Exploration of Disulfide Bridge and N-Glycosylation Contributing to High Thermostability of a Hybrid Xylanase

Title:Exploration of Disulfide Bridge and N-Glycosylation Contributing to High Thermostability of a Hybrid Xylanase

Volume: 21 Issue: 7

Author(s): Zhongbiao Tan, Cunduo Tang, Minchen Wu, Yao He, Die Hu and Junqing Wang

Affiliation:

Keywords: Disulfide bridge, N-glycosylation, site-directed mutagenesis, thermostability, xylanase.

Abstract: A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase AEx11A revealed that a disulfide bridge (Cys⁵-Cys³²) and an N-glycosylation site (Asn⁴²) were imported into AEx11A by N-terminal substitution of AoXyn11A with EvXyn11^TS. Two mutant genes AEx11A ^C5T and AEx11A ^N42Q were constructed by mutating Cys⁵- and Asn⁴²-encoding codons of AEx11A into Thr⁵- and Gln⁴²-encoding ones, and heterologously expressed in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11A^C5T (reAEx11A^C5T) was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3 and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A and reAEx11A^C5T in pH characteristics and kinetic parameters. Furthermore, both reAEx11A^N42Q and reAEx11A displayed no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the Nglycosylation had no effect on.

Cite this article as:

Tan Zhongbiao, Tang Cunduo, Wu Minchen, He Yao, Hu Die and Wang Junqing, Exploration of Disulfide Bridge and N-Glycosylation Contributing to High Thermostability of a Hybrid Xylanase, Protein & Peptide Letters 2014; 21 (7) . https://dx.doi.org/10.2174/0929866521666140328113126

DOI https://dx.doi.org/10.2174/0929866521666140328113126	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305