Eco-Friendly Alkaline Protease: Production and Application in Detergents
Abstract
An alkalophilic, halotolerant bacterial strain ASM1 isolated from agricultural soil was found to be capable of producing extracellular protease enzyme. Proteolytic strain was identified as Bacillus cereus and nucleotide sequence has been submitted in NCBI database under accession number KJ600795. Optimum enzyme production in terms of specific activity 9.58 U/mg of total protein was obtained at 35°C; pH, 9.0; 1 % glucose as C-source and 35 g/l beef extract as N-source after 48 hours of incubation in a defined medium inoculated with 2% inoculum size. Bacterial isolate was capable of tolerating up to 12.5% NaCl without requiring salt for physiological activities. Bacterial crude enzyme was purified by 6 folds with 25% yield and specific activity of 57.9 U/mg protein by two step purification i.e. ammonium sulfate precipitation and gel-filtration chromatography. Thermostability studies revealed retention of 60% proteolytic activity upto 55°C. Moreover enzyme remained stable in the pH range of 6-11. PMSF (phenylmethylsulfonyl fluoride) inhibited enzyme activity categorizing the enzyme as a serine protease. Enzyme remained stable in presence of 8 different metals, however activity declined in the presence of 20 mM Fe2+ ions. Enzyme retained substantial stability in the presence of solvents, surfactants, commercially available detergents, and NaCl. Enzyme exhibited efficacious de-staining of fixed blood stains in the washing test at room temperature, without requiring additional energy. This particular type of protease enzyme is of immense importance due to its alkaline-halotolerant profile at mesophilic temperature range which is a great deal for revolutionizing detergents’ industry.
Introduction
Proteases are hydrolytic enzymes that catalyze hydrolysis of proteins by addition of water across the peptide bonds into smaller polypeptides and free amino acids (Beg and Gupta, 2003). Proteases are ubiquitous in nature playing important physiological roles, in all domains of life (Barrett et al., 2001; Burhan et al., 2003). Microbial proteases constitute one of the three commercially significant groups of enzymes, contributing more than 60% of share in the global enzyme market (Chu, 2007; Huang et al., 2003; Jayakumar et al., 2012; Jon, 2008). Proteases constitute a very diverse group of biocatalysts with members having different substrate specificities; nature of catalytic sites; evolutionary relationship in amino acids’ sequence; catalytic mechanisms and varying activity-stability profiles on broad range of temperature and pH (Rai and Mukherjee, 2010; Rao and Narasu, 2007; Rawlings et al., 2012).
Bacterial bio-factories hold much more temptation for exploitation than other enzyme producers due to the ease of handling and production in a limited time and space with less complicated purification steps. Besides that bacteria are susceptible to artificial genetic manipulations and are able to survive under diverse and extreme environmental conditions (Burhan et al., 2003; Khademi et al., 2013; Rao and Narasu, 2007; Rao et al., 1998). Genus Bacillus is considered as the most significant source of bulk amounts of industrially important neutral and alkaline proteases which are highly stable at temperature and pH extremes (Beg and Gupta, 2003; Gupta and Khare, 2007; Venugopal and Saramma, 2006; Yang et al., 2000).
Proteases active and stable in the alkaline pH range are referred as alkaline proteases. Active site of alkaline protesaes may contain serine residues or metal ions (Khan, 2013). Alkaline proteases with serine residues on catalytic site are referred as Serine Alkaline Proteases (SAPs). Optimum pH for production and activity of serine proteases ranges between pH 7.0-12.0. Some SAPs are endowed with additional characteristic of halotolerance which makes them perfect tool for utilization in various industrial processes (Joo and Chang, 2005; Joshi et al., 2007; Maurer, 2004; Purohit and Singh, 2011; Singh et al., 2010). Stability studies in presence of salts, metal ions, surfactants, oxidants and solvents help in prospecting probable use of enzyme in industry (Gupta and Khare, 2007; Joo et al., 2003; Zambare et al., 2014). Alkaline proteases are majorly used as additives in the commercial detergents (Maurer, 2004). Different industries especially leather and detergent industries require efficacious, environment friendly and economical approaches for degradation of unwanted proteins (Hameed et al., 1996; Huang et al., 2003; Wang et al., 2007).
Protease production can be enhanced by optimization and manipulation of fermentation methods and conditions; cloning and modulation of genes expression and protein engineering (Gupta et al., 2002a; Gupta et al., 2002b). To achieve high protease production rates, understanding of strategies for protease production and broad range application in the industrial processes hold central importance. Aim of this study was to isolate, characterize and optimize proteolytic strain present in soil biome for enhanced enzyme production. Moreover, biochemical characterization and stability studies of the enzyme were aimed to determine possible eco-friendly application of enzyme in detergent industry.
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