Expression Of Xylanase Gene In PGAPz-Pichia Yeast Expression System

Plant cell wall contains a wealth of hemicellulose (hemicellose) resources which is only second to cellulose as renewable natural resources. Xylan (xylan) is the main component of hemicellulose which widely resides in corn, rice bran, wheat bran, bagasse, straw and other agricultural products. The complex structural xylan is a poly-five-carbon including a sugar a xylose unit connected by (3-1,4glycosidic bond and with many short substituent branched-chain in nature which mainly exists in the form of pecilo-xylan. Xylan is less utilized because its complete degradation need a join action of a variety of hydrolytic enzymes involved and its main chain and side chain contains a variety of substituents which are difficult to be decomposed. Xylanase is the main enzyme for degradation of xylan, which is a composite of enzymes constituted by a variety of enzymes. Xylanase has the main effects of catalyzing hydrolysis of xylan beta-1,4-glycosidic bond randomly, reducing the polymerization degree of xylan and degradating polysaccharide into oligosaccharides xylose. XOS has high added value, and it can be used as a functional food additive or in many other areas of the feed and so on, which has a very attractive market prospects. However, the studies on xylanase started very late in our country, and there are many problems in the industrial production.In recent years, with the development and application of cloning technology, the research of xylanase gene has made tremendous progress, the xylanase gene was transferred into a suitable host bacteria for expression which has made some progress and build a new expression vector. Xylanase is used in pulp and paper, food, textile, feed, energy industry and other industries.In this paper, Aspergillus niger xylanase B gene was inserted into the new carrier pGAPz, and the recombinant expression carrier pGAPz-xyn B was transferred into Pichia pastoris X-33by electroporation and integration with the genome, building the engineering Pichia Yeast, and successfully expressed xylanase secretion with pGAPz-Pichia yeast expression system. that the xylanase can be secreted into the matrix and had activities was proved by the method of DNS for the determination of the activity of the supernatant enzyme and cell detritus. The xylanase activity of culture medium supernatant after centrifugation to remove bacterial was7.81IU/ml, and the xylanase activity of collected bacteria by sonication was3.28IU/ml, which proved that xylan enzyme can be secreted into the matrix with large proportion and with activity. In addition, efficiently expressing fermentation medium has been screened on the basis of the original medium through the optimization of carbon and nitrogen sources, inorganic salts, metal ions, trace elements, The built engineering Pichia Yeast was cultured under the conditions of37℃,200rpm for about48hours in the shake flask contained main medium which contained30g/L wheat bran as a carbon source,20g/1beef extract as nitrogen source,5g/1(NH4)2SO4as inorganic salts,0.1%ferrous sulfate as a metal ion,5g/l dipotassium hydrogen phosphate and sodium dihydrogen phosphate medium, as the composition of the phosphate buffer and Tween80(0.01%), the xylanase enzyme activity of supernatant solution was up to11.38IU/ml which improved significantly, as45%compared to the first medium, which provides a reference for further research.