Molecular Cloning and Expression of the Trichoderma harzianum C4 Endo-β-1,4-Xylanase Gene in Saccharomyces cerevisiae

An endo-beta-1,4-xylanase (beta-xylanase) from Trichoderma harzianum C4 was purified without cellulase activity by sequential chromatographies. The specific activity of the purified enzyme preparation was 430 units/mg protein on D-Xylan. The complementary DNA (cDNA) encoding beta-xylanase (xynII) was amplified by PCR and isolated from cDNA PCR libraries constructed from T harzianum C4. The nucleotide sequence of the cDNA fragment contained an open reading frame of 663 bp that encodes 221 amino acids, of which the mature protein is homologous to several beta-xylanases II. An intron of 63 bp was identified in the genomic DNA sequence of xynII. This gene was expressed in Saccharomyces cerevisiae strains under the control of adh1 (alcohol dehydrogenase 1) and pgk1 (phosphoglycerate kinase 1) promoters in 2 mu-based plasmids, which could render recombinants able to secrete beta-xylanase into the media.