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Cloning, Characterization of Pichia etchellsii $\beta-Glucosidase$ II and Effect of Media Composition and Feeding Strategy on its Production in a Bioreactor

Sethi Benu    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Jain Monika    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Chowdhary Manish    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Soni Yogesh    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Bhatia Yukti    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Sahai Vikram    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology   ); Mishra Saroj    (Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology  );
  • 초록

    The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$ , from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$ , the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$ . Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

  • 주제어

    Pichia etchellsi .   recombinant Escherichia coli .   fed-batch cultivation.  

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