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Cloning, characterization ofPichia etchellsii β-glucosidase II and effect of media composition and feeding strategy on its production in a bioreactor

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Abstract

The cloning and expression of β-glucosidase II, encoded by the geneßglu2, from thermotolerant yeastPichia etchellsii intoEscherichia coli is described. Cloning of the 7.3 kbBamHI/SalI yeast insert containingßglu2 in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid intoE. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At 50°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−1. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

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Authors and Affiliations

  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz-Khas, 110016, New-Delhi, India

    Benu Sethi, Monika Jain, Manish Chowdhary, Yogesh Soni, Yukti Bhatia, Vikram Sahai & Saroj Mishra

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  1. Benu Sethi
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  2. Monika Jain
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  3. Manish Chowdhary
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  7. Saroj Mishra
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Correspondence to Saroj Mishra.

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Sethi, B., Jain, M., Chowdhary, M. et al. Cloning, characterization ofPichia etchellsii β-glucosidase II and effect of media composition and feeding strategy on its production in a bioreactor. Biotechnol. Bioprocess Eng. 7, 43–51 (2002). https://doi.org/10.1007/BF02935879

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