Bhuvan Shankar Vadala1, Sumedh Deshpande1, Anjali Apte-Deshpande2. 1. Central Dogma Pvt. Ltd, A4, Gulmohar Residency, Baner Road, Baner, Pune, Maharashtra, 411045, India. 2. Central Dogma Pvt. Ltd, A4, Gulmohar Residency, Baner Road, Baner, Pune, Maharashtra, 411045, India. anjali@centraldogma.co.in.
Abstract
BACKGROUND: Cellulases 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. RESULTS: Extracellular 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. CONCLUSION: Soluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.
BACKGROUND: Cellulases 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. RESULTS: Extracellular 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. coliexpression 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. CONCLUSION: Soluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.
Entities:
Keywords:
B. subtilis; Cellulase; Recombinant expression; pET21a
Authors: H Okada; K Tada; T Sekiya; K Yokoyama; A Takahashi; H Tohda; H Kumagai; Y Morikawa Journal: Appl Environ Microbiol Date: 1998-02 Impact factor: 4.792