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. 2001 Feb;90(2):248-55.
doi: 10.1046/j.1365-2672.2001.01237.x.

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli

P Basaran  1 , Y D Hang, N Basaran, R W Worobo
Affiliations

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli

P Basaran et al. J Appl Microbiol. 2001 Feb.

Abstract

Aims: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543.

Methods and results: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively.

Conclusion: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail.

Significance and impact of the study: The results of this study can be used to develop better xylanase-utilizing yeast strains.

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