N-糖基化对来自黄隐球酵母的α-淀粉酶Amy1生化特性的影响。

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

作者信息

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

机构信息

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.

出版信息

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

DOI:10.1016/j.carres.2009.06.006

PMID:19570529

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.

摘要

黄隐球酵母在含淀粉培养基中生长时会分泌一种糖基化的α-淀粉酶（Amy1）。研究了N-糖基化对这种糖蛋白的分泌、酶活性和稳定性的影响。向培养基中添加浓度高于0.5μg mL⁻¹的衣霉素（TM）会影响黄隐球酵母的生长。在0.5μg mL⁻¹ TM存在下黄隐球酵母生长后，细胞内部分的Amy1活性增加了55%。SDS-PAGE和凝胶活性检测表明，天然酶和去糖基化酶的表观分子量分别为68 kDa和64.5 kDa。N-糖基化过程既不影响最适pH也不影响最适温度。天然和非糖基化α-淀粉酶的Kₘ值分别为0.052和0.098 mg mL⁻¹，Vₘₐₓ值分别为0.038和0.047 mg min⁻¹。然而，非糖基化形式对蛋白水解酶胰蛋白酶和高温失活更敏感。此外，非糖基化酶的活性受Hg²⁺和Cu²⁺影响，这表明N-糖基化参与了Amy1的折叠。