Some Studies on the Production, Purification and Characterization of Proteases from Thermophilic Actinomycetes

Abstract

Proteases are ubiquitous in nature being found in a diversity of animals, plants and microorganisms, these enzymes constitute one of the most important groups of industrial enzymes, accounting for more than 65% f the total industrial enzyme market. Proteases "- from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Six strains of thermophilic actinomycetes (MST-l, MST-2, MST-3, MST-4, MST-5 and MST-6) were isolated from desert soils of district Rahim Yar Khan (Pakistan) and screened for the production of thermophilic proteases. Strain MST -3 was selected for further studies on the basis of maximum zone of hydrolysis on skim milk agar plate and identified as Streptomyces sp. from biochemical characterization and on the basis of 16S rRNA technique .. Optimization of various parameters was done and maximum production of enzyme was obtained at 50 °e, pH 8.0 and 150 agitation speed by 5% inoculum of 96 h in submerged fermentation experiments. Sucrose was found to be the best carbon source with 190.72 U/mg protein followed by starch (22.4 U/mg) while gelatin and malt extract proved to be the best nitrogen sources with 116.l8 and 93.54 U/mg protein, respectively. L-glutamine was the best used amino acid with 259 U/mg protein for maximum production of protease while maximum growth of the organism was obtained by L -lysine. A combined effect of optimum carbon and nitrogen sources was studied in the form of twenty three combinations in the basal medium. Maximum production of thermophilic protease was recorded in Medium- 20 comprising of l.5% starch, 0.5% potassium nitrate, 0.5% malt extract, 0.3% gelatin, 0.5% sodium chloride, 0.5% casein enzymatic hydrolysate Amine A and 0.09% calcium chloride. The production of enzyme remained independent of growth of the strain during all the experiments. The fermentation of extracellular thermophilic protease was upscaled to biofermenter of 3L capacity where maximum production was observed at 192 h (338.45 U/mg protein). Enzyme production was also studied by immobilized cells of Streptomyces sp. MST -3 in alginate beads resulting in higher yields as compared to free cells. The immobilized cells were also checked for their reusability in repeated batch cultivation for five cycles, duration of each cycle was 72 h. Successful production of protease was recorded during all cycles with maximum activity at 84 h (cycle II) . After production, purification of enzyme was carried out and crude enzyme was precipitated by 50% (NH4)2S04. Precipitates so obtained were dialyzed, lyophilized and processed for purification through DEAE-Cellulose anion-exchange chromatography and Sephadex G75-120 gel filtration. Both chromatographic procedures revealed distinct peak with active proteolytic activity. Molecular weight of the purified and lyophilized enzyme was found to be 35 kDa through SDS PAGE. Characterization studies indicated that optimum pH for the activity of crude and purified enzymes were 11.0 and 12.0 with residual activities of 120.58% and 15l.3%, respectively. Maximum stabilities of crude (126.44%) and purified enzyme (175.l %) were recorded at pH 12.0 and 10.0. respectively. The optimum temperature for crude and purified enzyme was found to be 60 °C and both type of enzymes were highly stable from 20 to 60 °C with increased activities following a decline in stability at 65°C with significant residual activities i.e. 78%- (crude) and 97% (purified enzyme). The complete loss of activity by PMSF classified protease by Streptomyces sp. MST -3 as a serine protease. Lead acetate and calcium chloride showed a stimulatory effect on enzyme activity with 113% and 106.6% residual activities, respectively. Na+ (69.2%), Mn2+ (82%), Mg2+ (82%) and Ni2+ (65.66%) showed slight inhibition without a significant loss of activity. However, a reduction in enzyme activity was observed by Cu2+ (4 2.5 %), Fe3+ (36.3 5~<'), Cd2+ (4.83%), C02+ (23.l7%) and Zn2+ (40.47%). Enzyme activity was completely lost by silver (Ag2+). Stability of purified protease against different solvents was determined in reaction mixture in the presence of 10, 20, 30, 40 and 50% concentrations of each solvent at 60°C for 1 h. The protease showed a high stability and enhanced activity against 10% ethylene glycol (37l.37%), 10-20% butanol (600-105.8%),10-50% ethanol (472,268,248,223 and 196.2%), 20-30% methanol (158.3 -169%), 10-20% chloroform (158-232.3%) and 30-40% acetone (193.43-355.57%), respectively. Also a high stability was observed in case of 30% butanol (85.96%), 40% methanol (84.7%) and 30% chloroform (80.28%). The enzyme activity was increased by increasing the concentration of solvents in case of methanol, chloroform, acetone, DMSO and benzene. Reduction in protease activity was observed by different concentrations of acetonitrile, hexane, DMSO, benzene, pantene and iso-propanol at various concentrations with maximum residual activities of 36.9%, 58.54%, 35.1%, 24.7%, 30.3% and 33.23%, respectively. Protease MST-3 was found to be stable in the presence ofl, 3 and 5% SDS, Tween 20, Tween 80 and hydrogen peroxide for 1 h at 60°C, residual activity was enhanced (380.44% and 104%) with 3% and 5% SDS, respectively. An increase in protease activity was observed by increasing the concentrations of hydrogen peroxide, Tween 20 and Tween 80. Protease has the Km value as 5.26 mg/ml and V max as 1828.0 U/mL with azocasein used as substrate. The crude enzyme was found to be compatible with commercial detergents like Ariel enzymax, Brite total, Surf excel and Express power with residual activities of 79.36%, 62%, 73.57% and 77.11%, respectively, suggesting its successful use as an additive in detergent formulations. Washing test of protease preparations with detergent (Express power) resulted in the complete removal of blood stain from the cotton cloth. Crude enzyme was found to be capable ofdigestion of natural substrates like blood clot, coagulated and uncoagulated egg white. The dehairing ability of enzyme was shown by easy removal of hair from cow's skin after an incubation of 1 h recommending its utility in leather industry