Abstract
An endospore forming Gram positive bacterium (MBCU4) was isolated from a vermicompost amended soil, and confirmed as Bacillus subtilis through the 16S rRNA sequence analysis. An extracellular chitinase was detected from this strain of B. subtilis under specific environmental condition. An attempt was made to purify the enzyme by ammonium sulfate precipitation followed by DEAE sepharose CL-6B column chromatography. The purified enzyme was demonstrated as a single band, having the molecular weight 31kDa on SDS PAGE analysis and its activity in the gel was determined by clear zone on zymogram. Further characterization of the isolated enzymes has showed that this enzyme is most active at pH 6.0 and at the optimized temperature of 50 0C. The purified chitinase exhibited high degree of antifungal activity particularly by degrading their cell wall components of plant pathogens Macrophomina phaseolina (69.0%) and Rhizoctonia solani (52.0%). It infers that the chitinase produced by B. subtilis could play an important role for biopesticidal activity.
Highlights
The endospore forming genus Bacillus is one of the most widely researched and commercialized biocontrol agents (Paulitz and Belanger, 2001)
Their biocontrol mechanisms include production of antibiotics and extracellular hydrolytic enzymes such as chitinase, laminarinase, lipase, and protease. These hydrolase enzymes contribute to degradation of fungal cell wall (Korsten et al, 1993; Paulitz and Belanger, 2001; Helisto et al, 2001) suitable for control of phyopathogenic fungi. Among those hydrolases chitinases considered as key hydrolytic enzymes in the lysis of cell walls of fungi, and play important factors in biological control (Guthrie et al, 2005)
Our chitinolytic zymography assay used in this study revealed a high resolution, excellent linearity, and range of calibration to determine molecular masses of chitinases
Summary
The endospore forming genus Bacillus is one of the most widely researched and commercialized biocontrol agents (Paulitz and Belanger, 2001) Their biocontrol mechanisms include production of antibiotics and extracellular hydrolytic enzymes such as chitinase, laminarinase, lipase, and protease. Zymography in protein chemistry provides reliable identification of enzymes like chitinase, based on the molecular mass of their active forms after gel electrophoresis (Grudkowska et al, 2013). It is based on visualization of areas where the specific substrate is digested by the enzyme of interest. The present study attempts to identify and characterize chitinase from newly isolated B. subtilis MBCU4 using zymography technique to enhance resolution and for molecular size determination, and evaluate the antifungal activities of the isolated chitinase against common fungal pathogens
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