Study On Cloning And Overexpression Of Gene Encoding Endo-β-1, 4-xylanase From Trichoderma Reesei In Pichia Pastoris

Xylan is one component of Hemicellulose,which is only less cellulose in plant cell wall. Hemicellulose is as 35%as the dry weight,of which xylan is the most.Because of it,the nutrient can be protected from the surface of animal digestive juice so that the digestion and the absorption were affected,which decrease the nutrient value.Xylanase hydrolyzes 1,4-β-D-xyloside bond by internally tangenting and the hydrolization products are xylan and hex-xylan mostly.With the application of xylanase in the field of paper making,food and forage,it becomes an important production of enzyme preparation.Xylanase can degradate xylan specially.It can be used in biotransformation,food,forage and medicine ect.But there are some problems,such as less production,which can be resolved by genetic engineering. In this study,xylanase gene xyn2 was cloned from Trichoderma reesei) QM941.E.coli expression vector and Pichia expression vector were constructed and were transferred into E.coli and Pichix ,which were analyzed of enzymology,setting the basis of large scale introduction.The main esults are as follows:Xylan was expressed from Trichoderma reesei by xylan revulsive.The best induced time was made to 72 hours,and the maximum enzyme activity was 6.32IU/ml.The best pH was 4.0 and the best temperature was 50℃.Xylanase gene xyn2 was cloned by RT-PCR,whose sequence was the same with NO.U24191 (GenBank).Xylanase gene was cloned by PCR.An intron was found in xylanase gene through Blast with cDNA sequence.Ecoli expression vector pEK1 was constructed and were transferred into Ecoli BL21,which were induced by IPTG 5 hours.The maximum enzyme activity was 42.28IU/ml.The best pH was 6.5,and the best temperature was 45℃.Pichia expression vector pPIC9-xyn2 was constructed.It was cultured in shake-flask for 96 hours and the maximum enzyme activity was 208.12IU/ml,about 33 times more than original strain. The best pH was 5.0,and the best temperature was 55℃.The promoter of pPIC9 vector was reconstructed so that it can be constitutive expression, which was named pPIC9Z.The constitutive expression vector pPIC9Z-xyn2of xyn2 gene was construced.It was transferred into Trichoderma reesei and the transformats were obtained.