Molecular Evolution In Vitro And Bioinformatics Analysis Of The Thermophilic Xylanase

Xylanase (E.C 3.2.1.8) is a widespread group of enzyme which can catalyze the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan. Xylanse has a wide application in industrial processes. It can be used in biobleachin of kraft pulps,fruit and vegetable processing, animal feeding and so on. The thermotable xylanase has atttacted widely attention because of its thermostabilization.In this study, xylanase gene was obtained from Aspergillus niger C3486 by PCR. To achieve secretive expression, pET32a (+) was used as expression vector, and the cloned gene was expressed in E. coli BL21 (DE3). After induction by IPTG, a large quantity of xylanase was produced but without enzyme activity. And some progress have made in the fugal expression system: the xylanase gene was then transformed into the Pichia.pastoris GS115, the enzyme produced from the Pichia.pastoris GS115 showed xylanase activity.Moreover, the total RNA was obtained, after RT-PCR, the gene was transformed into the pET28a as well, and the enzyme named Xyn-3 produced from the E.coli BL21 (DE3) also showed xylanase activity, and through the compare between the DNA and cDNA sequence, we know that the xylanase DNA contains an intron about 50 bp.With the analysis of bioinformatics, this xylanase contains 187 amino acids with a preceding peptide. Moreover, it has a typical conserve domain of xylanase. Perdicted by web site on line. The xylanase is globulin, and its super structure contains manyβ-sheet.And then, the xylanase gene from A.niger and T.reesei was mixed for co-evolution by the technology of Error PCR and DNA Shuffling, and the xylanase with higher thermostability were obtained.