Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product.

A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned. This represents the first report of a key component of the cellulase regulon from this organism. DNA sequencing revealed that cbh2 has an open reading frame of 1377 bp, which encodes a putative polypeptide of 459 amino acids, and is interrupted by seven introns. The deduced amino acid sequence revealed that cbh2 has a modular structure with a predicted molecular mass of 47 kDa and consisting of a fungal type carbohydrate binding module separated from a catalytic domain by a proline/serine/threonine rich linker region. The deduced protein is homologous to fungal cellobiohydrolases in Family 6A of the glycosyl hydrolases. Profiles of cbh2 expression in T. emersonii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level. Expression of the T. emersonii cbh2 gene is induced by cellulose, xylan, xylose, and gentiobiose and clearly repressed by glucose. Putative regulatory element consensus sequences have been identified in the upstream regulatory sequence of the cbh2 gene including the catabolite repressor element and the activator of cellulase expression (Ace) binding sites. High sequence identity (67%) between the catalytic domain of Cel 6A from Trichoderma reesei and the T. emersonii cbh2 gene product allowed structure prediction for the 3D model of the T. emersonii catalytic domain to be a variant of the classical TIM alpha/beta fold.

Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-D-glucans from the aerobic fungus talaromyces emersonii CBS 814.70.

A novel endoglucanase active on 1,3-1,4-beta-D-glucans was purified to apparent homogeneity from submerged cultures of the moderately thermophilic aerobic fungus Talaromyces emersonii CBS 814.70. The enzyme is a single subunit glycoprotein with M(r) and pI values of 40.7 +/- 0.3 kDa and 4.4, respectively, and an estimated carbohydrate content of 77% (w/w). The purified beta-glucanase displayed activity over broad ranges of pH and temperature, yielding respective optima values of pH 4.8 and 80 degrees C. This enzyme was markedly thermostable with 15% of the original activity remaining after incubation for 15 min at 100 degrees C. Substrate specificity studies revealed the identity of the enzyme to be a 1,3-1,4-beta-D-glucanase. Identical K(m) values (13.38 mg.ml(-1)) were obtained with lichenan and BBG, while the V(max) value with lichenan (142.9 IU.mg(-1)) was approximately twice the value obtained with BBG (79.3 IU.mg(-1)). Time-course hydrolysis of barley-beta-glucan did not proceed linearly with respect to time indicating an 'endo' or more processive action for the enzyme. HPAEC fractionation of the products of hydrolysis yielded a range of oligosaccharides, with cellobiose, cellotriose and cellotetraose being the predominant oligosaccharide products.