GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509
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A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The Km and kcat values of GSxyn for ρNPX were 0.48 mM and 36.64 s−1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The Km and kcat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s−1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The Km and kcat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s−1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.
KeywordsGH52 xylosidase Xylanase Geobacillus stearothermophilus Biotechnology Protein engineering
This study was supported by grants from the National Natural Science Foundation of China (NO. J1103510).
The authors declare that the experiments comply with the current laws of the country in which they were performed.
Conflict of interest
The authors claim that they have no competing interests.
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