The influence of N-glycosylation on biochemical properties of Amy1, an alpha-amylase from the yeast Cryptococcus flavus.
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.
de Barros
Manoel Cardoso
MC
Laboratório de Bioquímica, Faculdade de Ciências Biológicas, Universidade de Rio Verde-Fesurv, Fazenda Fontes do Saber-s/n, Rio Verde, Goiás, GO, CEP 75901-970, Brazil.
do Nascimento Silva
Roberto
R
Ramada
Marcelo Henrique Soller
MH
Galdino
Alexsandro Sobreira
AS
de Moraes
Lídia Maria Pepe
LM
Torres
Fernando Araripe Gonçalves
FA
Ulhoa
Cirano José
CJ
D049109:Cell Proliferation / Q000187:drug effects; D003454:Cryptococcus / Q000166:cytology / Q000201:enzymology*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D014415:Tunicamycin; D000516:alpha-Amylases; D009584:Nitrogen