A Unique Disulfide Bridge of the Thermophilic Xylanase SyXyn11 Plays a Key Role in Its Thermostability

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X. Yin^1#, Y. Yao^2#, M. C. Wu^3*, T. D. Zhu¹, Y. Zeng², and Q. F. Pang³

¹School of Biotechnology and Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, China; fax: +086-510-8532-9042; E-mail: yinxinat2008@126.com

²School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, China; fax: +086-510-8532-9042; E-mail: yaoyaosunshine@sina.cn

³Wuxi Medical School, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, China; fax: +086-510-8532-9042; E-mail: biowmc@126.com

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received November 30, 2013; Revision received December 6, 2013

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Based on the hyperthermostable family 11 xylanase (EvXyn11^TS) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5–Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11^C5T-encoding gene, Syxyn11^C5T, was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11^C5T, were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11^C5T decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.

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KEY WORDS: xylanase, thermostability, disulfide bridge, computational prediction, site-directed mutagenesis

DOI: 10.1134/S0006297914060066

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