Cloning and Molecular Analysis of cDNA Encoding a Carboxymethylcellulase of the YeastCryptococcus flavus

摘要

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeastCryptococcus flavuswas cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with theN-terminal amino acid sequence of secreted CMCase 1. No potentialN-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that theC. flavusCMCase 1 is partially homologous to theTrichoderma reeseiendoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced intoE. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.