Journal of Pure and Applied MicrobiologyVol. 9 No. 4

Cloning, Gene Expression and Characterization of Xylanase Coding Gene from Mesophilic Filamentous Fungus Trichoderma reesei (Hypocreaceae ) in E.coli

V.G. Saravana Kumar* and Jegatheesan Kalirajan

Department of Biotechnology, St. Michael College of Engineering & Technology, Kalayarkoil, Sivagangai ? 630 551, India.

Received on 10 June 2015 and accepted on 06 August 2015

 

ABSTRACT

This research paper describes an efficient bacterial transformation system for production of Xylanase enzyme from Xyn2 gene collected from Trichoderma reesei. Xylanase coding gene was collected from Genbank and artificially synthesized and cloned into E. coli cells. Artificial nucleotide gene sequence showed that the 840 long DNA fragment of Xyn2 gene had open reading frames encoding polypeptides of 229 amino acid residue. The Xyn2 gene made by two exon, first one start from 59 and end in 348bp and second one tart from 411 and end in 810bp and one intron present in the sequence, it start with 349 and end in 410 bp. Xylanase encoding gene-Xyn2 of Trichoderma reesei ligated into the pUC19 vector and numbered as GS57308 PUC19-Xyn2 system. The ligation mixture contained Xyn2-pUC19 DNA was then transformed into E. coli BL 21by CaCl2 method. The potent positive clone was identified by Blue -White colony screening, Congo red staining and RBB?Xylan assay. The positive clone of E.coli containing the Xyn2-pUC 19 gene was cultured for production Xylanase enzyme. The crude enzyme extract was identified and purified by SDS PAGE electrophorosois with coomasive blue staining. The Zymogram assay was performed for the qualitatively testing the presence of xylanase in the crude extract. The cloned xylanase Gene2 from T.reesei in E.coli showed highest enzyme activity of 176 Uml-1. The cloned Xyn2 gene for xylanase enzyme from T.reesei into E.coli could be a model system for gene expression, secretion, and purification of xylanase enzyme and it can be utilized for large-scale production of xylanase2 from E. coli transformant.

Keywords : pUC19 vector; transformant; Zymogram; RBB-Xylan assay; congo red.