| Xylan is a kind of natural polysaccharides, is the main component of cellulose and it is a common polysaccharide in the daily life. In the crop, for example corncob, cornstalk, Is a kind of abundant polysaccharide, but the utilization rate is not very good. Its chemical structure is very complex, in different species it have different structures, even the same plant, in different parts, the structure is different. Mainly because it have rich side chain, just because of this it have the diversity of the function and structure, in the substituent group, we found that most of this is Etherification, esterification, and complex derivatives. Rich side chain groups, but the main chain have homology, its main chain are connected by β-1, 4 glycosidic bond of D- xylose residues, Usually the main chain of xylose residue is less, branched chain the more so the temperature stability stability is worse, conversely, the better. The solubility of xylose is special in general, xylan is soluble in water, but there are a lot of xylan is not soluble in water, but soluble in alcohol solution. This is one of xylan classification basis. As a kind of natural polysaccharide, the role of the hydrolysate is becoming more and more attention, Especially the xylose molecules through complete hydrolysis, have good development prospect in the medical, food and other industries, and xylan is relatively easy, cheap, usually in some crop residues can be obtained, is a very suitable in the production of xylose raw materials.Xylanase is a kind of glycoside hydrolase, we can find it in the glycoside hydrolase family, for example GH10, GH11, GH7, GH8, GH43. But most of the xylanase belongs to GH10 and GH11, and people spent more experience also. Xylanase enzyme system is a kind of comprehensive, Mainly includes Endo-β-1,4-Xylanase, β-1,4 wooden glycosidase, and can make the oligomeric xylose hydrolysis return to xylose thoroughly Out-β-1,4-Xylanase, the narrow sense of xylanase is Endo-β-1,4-Xylanase, as a member of the glycoside hydrolase, xylanase has strong ability of hydrolysis of xylan, and they are widely used in the production and living, such as pulp and paper industry, paper deinking industry, medical industry, food processing industry, etc. Even in the wine industry has its place, because the cost is high, the application did a lot of limitations.The application of genetic engineering technology provides a platform for the application of xylanase and other enzyme products, because in the traditional industry, most of the strains are natural mutation or wild strains, genetic engineering reference that can using the method of artificial mutant strains to achieve the purpose of increase production. The experimental purpose is to fitrate more excellent strains, enhance the yield of xylanase through heterologous expression. By eukaryotic protein modification effect, its pH and temperature stability can also be improved. The experimental method of this paper is through NCBI database access to information, synthesis the Halopiger xanaduesis xylanase gene through PTDS method, use of pichia codon bias and xylanase protein sequence structure synthesis the expression vector. The secretory expression vetor through the BglⅡ enzyme digestion,electroporation into Pichia pastoris, we screening and get positive clones.the research included the optimum pH, pH stability, optimum temperature, temperature stability,metal ions and some compounds affect the activity of enzyme. The experment results show that: Xylanase maximum reaction rate of 219 umol/min·mg, purified enzyme liquid concentration to 200 μg/m L. Enzyme liquid Km value of 2.2 mg/ml. the recombination xylanase optimum pH value is 6.0,the enzymology properties stable between 4.5 to 8.0, the optimum temperature is 60℃, the enzymology properties stable when the temperature between 20℃-65℃, the inhibition of the enzyme is Mn2+, Cu2+, the facilitation of the enzyme is Fe2+,Cr2+, SDS, DTT. |
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