Screening, Isolation, Purification and Characterization of Thermophilic Amylase & Haloalkaliphilic Lipase

Abstract

Flavobacterium thermophilum, a thermophilic bacterium was obtained from MTCC and Halomonas shengliensis, a haloalkaliphillic bacterium isolated from saline soil of Khambhat was obtained from Dr. Vijay Kothari. Flavobacterium thermophilum is a Gram-Negative, unusally elongated bacillus and Halomonas shengliensis are Gram-Negative, short rods. The organisms gave positive results upon screening for extracellular Amylase and Lipase, respectively. Amylase activity was initially confirmed through starch-iodine assay and later confirmed through zymography. Quantitative estimation of amylase was carried out through modified DNSA assay. Partial purification of Amylase was done by anion-excahnge chromatography. Lipase screening was carried out using modified HM (Heterotrophic microorganism) media. Quantitative estimation of lipase activity was carried out spectrophotometrically by monitoring p-nitro phenol released by lipase from hydrolysis of p-nitro phenyl acetate. Purification of lipase was carried out by anion-exchange chromatography. Partial characterization of lipase was done. Molecular weight of lipase was found to be about 41 kDa. Lipase activity as a function of temperature showed that 80˚C is the optimum temperature for its activity, a considerably high value, considering the fact that the organism is not a thermophilic one. PMSF was found to have a major inhibitory effect on lipase activity, indicating that it has a serine residue in its active site. Further characterization of lipase is under progress.