Screening, isolation, purification and characterization of thermophilic and halophilic extremozymes

Abstract

We have carried out screening, isolation, and characterization of four different extracellular industrially important enzymes, Amylase, Protease, Lipase and, Pullulanase from haloalkaliphilic soil organism Virgibacillus marismortui which was isolated from saline soil of Khambhat and a thermophile Thermus thermophilus. It was observed that Virgibacillus marismortui secrete proteases which was confirmed by obtaining zone of hydrolysis in milk agar plates. We also found out that maximum protease production occurs in late logarithmic and/or stationary phase. Further protease activity was confirmed by zymographs. It was observed that these organisms do not produce amylase in normal culture however when grown on a minimal media with starch as sole carbon source these organisms were able to grow by producing amylases. The extracellular amylase activity was confirmed by DNSA test and starch iodine test. The crude amylase activity was estimated by DNSA test of ammonium sulphate precipitated sample. When grown on pullulan as sole carbon source, the organism was able to grow which indicates that these organisms are able to hydrolyse pullulan and produce maltotriose which can be detected by DNSA test. After ammonium sulfate precipitation and dialysis of the sample its activity was again checked and crude activity was determined. We also tried to grow these organisms in different salt concentrations like 6%, 10%, 15% and 25% NaCl and determined effect of salt concentration on growth and enzyme production. We also carried out screening of thermophile Thermus thermophilus for production of amylase and pullulanase.