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

Carbohydr Res. 2009 Sep 8;344(13):1682-6. doi: 10.1016/j.carres.2009.06.006. Epub 2009 Jun 6.

Abstract

The yeast Cryptococcus flavus secretes a glycosylated alpha-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 microg 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 microg 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 K(M) values of native and non-glycosylated alpha-amylases were 0.052 and 0.098 mg mL(-1), and V(max) 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 Hg(2+) and Cu(2+) suggesting that N-glycosylation is involved in the folding of Amy1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Proliferation / drug effects
  • Cryptococcus / cytology
  • Cryptococcus / enzymology*
  • Extracellular Space / drug effects
  • Extracellular Space / metabolism
  • Glycosylation / drug effects
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism
  • Nitrogen / metabolism*
  • Tunicamycin / pharmacology
  • alpha-Amylases / chemistry
  • alpha-Amylases / isolation & purification
  • alpha-Amylases / metabolism*

Substances

  • Tunicamycin
  • alpha-Amylases
  • Nitrogen