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Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris

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Abstract

A β-glucosidase gene (bgl) from Aspergillus oryzae GIF-10 was cloned, sequenced and expressed. Its full-length DNA sequence was 2,903 bp and included three introns. The full-length cDNA sequence contained an open reading frame of 2,586 nucleotides, encoding 862 amino acids with a potential secretion signal. The A. oryzae GIF-10 bgl was functionally expressed in Pichia pastoris. After 7-day induction, protein yield reached 321 mg/mL. Using salicin as the substrate, the specific activity of the purified enzyme reached 215 U/mg. The purified recombinant β-glucosidase was a 110-kDa glycoprotein with optimum catalytic activity at pH 5.0 and 50 °C. The enzyme was stable between 20 and 60 °C, and retained 65 % of its activity after being held at 60 °C for 30 min. The recombinant β-glucosidase was relatively stable in a broad range of pHs, from 4.0 to 6.5. It showed broad specific activity, hydrolyzing a range of (1-4)-β-diglycosides and (1-4)-α-diglycosides, and Mn2+ stimulated its activity significantly.

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Acknowledgments

We gratefully acknowledge financial support for this work from the National Natural Science Foundation of China (Grant No. 30671530). The method for high-throughput screening of the mutant library is currently being examined for a patent application for an invention (Application No. 201110089103.6).

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Authors and Affiliations

  1. College of Life and Physical Science, Sichuan Agricultural University, Yaan, 625014, China

    Zizhong Tang, Shan Liu, Haijun Jing, Rong Sun, Moyang Liu, Hui Chen, Qi Wu & Xueyi Han

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  1. Zizhong Tang
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  2. Shan Liu
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  3. Haijun Jing
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  4. Rong Sun
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  5. Moyang Liu
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  8. Xueyi Han
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Correspondence to Hui Chen.

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Tang, Z., Liu, S., Jing, H. et al. Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris . Mol Biol Rep 41, 7567–7573 (2014). https://doi.org/10.1007/s11033-014-3644-1

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  • DOI: https://doi.org/10.1007/s11033-014-3644-1

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