文献类型：期刊文献

英文题名：Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris

作者：Guo, Meijin[1,2]; Hang, Haifeng[1]; Zhu, Taicheng[1]; Zhuang, Yingping[1]; Chu, Ju[1]; Zhang, Siliang[1]

机构：[1] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China; [2] Department of Biotechnology, Jiangxi Agricultural University, 10 Yingshang Road, Nanchang, 330045, China

年份：2008

卷号：42

期号：4

起止页码：340

外文期刊名：Enzyme and Microbial Technology

收录：EI(收录号：20080811104680)

语种：英文

外文关键词：Amino acids - Biochemical engineering - Glycosylation - pH - Thermodynamic stability

摘要：Recombinant phytase expressed in Pichia pastoris FPHY34 is a typical glycoprotein, whose gene possesses 10 potential N-glycosylation sites without O-glycosylation. Effects of glycosylation on phytase's biochemical characterization were investigated. Secreted phytase's micro-heterogenious glycosylation phenomenon was observed and verified by SDS-PAGE, N-terminal amino acid sequencing and mass spectrum analyses. Deglycosylation of recombinant phytase reduced the molecular weight from 83 to 63 kDa, which indicates the presence of total carbohydrate content of approximately 24.1%. According to image analysis of 2-dimension gel electrophoresis, the range of isoeclectric point of phytase covers nearly 1 pH value rather than a specific point, which might be due to heavy and complex glycosylation. The phytase expressed by P. pastoris still retained 40% and 30% activities at 80 and 90 °C for 10 min, respectively. However, upon deglycosylation in vitro by peptide-N-glycohydrolase F (PNGase F), thermostability of deglycosylated phytase significantly declined after 10 min treatment at 40 and 50 °C. Optimum pH of phytase tested was shifted from pH 5.0 to 2.5 by deglycosylation, although there exhibit bi-humps pH optima at both before and after PNGase F digestion cases. ? 2007 Elsevier Inc. All rights reserved.