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

ISSN: 1875-5305 (Online)
ISSN: 0929-8665 (Print)


Volume 21, 12 Issues, 2014


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Protein & Peptide Letters

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

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

Affiliation: Wuxi Medical School, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, P.R. China.

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 EvXyn11TS. 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 AEx11AC5T (reAEx11AC5T) 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 reAEx11AC5T in pH characteristics and kinetic parameters. Furthermore, both reAEx11AN42Q 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.

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

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Article Details

Volume: 21
Issue Number: 7
First Page: 657
Last Page: 662
Page Count: 6
DOI: 10.2174/0929866521666140328113126
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