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Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris

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Abstract

Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.

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Acknowledgments

This research was supported by Shahid Beheshti University and National Iranian Oil Refining and Distribution Company. We thank Dr. J. Amani for a critical reading of the manuscript.

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Authors and Affiliations

  1. Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Ali Akbarzadeh, Mostafa Motallebi & Mohammad Reza Zamani

  2. Nanobiotechnology Engineering Laboratory, Faculty of Energy Engineering and New Technologies, Shahid Beheshti University, GC, Tehran, Iran

    Ali Akbarzadeh, Seyed Omid Ranaei Siadat & Sakineh Moshtaghi

  3. Protein Engineering Laboratory, Protein Research Center (PRC), Shahid Beheshti University, GC, Tehran, Iran

    Seyed Omid Ranaei Siadat

  4. Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, Tehran, Iran

    Mohammad Barshan Tashnizi

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  1. Ali Akbarzadeh
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  2. Seyed Omid Ranaei Siadat
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  3. Mostafa Motallebi
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  4. Mohammad Reza Zamani
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  6. Sakineh Moshtaghi
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Correspondence to Seyed Omid Ranaei Siadat.

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Akbarzadeh, A., Ranaei Siadat, S.O., Motallebi, M. et al. Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris . Appl Biochem Biotechnol 172, 2253–2265 (2014). https://doi.org/10.1007/s12010-013-0672-6

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  • DOI: https://doi.org/10.1007/s12010-013-0672-6

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