Development of Bioprocess for Improved Production of Alkaline Protease by Mutant Strain of Aspergilllus flavus in Solid State Fermentation using Agricultural Wastes

摘要

A mutant of Aspergillus flavus MTCC 9952, developed by chemical mutagenesis, produced 37% higher alkaline protease over wild type after optimization of fermentation conditions. The agro-industrial wastes wheat bran and corn cob in the ratio of 1:1 with initial moisture content of 50% were found to be most suitable substrates for protease production under solid state fermentation. Maximum alkaline protease was produced when culture was incubated at 37°C with initial medium pH 9.0 in 48 h with inoculum size of 1x108 spores/gram IDS. Supplementation of fructose (200 mg/g IDS) as carbon and malt extract (100 mg/g IDS) as nitrogen source with other ingredients like di-ammonium hydrogen phosphate (20 g/g IDS) and CuSO4 (0.5 mg/g IDS) considerably increased the protease production. An inexpensive and readily available agro by-product can be used for production of industrially important enzymes. Introduction Proteases are important group of enzymes from an industrial perspective and cater for the requirement of nearly 60% of the world enzyme market (Kalisz, 1998). Alkaline proteases execute a large variety of complex physiological, metabolic and regulatory functions which is evident from its occurrence in all forms of living organisms (Wandersman, 1989). Furthermore, these enzymes are of great interest from a biotechnological angle and are being investigated not only in scientific areas like protein chemistry and protein engineering, but also find application in detergent, food, pharmaceutical and tannery industries (Kumar and Takagi, 1999). In the last few decades, the exponential increase in the application of proteases in various fields instigated the research in both qualitative improvement and quantitative enhancement of proteases. To achieve commercially viable production levels, it requires strain-improvement programs as the production level of the enzymes in naturally occurring strains is, in most cases, too low for commercial exploitation (Verdoes et al., 1995). In recent years the genetic engineering and targeted mutagenesis have been emerged as attractive tool for strain improvement, though the classical mutagenesis methods (physical and chemical) in combination with mutant selection and medium optimization forms a prevailing strategy to obtain overproduction of the enzyme. On an industrial scale, extracellular alkaline proteases are produced using complex media containing cost effective substrates responsible for 30 to 40% of the production costs (Kumar and Parrack, 2003). For potential industrial applications, organisms growing on economical substrates are requisite (Shikha et al., 2007). To this end, solid state (substrate) fermentation (SSF) offers utmost possibilities wherein agro-industrial wastes (wheat bran, rice bran, mustard oil cake, coconut oil cake etc.) are used for the production of value added products by using microorganisms. In addition, SSF poses several economic and engineering advantages over submerged fermentation (SmF) such as (a) low moisture content, preventing bacterial contamination, (b) simplicity of equipment (Hesseltine, 1987), (c) amenability to use upto 20-30% substrate, in contrast to the maximum of 5% in SmF process (Pamment et al., 1978) (d) high volumetric productivity (e) lower downstream processing charges (Pandey et al., 2000). Keeping in view the incessant escalating demand of alkaline protease, the present study has been focused on the development of a potential mutant strain and optimization of fermentation parameters in SSF to obtain elevated level of protease production. ?Experimental: Materials and Methods Microorganism and culture conditions A protease producing fungal strain was isolated in our laboratory from the soil sample of dairy industry and identified as Aspergillus flavus MTCC 9952 at Institute of Microbial Technology, Chandigarh, India. The culture was routinely maintained on malt extract-glucose-agar slants (containing g/L: malt extract, 20; glucose, 20; KCl, 0.5; MgS