SCREENING AND CHARACTERIZATION OF AN EXTRACELLULAR PROTEASE ENZYME FROM A HALOPHILIC BACTERIUM

Abstract

In aqueous medium Proteases hydrolyze peptide bonds but in non-aqueous they catalyze the reverse reaction and catalyze the synthesis of peptides. For Industrial applications organic solvent stable proteases are required. Most of the salt stable halophilic protein can work well in non-aqueous conditions. Thus there is a continuous demand of new microbes which can produce novel enzymes. In the present study four soil and one salt samples were collected from Sambhar salt lake Rajasthan. Complex halophilic medium with 4 M NaCl at pH 9.0 and 45 ºC were used to study bacterial diversity of the samples. Sixteen purified cultures were obtained from four soil and one salt samples. These isolates were colorless transparent to red orange in color and showed rod, cocci and pleomorphic type of cellular morphology when seen under 100 X magnification of phase contrast microscope. On the basis of salt requirement isolates were extreme halophiles, borderline extreme halophiles and moderately halophilic. All the isolates showed better growth in NaCl medium as compared to that in KCl medium. All the isolates can grow at a wide range of temperature and pH and some of them had potential to produce industrially important hydrolytic enzymes. Extremely halophilic and borderline extremely halophilic isolates showed resistance towards many antimicrobial agents while all the moderately halophilic isolates were found to sensitive for them. Moderately halophilic isolates were found to utilize more carbon sources in comparison to extremely halophilic and borderline extremely halophilic isolates. Five isolates out of sixteen were found to produce extracellular protease. On the basis of maximum protease activity isolate MSL-1 was selected and further characterize. 16S rRNA gene sequence of MSL-1 showed 98 % similarity with Halobiforma lacisalsi, 97 % with Hbr. haloterrestris and 96 % with Hbr. nitratireducens. Thus the strain MSL-1 phylogenetically belongs to genus Halobiforma and named as Halobiforma sp. strain BNMIITR. It had six different types of lipids with two glycolipids and one phospholipid. Major cellular fatty acids were C 14:0, C 15:0 iso, C 15:0 anteiso, C 16:0, C 17:0 iso, C 17:0. Further protease production was affected by nutritional factors and culture conditions. NaCl was found most suitable salt for protease production at pH 10.0 and 45 ºC temperature. Protease production was considerably increased in the presence of low cost agro industrial byproducts like soybean husk, soybean flour, chick pea flour and chick pea husk. Protease production was catabolically repressed by many amino acids with positive effect of proline. Protease production was reduced by many surfactants while it was completely inhibit by SDS. Enzyme purification was done by solvent precipitation followed by HIC. Enzyme was a monomer with a molecular mass 21 kDa. Protease of Halobiforma sp. strain BNMIITR can work at wide range of pH (6-12). Enzyme also showed broad range of substrate specificity (caseinolytic, esterase and elastase activities). Enzyme was very stable in presence of many metals, surfactants and oxidizing agents but activity was reduced in the presence SDS and EDTA with complete inhibition by PMSF thus a serine kind of protease. Thermal stability of the enzyme was affected by salt concentration. Enzyme was stable in polar as well as non polar solvents with considerably high stability in nonpolar solvents at 30 oC. Enzyme was stable and active in the presence of various commercially available detergents. Interestingly enzyme activity was increased in the presence of DMF and DMSO.