The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris
The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris
复制标题
DOI：
10.1016/j.enzmictec.2013.09.014
复制
发表时间：
2014-01-10
影响因子：
3.4
通讯作者：
Yu, Xiaobin
中科院分区：
文献类型：
--
作者：
Han, Minghai;Wang, Xinfeng;Yu, Xiaobin
铜绿假单胞菌弹性蛋白酶（PAE）由铜绿假单胞菌（P. aeruginosa）产生，是一种在有机溶剂中用于肽合成的有前景的生物催化剂。由于铜绿假单胞菌是一种机会致病菌，为了工业应用，该酶已在安全且高效的宿主巴斯德毕赤酵母（P. pastoris）中异源过表达。重组弹性蛋白酶（rPAE）包含三个潜在的N -糖基化位点（Asn - Xaa - Ser/Thr共有序列），并且是异质性N -糖基化的。为了研究N -糖基化在rPAE的活性、稳定性和表达中的作用，使用定点突变技术对这些潜在的N -糖基化位点（N43、N212和N280）进行突变。具体来说，将天冬酰胺（Asn，N）残基转变为谷氨酰胺（Gln，Q）。然后比较了非糖基化和糖基化rPAE的酶活性和稳定性。结果表明，N -糖基化对其活性的影响不显著。rPAE的非糖基化和糖基化异构体在水介质中水解酪蛋白时显示出相似的动力学参数，并且在50%（v/v）二甲基亚砜中催化二肽合成时，它们表现出相同的底物特异性和活性，并产生相似的产量。然而，N -糖基化提高了rPAE在水介质和50%（v/v）有机溶剂中的稳定性。在70℃时，糖基化和非糖基化形式的rPAE的半衰期分别为32.2分钟和23.1分钟。任何潜在N -糖基化位点的突变都对其在巴斯德毕赤酵母中的表达有害。与野生型相比，N43Q突变体的表达降低了23.9%，N212Q突变体降低了63.6%，N280Q突变体降低了63.7%。此外，这些位点的联合突变导致突变体的酪蛋白水解活性进一步降低。这些结果表明，所有的N -糖基化位点对于rPAE的高水平表达都是必需的。（C）2013爱思唯尔公司。保留所有权利。
The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70 degrees C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE. (C) 2013 Elsevier Inc. All rights reserved.