Full Length Research Paper

Cloning, sequencing and expression of a novel xylanase cDNA from a newly isolated Aspergillus awamori in Pichia pastoris

Yinan Yang^1,2, Kuixian Shan², Lifeng Ping³* and Jingmei Lu¹*

¹School of Life Sciences, Northeast Normal University, Changchun, 130024 P. R. China.

²College of Horticulture, Jilin Agricultural University, Changchun, 130118 P. R. China.

³Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021 P. R. China.

*Corresponding author. E-mail: jingmeilu@yahoo.com. Tel: 86-431-84656798.

Accepted 30 October, 2008

A strain SH 2016, capable of producing xylanase, was isolated and identified as Aspergillus awamori, based on its physiological and biochemical characteristics as well as its ITS rDNA gene sequence analysis. A xylanase gene of 591 bp was cloned from this newly isolated A. awamori and the ORF sequence predicted a protein of 196 amino acids with a molecular mass about 21 kDa. An expression plasmid carrying the gene under the control of the methanol regulated alcohol oxidase gene (AOX1) promotor was introduced into Pichia pastoris, and xylanase gene was successfully expressed into the medium using methanol as inducer. Xylanase with 6 his tags was purified using Ni2+-NTA column. The characteristics of purified xylanase were investigated.

Key words: Aspergillus awamori, cDNA, cloning, xylanase, eukaryotic expression, purification and characterization.