Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.
The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed.
Guinness Brewing Worldwide Research Centre, St. James's Gate, Dublin, Ireland.
B T
BT
Kinsella
A
A
Larkin
M
M
Bolton
B A
BA
Cantwell
D015139:Blotting, Southern; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005800:Genes, Fungal; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
D000520:alpha-Glucosidases