Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 182, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2021.105843
Keywords
Thermostable beta-glucosidase; Signal peptide screening; Bacillus; Extracellular secretion
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Funding
- Department of Biotechnology, New Delhi, India
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This study aimed to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus in B. subtilis, resulting in a significant improvement in the yield of PfuBGL. The research demonstrated the high potential of utilizing B. subtilis as a host organism for industrial production of various proteins/enzymes.
Commercial applications of beta-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 +/- 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 +/- 0.21 pNP U/ml with specific activity of 35.52 +/- 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.
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