Abstract
BackgroundCellulases are well known for their various industrial applications. They are naturally produced by different species of bacteria and fungi. Fermentation process of cellulase producers has limitation due to the high substrate cost required for cellulase induction and challenges to maintain the suitable condition for the respective cellulase production. Recombinant cellulase production could be the potential solution to these problems. In the current study, we investigated recombinant cellulase expression in Escherichia coli using cellulase gene from Bacillus subtilis. ResultsExtracellular cellulase production from B. subtilis strain was first confirmed on CMC agar and then the cellulase gene (1500 bp) was amplified from this strain and was further cloned in pET21a expression vector. In initial experimental studies, recombinant cellulase expression was achieved in inclusion bodies through shake flask level fermentation of transformed E. coli expression host BL21DE3. Attempts were made to express this 55 KDa His tagged recombinant cellulase into soluble form by modifications in fermentation conditions. Partially purified recombinant cellulase was obtained using Ni-NTA affinity chromatography. The activity of the purified enzyme was confirmed by 3,5-dinitrosalicylic acid (DNS) qualitative assay. ConclusionSoluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.
Highlights
Cellulases are well known for their various industrial applications
Gene amplification and cloning Available strain of B. subtilis was tested for cellulase activity on carboxymethyl cellulose (CMC) agar and cellulase gene was amplified from its genomic DNA using forward and reverse primers mentioned earlier in a 25-μL reaction volume
Inherent cellulase activity of Bacillus subtilis Cellulase activity was ensured before cloning and recombinant expression in the bacterial system
Summary
Cellulases are well known for their various industrial applications. They are naturally produced by different species of bacteria and fungi. The major industrial application of cellulases is in the textile industry to produce the stone-washed look of denims and for the household laundry detergents for refining fabric softness and brightness [2]. Cellulases are produced in large scale using fungi, bacteria or actinomycetes. The high cost of cellulases is mainly due to the substrates used in the production, and slow growth rate of fungi [1]. Bacillus spp. is highly studied for its diverse range of cellulases that are stable under extreme conditions [8, 9].
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