragment immunospecific to a tumor cell or cancer cell antigen, epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, or folic acid. Also provided is a method for treating cancerous tumors comprising administering the composition to a patient that has a cancerous tumor.

The invention provides chitoanase. An amino acid sequence of the chitoanase is SEQ ID NO: 1. The enzyme has good thermal stability; the optimal reaction temperature is 50 DEG C and the optimal pH (Potential of Hydrogen) is 8.0; the specific enzyme activity reaches 330U/mg. The chitoanase belongs to an internal enzyme digestion and external enzyme digestion mixed type and a substrate, which can bedegraded at minimum, is chitobiose and the chitosan can be hydrolyzed glucosamine and chitobiose. The chitoanase provided by the invention is hopefully used for producing the chitosan oligosaccharideand has practical application value.

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial strain for producing chitosanase and a method for preparing chitosan oligosaccharide by using the strain. The chitosanase production strain is the Bacillus sp., which is deposited with China General Microbiological Culture Collection Center (CGMCC) on November 26, 2014 and is assigned the accession number of CGMCC No.10066. The use of the chitosanase production strain shortens the time of fermentation and enzyme production and improves the quality and production efficiency of the chitosan oligosaccharide.

The invention relates to novel recombinant chitosanase and the applications thereof, as well as a method for preparing the recombinant chitosanase and the nucleic acid, vectors, host cells and the like used in the method. The recombinant chitosanase is shorter than full-length chitosanase, can be high effectively expressed through genetic engineering method with the expression amount higher than 500mg/L and carry out separation and purification by utilizing simplified purification technology; and the specific activity of the enzyme thereof can reach 700U/mg, thus having low production cost and being suitable for popularization in practical use.

A hemicellulase is the fermented product of Bacillus pumilus hemicellulase No.A1 (CCTCC M201023) and can be used in preparing chitoligose. A process for preparing the said chitoligose includes such steps as zymolyzing chitosan with the said hemicellulase, centrifugal filtering, flocculating, vacuum concentrating, filtering, decolouring, deodouring, super filtering and classifying and drying. Its advantages are high zymolyzing efficiency and high content of active components in product.

The invention relates to a controlled release delivery compositions and methods of using them for pathologies associated with Otorhinolaryngology and Head and Neck. Specifically, the invention relates to regulating drug delivery by the use of chitosan based matrices together with chitosanases.

The invention discloses a method for preparing Chitosan oligosaccharide, which is characterized in that: dissolving Chitosan oligosaccharide into acetate solution, adding Chitosan oligosaccharide enzyme for hydrolysis; condensing got hydrolysate, depositing with ethanol, drying or spraying for drying; said Chitosan oligosaccharide enzyme is Chitosan oligosaccharide enzyme liquid or powder; the ratio between volume of said Chitosan oligosaccharide enzyme liquid and weight of Chitosan oligosaccharide is 0.1-2 l :1 kg; and the weight ratio between powder and Chitosan oligosaccharide is 0.5 g- 10 g: 1 kg. The Chitosan oligosaccharide prepared in this invention is characterized by good water solubility, high productivity and low cost.

The invention provides a recombinant thermostable chitosanase-producing engineered Pichia pastoris GS115/HBCSN and a production method of thermostable chitosanase. The invention optimizes Aspergillus fumigatus chitosanase gene (GenBank accession number AY190324) based on the codon bias of Pichia pastoris, and the optimized nucleic acid sequence shows a 70% homology to the original nucleic acid sequence. The chitosanase produced by the recombinant Pichia pastoris can be secreted out of cells in a soluble form, with expression level of 3 mg/mL, and enzyme activity of 25,000U/mL. The chitosanase after glycosylation modification in Pichia pastoris has excellent thermal stability, which is proven by that the residual activities after treating at 80DEG C for 20 min, treating at 90DEG C for 40 min and treating at 100DEG C for 20 min are 56%, 87% and 87% respectively. The inventive thermostable chitosanase is especially suitable for large-scale industrial degradation of chitosan and environmental pollution reduction.

The invention discloses a technique for producing water-soluble functional chitosan. The technique comprises the steps that: chitosan is added into H2O2, and the mixture is mixed evenly, microwave-heated for a proper time, and is cooled; the chitosan treated by microwave is added into the solutions of acetic acid or hydurochloric, and is stirred until the chitosan is fully dissolved; chitosan solution is prepared after pH is adjusted to 5 to 6; non-specific chitosanase is added into the chitosan solution, and zymohydrolysis, enzyme-annihilation, enzymolysis liquid filtering and centrifugalization are carried out to remove precipitate, and then a clear solution is obtained; hyperfiltration is carried out on the clear solution, and nanofiltration is carried out to the hyperfiltrated solution to remove salinity and micromolecule monosaccharide; filtrate is sprayed and dried, and then the product is obtained. The technique organically integrates microwave degradation, oxidative degradation and zymohydrolysis and adopts non-specific enzyme to solve ab enzyme preparation supply problem; the controlling parameters of zymohydrolysis is comparatively moderate; the technique is applicable to the scale production of water soluble functional chitosan.

The invention discloses bacillus circulans chitoanase as well as a preparation method and application thereof. According to the preference of pichia pastoris codons, a chitoanase coding gene sequencein bacillus circulans is obtained by utilizing a whole gene synthesis method; an optimized nucleic acid sequence is shown as SEQ ID NO. 2. Furthermore, efficient secreting expression is carried out onan optimized chitoanase coding gene by utilizing a pichia pastoris expression system to obtain the bacillus circulans chitoanase; an amino acid sequence of the bacillus circulans chitoanase is shownas SEQ ID NO. 1. The chitoanase disclosed by the invention has relatively high hydrolysis activity on a chitosan substrate and a crude enzyme solution produced by shake-flask fermentation has a hydrolysis capability of degrading 5g of chitosan by utilizing 1mL of the crude enzyme solution (with the protein content of about 0.77mg); when the chitosan with the same amount is degraded, about 150mg ofneutral proteinase obtained from bacillus subtilis is needed; the efficiency is theoretically improved by about 200 times; the bacillus circulans chitoanase has a very good industrial application prospect.

The invention provides a chitosanase EAG1 mutant with an amino acid sequence shown in SEQ ID NO:1. The chitosanase EAG1 mutant provided by the invention, compared with chitosanase EAG1, has more excellent thermal stability and higher catalytic efficiency and has capability of rapidly decomposing chitosanase. Through efficient heterologous expression on engineered yeast containing a mutant gene, large-scale preparation of the chitosanase EAG1 mutant can be realized.

The invention discloses rhizosphere bacterium gynuella sunshinyii-sourced chitosanase (GsCho46A) and an encoding gene and application thereof. The chitosanase is a) or b) or c): a) protein of which the amino acid sequence is encoded by 1st-266th amino acid residues in SEQ ID NO.2; b) fusion protein obtained by connecting labels and/or a label to the N end and/or the C end of the protein shown by the 1st-266th amino acid residues in the SEQ ID NO.2; c) protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown by the 1st-266th amino acid residues in the SEQ ID NO.2 and having the same function. The chitosanase disclosed by the invention has excellent enzymatic properties: the activity under a cryogenic condition is relatively high and a requirement on enzymatic hydrolysis equipment is relatively low; through temperature control, high-polymerization chitooligosaccharide with the degree of polymerization (DP) 2-7 can be efficiently prepared; the reaction condition is mild, the energy consumption is low, and no pollutant is produced; during enzymatic preparation of the chitooligosaccharide, the chitosanase has an important application value and an important economic value.

This invention discloses a method for preparing immobilized chitosanase by chitin-glutaraldehyde crosslinking and adsorption. The method comprises: refining chitin carrier, crosslinking with glutaraldehyde, extracting chitosanase, and immobilizing. The method overcomes such problems as low adsorption strength of the carrier, easy desorption and easy deactivation of the enzyme faced by traditional methods that utilize crosslinking or adsorption only. Besides, the also has such advantages as long half-life, and little influence of environment to the immobilized system.

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 preparation method of a special dust suppressant for the soil surface, and belongs to the technical field of environmentally-friendly materials. The preparation method comprises the following steps: mixing and swelling polyvinyl alcohol with water, and performing heating and stirring for dissolving to obtain a polyvinyl alcohol solution; mixing and swelling chitosan withwater, and performing heating and stirring for dissolving to obtain a chitosan solution; mixing and swelling sodium alginate with water, and performing heating and stirring for dissolving to obtain asodium alginate solution; mixing the polyvinyl alcohol solution, the chitosan solution, the sodium alginate solution, a cross-linking agent, chitosanase and algae according to a certain weight part ratio, and carrying out ultrasonic dispersion to obtain a dispersion; carrying out spray drying on the dispersion at a low temperature to obtain coated microcapsules; and stirring and mixing the coatedmicrocapsules, isocyanate, water, a surfactant and nano-metal oxide to obtain the special dust suppressant for the soil surface. The special dust suppressant for the soil surface has the advantages ofexcellent water retention property, excellent water stability and excellent wind erosion resistance.

The invention provides a display system of a chitosanase cell surface. A fused gene segment is obtained by fusing a gene csn and a gene inaQN, wherein the gene csn is coded with chitosanase and the gene inaQN of an N-terminal structural domain of an ice nucleating protein is responsible for transmembrane transport and has an anchoring function. The fused gene segment is expressed on the surface ofa host cell, and the display system of the chitosanase cell surface is obtained. The nucleotide sequence of the gene csn is showed as SEQID NO.1. The nucleotide sequence of the gene inaQN is showed as SEQIDNO. 2. The invention further provides a preparation method of the display system of the chitosanase cell surface. The display system of the chitosanase cell surface can directly react with thechitosan in a solution, tedious steps of protein purification are saved, and cost of producing low-molecular-weight chitosan and oligomeric chitosan is reduced greatly. The display system of the chitosanase cell surface has great application prospects in the field of low molecular weight chitosan and oligomeric chitosan preparation.

The invention provides chitosanase gene derived from soil metagenome library, and the DNA sequence of the chitosanase gene is shown in SEQID NO:1. The invention also provides chitosanase obtained by encoding of the chitosanase gene and recombinant plasmid including the chitosanase gene, and further provides escherichia coli engineering bacteria containing the chitosanase gene and an obtaining method thereof. The obtaining method of the chitosanase gene is simple, and the escherichia coli engineering bacteria containing the chitosanase gene is provided. The chitosanase can be obtained through high-efficiency expressionof the escherichia coli engineering bacteria. The bioinformatics analysis shows that the obtained chitosanase gene belongs to the forty-sixth family of glycoside hydrolase. The chitosanase gene has high catalytic activity and good thermal stability, with optimal action temperature being 55 DEG C and pH being 6.0, and has great industrial value and economic value.

The invention belongs to the field of biotechnology, and particularly relates to a bacillus subtilis for producing chitosanase and application thereof. The chitosanase produced by the bacillus subtilis has high yield and strong enzyme activity, and has a different protein structure from ordinary chitosanase, and provides a new idea for in-depth study of chitosanase molecule mechanism. The ordinarychitosanase has good activity only when the ordinary chitosanase is in weakly alkaline to weakly acidic conditions, and the chitosanase found in the invention has better performance in weak alkaline,neutral, weakly acidic and relatively strong acidic conditions. At the same time, the chitosanase discovered in the invention can not only act on soluble chitosan but also on insoluble high-poly chitosan, omitting pre-treatment of chitosan degradation; final reaction products are dipolyglucosamine and tripolyglucosamine, the bacillus subtilis for producing the chitosanase is simple in composition, is convenient for subsequent further processing applications, and greatly saves production costs.

The invention relates to genetic engineering modification, in particular to a chitosanase mutant and an application thereof. The chitosanase mutant is obtained by mutating tyrosine Y at the 116th siteof an amino acid sequence of chitosanase BC345 into alanine A. A section of amino acid rich in alanine A is added to the tail end of amino acid of the chitosanase BC345 mutant, or tyrosine Y at the 116th site of the amino acid sequence of the chitosanase BC345 mutant is mutated into alanine A, and a section of amino acid rich in alanine A is added to the tail end of amino acid of the chitosanaseBC345 mutant. The chitosanase mutant used in the invention is high in activity and resistant to high temperature. The chitosanase is reasonably modified, and the enzyme activity and the thermal stability of the chitosanase are greatly improved through site-specific mutagenesis and increase of flexible fragments, so that the chitosanase has the potential of industrial production of chitosan oligosaccharide.

The invention belongs to the technical field of gene engineering, and particularly relates to a chitosanase gene csnbaa, chitosanase and a preparation method and application thereof. The nucleotide sequence of the chitosanase gene csnbaa is shown as SEQ ID NO: 1, and the chitosanase gene csnbaa is obtained by comprehensively optimizing the sequence of chitosanase derived from Bacillus atrophaeus.The amino acid sequence of the chitosanase CsnBaa is as shown in SEQ ID NO: 2, so that not only are the types and source ways of chitosanase enriched, but also the fermentation enzyme activity can reach 4630 U/mL, and the chitosanase CsnBaa shows good stability in a relatively wide temperature range and a relatively wide pH range and has good application prospect and industrial value.

The invention belongs to the technical field of fertilizer, and particularly relates to biologic organic fertilizer and a preparation method thereof. The biologic organic fertilizer comprises biologic organic matter and microbial agent; the microbial agent includes bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and/or aspergillus niger; the biologic organic matter is prepared from, by weight, 10-20 parts of organic raw materials, 1-2 parts of zymophyte, 0.7-2 parts of bio-enzyme and 80-90 parts of water; organic raw materials include bean pulp, fishmeal, wool, corn starch and/or chitosan; the zymophyte includes bacillus subtilis, amyloliquefaciens, bacillus licheniformis and/or aspergillus niger; the bio-enzyme includes protease, lipase, amylase and/or chitosanase. The biologic organic fertilizer has the good water solubility, can be directly absorbed and utilized by crops and takes effect rapidly, and the bioavailability of the biologic organic fertilizer is effectively improved.

The invention relates to a chitosanase and a method for producing chitosan oligosaccharide by using same. The chitosanase is prepared according to the following steps: inoculating Bacillus sp. AMI09 which is separated from soil on the south coast of Kyongnam in South Korea and can produce chitosan oligosaccharide with high polymerization degree into an LB culture solution, then carrying out shaking culture to obtain a culture solution for inoculation, performing centrifugal separation of a great number of culture solution which is cultured in an MS culture medium to obtain supernatant, concentrating and washing the supernatant, performing ion exchange chromatography and gel filtration chromatography, and finally dialyzing with deionized water to obtain chitosanase. The heat-resistant chitosanase prepared by the strain can stably produce chitosan oligosaccharide with high polymerization degree and high hexanose content under the reaction condition of 60 DEG C.