The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL−1 affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL−1 TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The KM values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL−1, and Vmax values were 0.038 and 0.047 mg min−1, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg2+ and Cu2+ suggesting that N-glycosylation is involved in the folding of Amy1.

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