Cloning, sequence analysis and characterization of a novel i-glucosidase- like activity, MUGA from Pichia etchellsii

0-glucosidases (3-D-glucoside glucohydrolase, EC 3.2.1.21) comprise a heterogenous group of enzymes that are able to cleave the /3-glucosidic linkages of di-and/or oligosaccharides and other glucose conjugates. These enzymes are widely distributed in the living world and play pivotal roles in many biological processes. To study the biological role and useful biotechnological applications of these enzymes, we have reported on a number of p-glucosidases isolated from the thermo-tolerant yeast Pichia etchellsii. This organism grows optimally at 40-45°C and produces multiple 0glucosidases. Two enzymes namely, Bgl I and Bgl II were identified by way of expression in Escherichia coli. Two /3-glucosidases have been purified from the cell wall of the yeast, namely BGLI and BGLII and detailed analysis indicated these to be different from each other and from the other E. coli expressed enzyme. Our continued search in this yeast system has lead to identification of a novel hydrolytic activity. It resembled the reported 3-glucosidases in terms of its ability to hydrolyse MUG (methyl umbelliferyl 13D-glucoside) but did not hydrolyze pNPG (p-nitrophenyl (3-D-glucoside), which is a commonly used substrate for assay of these enzymes. The present work was undertaken to determine and analyze the nucleotide sequence encoding this activity, characterize the purified protein (biochemically and structurally), and evaluate its function in the yeast. Genomic DNA fragment encoding a noveli3-glucosidase-like activity of the yeast P. etchellsii was cloned and expressed in E. coli. Two MUG hydrolyzing clones were isolated, namely, pMG8: DH5a and pMG16: DH5a containing the same gene on different insert lengths. The sequencing of the 6.35 kbp yeast insert in pMG8 plasmid showed multiple ATG's with a single termination codon (TAG). An open reading frame