嗜热有孢圆酵母YK-1中α-葡萄糖苷酶的纯化及特性研究

Purification and characterization of alpha-glucosidase from Torulaspora pretoriensis YK-1.

作者信息

Oda Y, Iwamoto H, Hiromi K, Tonomura K

机构信息

Department of Food Science and Technology, Fukuyama University, Hiroshima, Japan.

出版信息

Biosci Biotechnol Biochem. 1993 Nov;57(11):1902-5. doi: 10.1271/bbb.57.1902.

DOI:10.1271/bbb.57.1902

PMID:7764339

Abstract

alpha-Glucosidase was partially purified 103-fold from a cell-free extract of Torulaspora pretoriensis YK-1 by column chromatography on Toyopearl HW55F, DEAE-Toyopearl 650M, hydroxylapatite and phenyl-Toyopearl 650M. Further purification by preparative polyacrylamide gel electrophoresis (PAGE) gave the homogenous protein, but the specific activity was reduced. The molecular weight of the enzyme was estimated to be 69,000 by SDS-PAGE and 60,000 by gel filtration. Optimum pH and temperature were 6.8 and 35 degrees C, respectively. The enzyme was inhibited strongly by AgNO3, HgCl2, sodium dodecyl sulfate, and N-ethylmaleimide. The Km (mM) for p-nitrophenyl alpha-D-glucopyranoside, maltose, maltotriose, isomaltose, methyl alpha-glucoside, and sucrose were 0.15, 150, 45, 17, 18, and 29, and Vmax (mumol/min/mg protein) for those substrates were 87, 0.23, 2.4, 9.0, 12, and 7.4, respectively. The N-terminal amino acid sequence of the enzyme was PEVKNHPETQPKWWKEATVY. The properties of alpha-glucosidase from T. pretoriensis YK-1 were similar to those from Saccharomyces cerevisiae.

摘要

通过在Toyopearl HW55F、DEAE-Toyopearl 650M、羟基磷灰石和苯基-Toyopearl 650M上进行柱色谱，从 Pretoriensis YK-1 酵母无细胞提取物中部分纯化了α-葡萄糖苷酶，纯化倍数为103倍。通过制备型聚丙烯酰胺凝胶电泳（PAGE）进一步纯化得到了均一的蛋白质，但比活性降低。通过SDS-PAGE估计该酶的分子量为69,000，通过凝胶过滤估计为60,000。最佳pH和温度分别为6.8和35℃。该酶受到硝酸银、氯化汞、十二烷基硫酸钠和N-乙基马来酰亚胺的强烈抑制。对硝基苯基α-D-吡喃葡萄糖苷、麦芽糖、麦芽三糖、异麦芽糖、甲基α-葡萄糖苷和蔗糖的Km（mM）分别为0.15、150、45、17、18和29，这些底物的Vmax（μmol/min/mg蛋白质）分别为87、0.23、2.4、9.0、12和7.4。该酶的N端氨基酸序列为PEVKNHPETQPKWWKEATVY。Pretoriensis YK-1酵母α-葡萄糖苷酶的性质与酿酒酵母的相似。