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

• Published in: Carbohydrate research Vol. 344; no. 13; pp. 1682 - 1686
• Main Authors: de Barros, Manoel Cardoso, do Nascimento Silva, Roberto, Ramada, Marcelo Henrique Soller, Galdino, Alexsandro Sobreira, de Moraes, Lídia Maria Pepe, Torres, Fernando Araripe Gonçalves, Ulhoa, Cirano José
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 08.09.2009
• Subjects: alpha-Amylases - chemistry; alpha-Amylases - isolation & purification; alpha-Amylases - metabolism; Cell Proliferation - drug effects; Cryptococcus - cytology; Cryptococcus - enzymology; Cryptococcus flavus; Extracellular Space - drug effects; Extracellular Space - metabolism; Glycosylation - drug effects; Intracellular Space - drug effects; Intracellular Space - metabolism; Nitrogen - metabolism; Tunicamycin; Tunicamycin - pharmacology; α-Amylase, N-glycosylation; Cryptococcus flavus; Tunicamycin; α-Amylase, N-glycosylation
• DOI: 10.1016/j.carres.2009.06.006
• ISSN: 0008-6215

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 K M values of native and non-glycosylated α-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.