Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena Muhammad
Ibrahim Rajoka
Keywords: Carbohydrates, cellobiohydrolase, Cellulomonas flavigena, exo-glucanase, enthalpy, entropy, fermentation, induction.
Cellulases and xylanases are concomitantly produced by soil cellulolytic fungi and bacteria in helping mobilization of soil organic matter to provide carbon and energy source to soil micro flora. These enzymes play a key role in preparing plant protoplasts for genetic manipulation for crop improvement. These are also implicated in baked foods, fruit processing, cloth cleaning, preparation of dehydrated vegetables and food products, preparation of essential oils, flavours, pulp and paper production, starch processing, preparation of botanical extracts, jams, baby foods, juices, degumming coffee extracts, improved oil recovery, yeast cell wall destruction, waste treatment, textile refining and preparation of feed for farm animals (Kubicek et al. 1993). Cellulases are generally considered to be synthesized in the presence of inducers. The inducers of cellulase formation are cellulose, cellulose derivatives, cellobiose, sophorose, xylan, pectin and lactose (Bahkali, 1992). The regulation of these enzymes synthesis is under the control mechanisms of induction and repression (Gadgil et al. 1995; Spiridonov and Wilson, 1998). Cellulase production
has attracted a world-wide attention due to the possibility of using
this enzyme complex for conversion of abundantly available renewable
lignocellulosic (LC) biomass for production of carbohydrates for
numerous industrial applications (Gadgil et al.
1995). Different fungi and bacteria have been used for production
of cellulases and xylanases. Cellulase system of C. flavegina
shows high activities of cellulases and xylanases during growth
on cellulosic substratesand end-product inhibition and thermal stability
at room temperature of Extensive screening of potential cellobiose (CBH) or filter paperase (FPase) inducers have shown that when Cellulomonas flavigena strainis grown in media containing monomeric saccharides, dimeric saccarides, carboxy methyl cellulose (CMC) or a-cellulose, the strain of C. flavigena has a shorter lag period and doubling time when grown on monosaccharides namely arabinose, xylose, glucose, galactose and fructose than those on disaccharides or polysaccharides. CBH is mainly present in the intracellular preparation. The organism synthesizes CBH to a measurable level from disaccharides, otherwise only basal level of this enzyme was produced. Similarly FPase is produced in very small quantity below the detection limits of the assays or can not be quantified due to the presence of soluble sugars which require dilution resulting in dilution of enzyme which can not be measured under the assay conditions. Following growth on mono- and di-saccharides, cell extract containing cellobiosidase are collected and assayed for cellobiosidase activity. Carbon sources which support rapid growth measured as qS (specific substrate utilization rate) are the worst repressors of enzyme synthesis. a-Cellulose supportsthe maximum activity, followed by other substrates. These results implicate that the quantity of cellobiosidase or FPase produced depended substantially on the type of innducer used. Among monosaccharides, only fructose supports production of greater quantity of cellobiosidase (Nochureet al. 1993). Among disaccharides, cellobiose is the best inducer, followed by maltose and lactose. b-Linked disaccharides namely cellobiose and lactose are better inducers than a-linked disaccharide, maltose. From soluble carbohydrates, low levels of CBH, is attributed to the organism's low requirement of the enzymes for growth and metabolism. There is greater enhancement in cellobiohydrolase productivity following growth on a-cellulose over that obtained from cellobiose. FPase gets immobilized on cellulosic substrates (10 ± 0.25%) and can be successfully eluted with Tween 80. Similarly some cells get adsorbed on the surface of insoluble substrates. These cells contain cellobiosidase activity and the values of cellobiosidase are also compensated to contain cell-bound cellobiosidase activity. During growth of the organism on different cellulosic substrates, reducing sugars accumulate in the growth medium as unmetabolized principles during enzymic degradation and induce FPase (Bagga et al. 1989; Okeke and Paterson, 1992). On the medium containing soluble carbon sources viz. cellobiose and CMC, the organism synthesizes low activities of FPase while it synthesizes a high level of enzyme during growth on cellulosic substrates. This is attributed to amount of carbohydrates accumulated from each carbon sources in the culture broth. Those substrates which release more carbohydrates were stronger repressors. Maximum specific activity of cellobiosidase and FPase of 4.0 IU/mg protein each on a-cellulose and 1.65 IU/mg protein on CMC medium and kinetic parameters of FPase formation are substantially higher than that supported by a fungus (Wood, 1988) and haemophilic organisms (Spiridonov and Wilson, 1998; Kalogeris et al. 2003). Among nitrogen sources, nitrates are good sources for synthesis of these enzyme components. Studies have led to conclude that the carbohydrate and nitrogen sources play a vital role in the production of CBH or FPase by Cellulomonas. Alpha-cellulose is the best carbon followed by LC substrates but the former is an expensive substrate, therefore, LC biomass could be used for commercial production of cellulases. The test organism may serve as a potential source of FPase. Efforts have been made to increase the enzyme production by manipulating these aspects only. The scope to increase the production by the use of genetical, biochemical and microbial engineering techniques to make use of full potential of this organism are to be studied. Gamma ray mutation followed by chemical mutagenesis may give mutant derivatives for improved cellulolysis (Gadgil et al.1995; Rajoka et al. 1998). Thermodynamic studies (Aiba et al. 1973) suggest that the cell system exerts defence (probably due to chaperone activity) against thermal inactivation.
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