Production and Partial Purification of Cellulase from a New Isolate, Penicillium verruculosum BS3

Aims: This study describes the production of cellulases by a newly isolated fungus, Penicillium verruculosum BS3 on lignocellulosic biomass with special emphasis on endoglucanase.
Study Design: Isolation, screening and characterization of cellulolytic fungus. Production of cellulase by submerged and solid state fermentation. Partial purification of cellulase by ammonium sulphate precipitation, dialysis and spin column. Characterization by Native PAGE and SDS-PAGE.
Place and Duration of Study: Enzyme Technology Laboratory, School of Biological Sciences, University of Calicut, Kerala - 673635, India, between October 2013 and November 2014.
Methodology: The lignocellulolytic fungus was characterized and identified by lacto-phenol cotton blue staining and 28S rRNA gene sequencing. Preliminary confirmation of cellulase production was done by iodine plate assay method. Production of cellulase was carried out on synthetic and natural raw substrates employing submerged and solid state fermentation strategies. The produced cellulase was purified by ammonium sulphate precipitation, dialysis and spin column followed by characterization using native-PAGE and SDS-PAGE.
Results: Primary screening on carboxymethyl cellulose (CMC) agar plates showed cellulase production potential of P. verruculosum BS3. Production of cellulases was quantitatively estimated on basal salt medium (BSM) supplemented with 1% CMC by submerged fermentation (SmF). Among cellulases, endoglucanase showed the highest yield (930.9 U/ml), which was focused on the subsequent studies. The CMC in BSM was replaced by the natural flour (1%) of banana, tapioca, potato or banana peel, of which tapioca flour supported the maximum production of endoglucanase (598.3 U/ml). In solid state fermentation (SSF), P. verruculosum BS3 was grown on sawdust supplemented with 10% banana flour as a solid support, moistened with BSM and the maximum production of endoglucanase (3214.1 U/gds) was observed on day 9 of incubation at 28ºC. The partially purified (9.1 folds purification with 2.1% yield) cellulase was then characterized by native PAGE and SDS-PAGE. Upon staining with Congo-red (1%), on the CMC (0.25%) impregnated native gel, the zymogram showed a major cellulase fraction as yellow opaque zone. From the SDS-PAGE profile, the approximate MW of cellulase was estimated as 17 kDa.
Conclusion: This study signifies the industrial importance of the cellulase produced by P. verruculosum BS3 on cheap agro-products, and that the low MW cellulase has added importance as it offers potentials for protein engineering.