Production of Alkaline Protease by Solvent-Tolerant Alkaliphilic Bacillus circulans MTCC 7942 Isolated from Hydrocarbon Contaminated Habitat: Process Parameters Optimization
In the present investigation, a newly isolated organic solvent-tolerant and alkaliphilic bacterial strain was reported from a hydrocarbon (gasoline and diesel) contaminated soil collected from the petrol station, Shirpur (India). The strain was identified as Bacillus circulans MTCC 7942, based on phenotype, biochemical, and phylogenetic analysis of 16S rRNA gene sequence. The capability of Bacillus circulans to secrete an extracellular, thermostable, alkaline protease and grow in the presence of organic solvents was explored. Bacillus circulans produced maximum alkaline protease (412？U/mL) in optimized medium (g/L): soybean meal, 15; starch, 10; KH2PO4, 1; MgSO4·7H2O, 0.05; CaCl2, 1; Na2CO3, 8; pH 10.0 at 37°C and 100？rpm. The competence of strain to grow in various organic solvents—n-octane, dodecane, n-decane, N,N-dimethylformamide, n-hexane, and dimethyl sulfoxide, establishes its potential as solvent-stable protease source for the possible applications in nonaqueous reactions and fine chemical synthesis. 1. Introduction The world enzyme market will grow up to $2.1 billion by 2016 [1]. Of these, alkaline protease alone accounts for 40% of the total world enzyme production, with growing applications in bakery, brewing, detergent, diagnostic reagents, feeds modification, leather finishing, laundry additives, pharmaceuticals, peptide synthesis, silk, silver recovery from X-ray/photographic film, soy processing, and waste treatment [2]. Majority of alkaline proteases produced by mesophilic microbes are effective in a narrow range of pH, temperature, and ionic strength, accordingly unsuitable for commercial purpose under demanding industrial conditions. Hence, alkaline proteases secreted by alkaliphilic bacteria are of potential interest in detergent industry due to their ability to withstand extremes of temperature (40–60°C), alkaline pH (9–11), high salt concentration, and other harsh conditions [3]. Alkaline protease that catalyses peptide bond formation in the nonaqueous media has greatly expanded potentials in green chemistry for fine chemical synthesis [4]. Alkaline proteases have expanded new possibilities such as (i) shifting of thermodynamic reaction equilibrium to favour synthesis, (ii) enhancement of bioavailability of hydrophobic substrates, (iii) total inhibition of water-dependent side reactions, (iv) alteration in enantioselectivity of reaction, (v) improving thermal stability, and (vi) facilitating the product recovery. Thus, the stability of proteases in organic solvents offers certain merits like (i) synthesis of various compounds, (ii)
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