CLONING AND CHARACTERIZATION OF A GENE ENCODING A CELL-BOUND, EXTRACELLULAR BETA-GLUCOSIDASE IN THE YEAST CANDIDA-WICKERHAMII

The ability of yeasts to ferment cellodextrins is rare. Candida wicker hamii is able to use these sugars for alcohol production because of a cell-bound, extracellular, beta-glucosidase that is unusual by not bei ng inhibited by glucose. A cDNA expression library in lambda phage was prepared with mRNA isolated from cellobiose grown C. wickerhamii. Imm unological screening of the library with polyclonal antibodies against purified C. wickerhamii cell-bound, extracellular beta-glucosidase yi elded 12 positive clones. Restriction endonuclease analysis and sequen ce data revealed that the clones could be divided into two groups, bgl A and bglB, which were shown to be genetically distinct by Southern hy bridization analyses. Efforts were directed at the study of bglB since it appeared to code for the cell-bound beta-glucosidase. Sequence dat a from both cDNA and genomic clones showed the absence of introns in b glB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and imm unoblotting of cell lysates from Escherichia coli bglB clones confirme d the presence of an expressed protein with an apparent molecular mass of 72 kDa, which is consistent with that expected for an unglycosylat ed form of the enzyme, Amino acid comparisons of BglB with other beta- glucosidase sequences suggest that it is a member of family 1 glycosyl hydrolases but is unusual in that it contains an additional 100 to 13 0 amino acids at the N terminus. This sequence did not have homologies to other known protein sequences and may impart unique properties to this beta-glucosidase.