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Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix Patriciarum
Author(s) -
You Chun,
Huang Qiang,
Xue Huping,
Xu Yang,
Lu Hong
Publication year - 2010
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22623
Subject(s) - thermostability , chemistry , hydrophobic effect , cysteine , xylanase , mutagenesis , dtnb , mutant , site directed mutagenesis , circular dichroism , hydrogen bond , biochemistry , homology modeling , stereochemistry , enzyme , organic chemistry , molecule , glutathione , gene
In this study, we employed directed evolution and site‐directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum , and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far‐ultraviolet circular dichroism analysis showed that the melting temperatures ( T m ) of the G201C and C60A–G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site‐directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A–G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well‐packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side‐chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself. Biotechnol. Bioeng. 2010;105: 861–870. © 2009 Wiley Periodicals, Inc.