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Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymic thermostability

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Abstract

Thermostable α-amylases are widely used in industry. The α-amylase from Bacillus licheniformis (BLA) with six potential glycosylation sites possessed excellent thermal and pH stability and high activity. Here, it was expressed in Pichia pastoris. The Pic-BLA-producing yeast without any antibiotics-resistant gene was cultivated in flasks and the amylase activity in fermentation supernatant reached 900 U/mL. The recombinant α-amylase Pic-BLA produced in P. pastoris was deeply glycosylated with 30% increase in molecular mass (MM). The deglycosylation treatment by Endoglycosidase H (Endo H) reduced the MM of Pic-BLA. Thermostability analysis showed that Pic-BLA and deglycosylated Pic-BLA were similar in heat tolerance. In order to eliminate the extra impact of Endo H, the BLA was also expressed in Escherichia coli to get non-glycosylated Eco-BLA. A comparative study between non-glycosylated Eco-BLA and glycosylated Pic-BLA showed no obvious difference in thermostability. It is speculated that the glycosylation has little effect on the thermostability of α-amylase BLA.

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Abbreviations

BLA:

Amylase from B. licheniformis

Pic-BLA:

BLA expressed in P. pastoris

Eco-BLA:

BLA expressed in E. coli

Endo H:

Endoglycosidase H

MM:

Molecular mass

GH:

Glycoside hydrolase

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31670069), Technical Innovation Special Fund of Hubei Province (2017ACA171), and 2016 Wuhan Yellow Crane Talents (Science) Program. Thanks very much to Dr. K.V. Arivizhivendhan for helping us revise the English.

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Authors and Affiliations

  1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, School of Life Sciences, Hubei University, Wuhan, 430062, Hubei, China

    Xinlin Hu, Xin Yuan, Nisha He, Pan Wu & Guimin Zhang

  2. Health Science Center, University of Tennessee Memphis, Memphis, TN, 38163, USA

    Tony Z. Zhuang

Authors
  1. Xinlin Hu
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  2. Xin Yuan
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  3. Nisha He
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  4. Tony Z. Zhuang
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  5. Pan Wu
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  6. Guimin Zhang
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Contributions

The experiments were conceived and designed by XH, PW and GZ. The experiments and data analysis were performed by XH and XY. The manuscript was prepared by XH, NH, TZZ, PW and GZ.

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Correspondence to Pan Wu or Guimin Zhang.

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Hu, X., Yuan, X., He, N. et al. Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymic thermostability. 3 Biotech 9, 427 (2019). https://doi.org/10.1007/s13205-019-1943-x

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  • DOI: https://doi.org/10.1007/s13205-019-1943-x

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