Bacillus Cellulase Molecular Cloning, Expression, and Surface Display on the Outer Membrane of Escherichia coli

Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry
Daehwan Kim, Seockmo Ku

Abstract

One of the main challenges of using recombinant enzymes is that they are derived from genetically-modified microorganisms commonly located in the intracellular region. The use of these recombinant enzymes for commercial purposes requires the additional processes of cell disruption and purification, which may result in enzyme loss, denaturation, and increased total production cost. In this study, the cellulase gene of Bacillus licheniformis ATCC 14580 was cloned, over-expressed, and surface displayed in recombinant Escherichia coli using an ice-nucleation protein (INP). INP, an outer membrane-bound protein from Pseudomonas syringae, was utilized as an anchor linker, which was cloned with a foreign cellulase gene into the pET21a vector to develop a surface display system on the outer membrane of E. coli. The resulting strain successfully revealed cellulase on the host cell surface. The over-expressed INP-cellulase fusion protein was confirmed via staining assay for determining the extracellular cellulase and Western blotting method for the molecular weight (MW) of cellulase, which was estimated to be around 61.7 kDa. Cell fractionation and localization tests demonstrated that the INP-cellulase fusion protein was mostly present in...Continue Reading

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Citations

Aug 3, 2020·Biotechnology and Bioengineering·Xiaoqiang JiaKang Wu
Feb 7, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Rafaela I S Ladeira ÁzarDaehwan Kim
Jan 2, 2021·International Journal of Biological Macromolecules· AlokikaBijender Singh
Jan 12, 2021·Journal, Genetic Engineering & Biotechnology·Bhuvan Shankar VadalaAnjali Apte-Deshpande

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Methods Mentioned

BETA
PCR
surface
electrophoresis

Software Mentioned

BLASTP
Clustal Omega

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