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Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme

  • Biotechnology Methods
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9KM vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9KM-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn2+, which caused a strong inhibition. The K m and V max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.

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Acknowledgments

This work was financially supported by the National Nature Science Foundation of China (No. 31101229) and the Fundamental Research Funds for the Central Universities of China (No. JUDCF11032). The authors are grateful to Prof. **anzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.

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Authors and Affiliations

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Jun-Qing Wang & Min-Chen Wu

  2. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Jun-Qing Wang, **n Yin & Cun-Duo Tang

  3. School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Min-Chen Wu, Shu-Juan Gao & **g-Tu Wei

  4. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Hui-Min Zhang & Jian-Fang Li

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  1. Jun-Qing Wang
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  2. **n Yin
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Correspondence to Min-Chen Wu.

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Wang, JQ., Yin, X., Wu, MC. et al. Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme. J Ind Microbiol Biotechnol 40, 75–83 (2013). https://doi.org/10.1007/s10295-012-1201-2

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  • DOI: https://doi.org/10.1007/s10295-012-1201-2

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