| Xylanase hydrolyzes 1,4-β-D-xyloside bond by internally tangenting and the hydrolization products are xylan and hex-xylan mostly。The polyxylose which contains xylohiose and xylotriose applicated foods domain for health care affect of low-grade fever,stability,moisture and propagate Bacillus bifidus. The polyxylose was made by enzymic degradation.The key point of producing polyxylose was gain more xylohiose and xylotriose but less xylose by screening xylanase which has higher activity and higher substrate specificity. The traditional gain xylanase which has higher endoxylanase activity but less xylosidase activity was screening strain and controlling the fermentation condition, the result was dissatisfactory. The effective method was expressing xylanase by genetic engineering.In this paper,basing on pPIC9-xyn2 vector, we design and build a new type of expressive vector by replacing and transformating the promoter and signal peptide, and this will establish the foundation for efficiently explore new methods of a high-level expression in Pichia pastoris , the nature of Xylanase enzyme ,and the conditions of shake-flask fermentation,the research contents and results are as follows:1. Vector constructionBasing on pPIC9-xyn2 vector, we design and build pGAPHα-xyn2,pGAPRHα-xyn2,pGAPHINU-xyn2,pGAPHαM-xyn2,pGAPHαE-xyn2 by replacing and reforming the promoter and signal peptide. In this series of vectors, xyn2 was regulated by GAP promoter, the expression of exogenous protein secretion signal peptide wasα-Factor signal peptide of Saccharomyces cerevisiae, INU and optimum designed signal peptide, then add enhanser in the latter two vector.2.Expression of xyn2 in Pichia pastorisWe cloned pGAPHα-xyn2,pGAPRHα-xyn2,pGAPHINU-xyn2,pGAPHαM-xyn2,pGAPHαE-xyn2 vector into Pichia pastoris by using electroporation method and obtain transformants, Transformants were picked randomly, and cultured in YPD. After cultureing 72h , determinated the activity of enzyme by DNS,the enzymatic activity of different recombinated-strain is 92 IU/ml,104 IU/ml,85 IU/ml,168 IU/ml,128IU/ml. The results show that, GAP promoter can express the exogenous gene,INU signal peptide and the transformed signal peptides are able to guide the secretion of foreign protein , and the enhancer can serve to strengthen the GAP. The analysis of Xylanase-SDS-PAGE shows that the size of molecular of xylanase is 21KDa.3. Reseach of recombinant xylanase characterXylanase were placed in different temperatures, pH, cation and other conditions to study that how optimum temperature, pH, thermal stability, acid-base stability, metal ions work on xylanase . The results show that the optimum temperature is 50℃,the optimum pH is 5.0, thermal stability is better when temperature is below 60℃,acid stability is better when pH is below7 and it will devitalize when pH is 9. There is promoting affection on the activity of xylanase by Fe2 +,Mg2 +,Zn2 +,Cu2 +,Fe3 + ,on the other hand, inhibition of xylanase by Mn2 +.4. Reseach of fermentational conditionThe recombinant bacteria were cultured under different conditions, such as pH, temperature, liquid volume, incubation time, Triton, EDTA, to find a suitable fermentative condition。And this will provide a basis for industrial production. The results show that the recombinant bacteria is at a ideally fermentative condition in the following conditions: the initial pH 5.0, incubation temperature of 28℃, culture time of 120h, liquid volume of 250ml loaded 75ml, Triton adding amount of 0.01%, EDTA adding amount of 5mM.5. Analysis of enzymolysis productThe enzyme product of Xylanase is analysed by TLC.The results show that the enzyme products of xylanase are,xylohiose,xylotriose and a small amount of xylose.
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