Abstract
An alkalophilc and thermophilic Bacillus sp. BHA that produced a thermostable alkaline protease was isolated from decaying protein substrates. The isolate was found to grow in pH range 7 - 11 with an optimum pH 9.0 and temperature up to 55℃. The activity of alkaline protease of Bacillus sp. BHA (68.98 APU/ml) was found higher than the standard strains of Bacillus amyloliquefaciens MTCC 610 (8.98 APU/ml) and Bacillus subtilis MTCC 8349 (12.14 APU/ml, used in this study, and was comparable (68.98 APU/ml, equivalent to 30.38 APU/mg) to the activity of the commercially produced standard protease procured from Novo Nordisk, Denmark (30.35 APU/mg). Hence, the proteolytic activity produced by this isolate was further investigated in batch and fed-batch process. Sucrose was the best carbon source for the production of protease activity by that isolate. Different organic nitrogen sources (casein, peptone and beef extract) at 1% (w/v) with varying levels of sucrose (1% - 4% w/v) initially repress enzyme synthesis. The duration and extent of repression decreased with increased concentration of sucrose. Maximum protease activity was found in basal medium with 4% (w/v) sucrose and 1% (w/v) yeast extract. Yeast-extract was thought to be an inducer of enzyme synthesis. Further, the basal medium was unique with respect to the enzyme production, as protease production was growth associated with the peak enzyme production being detected at the time of maximum growth. Interestingly, a rise in 34.2% (104.86 APU/ml) of protease activity was detected at incubation temperature of 50℃ and when culture filtrate was assayed at 60℃, signifying a high temperature stability of the produced protease by this isolate. Additional studies on the enzyme characterization were resulted in recognition of highly significant properties of the activity towards casein at pH 9.0 and stability at high temperature with retention of 96% the enzyme activity at 60℃. The parametric study under feed intervals had enabled improvement in the maximum protease activities attainable from batch cultures in excess of 21.78% and 26.32% via two feeding strategies. A small continual increase in enzyme activity (132.46 APU/ml during 24 h - 120 h) and enhancement in protease production in excess of 36.84% was observed by fed-batch process than the batch experiment.
Highlights
The global demand for industrial enzymes is estimated to be approximately $2 billion
Enzymes in laundry and cleaning products account for 40% of global industrial enzyme market of these alkaline proteases used primarily as detergent additive represents about 60% of the global detergent enzyme market for enzyme accounting for approximately 19% of total enzyme demand and more than 25% of industrial enzymes sales [4,5,6]
Erlenmeyer flasks (500 ml) having 50 ml of the medium consisted of basal salt solution (g/l); MgSO4·7H2O 1, CaCl2·2H2O 0.5 with sucrose 20 and yeast extract 5 was used for cultivation of cells in batch culture prior to initiation of fed-batch operation. 5% (w/v) inoculum incubated at 37 ̊C, 200 rpm for 48 h was used
Summary
The global demand for industrial enzymes is estimated to be approximately $2 billion. Protease constitutes one of the most significant groups of industrially important enzymes accounting for 60% of the total enzymes market [2]. Microbial alkaline proteases are reported to share the following properties-A serine residue at the active site, esterolytic activity, alkaline pH optima and sensitivity to organophosphorous reagents such as phenyl-methyl sulphonyl fluoride (PMSF) or Di-isopropyl fluorophosphates (DIFP), generally not inhibited by EDTA. They are not activated by metal ions or reducing agents. BHA under alkaline and high temperature favored the alkalophilic and thermostable characterization of the produced protease
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