Research Article
Effects of Nutrients on the Extracellular Lipase Production by Mutant Strain of Rhizopus oligosporous TUV-31
Government College Lahore, Pakistan
Athar Hussain
Government College Lahore, Pakistan
Lipases are hydrolytic enzymes extensively used in the hydrolysis of fat as well as in the synthesis of glycerides. Fungi like Aspergillus, Rhizopus, Mucor, Geotrichum, Penicillium, Candida and Syncephalostrum are potential sources of lipase production (Toide et al., 1996). Microbial lipases are both extracellular and intracellular (Haq et al., 1998; Pastou et al., 2000). Among different fungi, Rhizopus oligosporous shows higher productivity of lipase as reported by Toshihiko et al. (1989). Although lipases can be produced both by submerged and solid-state fermentation (Huang et al., 1995; Rivera et al., 1991; Pandey, 1992). Solid-state fermentation gave better results (Christen et al., 1998).
Lipases are extremely versatile enzymes, showing many interesting properties for industrial applications. They are currently applied in detergent formulations, for the removal of fatty stains (Egmond et al., 1996). Lipases can be applied in fur processing (Rezanka, 1991). The hydrolytic behaviour of lipases is applied in oil and fat hydrolysis (Bell et al., 1981). Lipases are used in bakery products for enhancing taste and aromatic properties of milk, butter cheese and yogurt (Mohsen et al., 1986). The specificity of lipases makes it possible to obtain compounds, which are difficult to prepare by conventional chemical methods (Rezanka, 1991). All these commercial applications make it a potential subject for this study.
Materials and Methods
Inoculum preparation: Spore suspension was used as inoculum. In this study, 5−7 days old culture was used and the spore suspension was prepared in sterilized 0.005% Monoxol. O.T. (Di-Octyl ester of sodium sulphosuccinic acid). Haemocytometer slide was used for counting of spores. Production of fungal lipase was studied by solid-state fermentation (Korn and Fujio, 1997).
Lipase activity: Lipase activity in the fermented meal was determined titrimetrically on the basis of olive oil hydrolysis, as reported by Kundu and Pal (1970).
Results and Discussion
Effect of carbon sources: Different carbon sources were used for extracellular lipase production by mutant strain of Rhizopus oligosporous Tuv-31 (Table 1). One percent additive was added to the substrate of almond meal. Glucose, sucrose, starch, olive oil, mustard oil and Tween 80 were used as carbon sources. Tween 80 was found to be the best carbon source, as compared to others because it gives maximum lipase production (61.87μ/g), since it was miscible with water and did not generally inhibit fungal growth (Martinez et al., 1993). Handelsman and Shoham (1994) also reported the production of extracellular lipase by addition of Tween 80 as best carbon source. Our studies are in accordance with Nahas (1988), highest yields of enzymes were obtained when Tween 80 were the carbon sources.
Effect of different concentration of Tween 80: Variation in the concentration (0.1-2%) of Tween 80 was also effective against lipase production. Maximum lipase level (65.17 μ/g) was obtained at 0.5% concentration of Tween 80 as it provided optimum amount of carbon (Table 2). Enzyme level however decreased with further increase in Tween 80 concentration. It might be due to the increase in fatty acid accumulation through hydrolysis of substrate, suppressing lipase synthesis. Sidhu et al. (1998) used 0.5% Tween 80 for the production of extracellular lipase. Espinosa et al. (1990) also reported that Tween 80 exerted a positive effect on enzyme production in a range that goes from 0.02 to 2.00%.
Effect of organic nitrogen sources: Different organic nitrogen sources were used for extracellular lipase production by mutant strain of Rhizopus oligosporous Tuv-31 (Table 3). For this purpose 1% of different organic nitrogen sources such as urea, peptone, yeast extract, soybean meal, nutrient broth and casein were added to the substrate. Soybean meal gave maximum (69.65μ/g) production of lipase, was found to be the best organic nitrogen source. As soybean meal supported good growth of Rhizopus oligosporous, thus there was an increased enzyme production. Results are also in accordance with Huang et al. (1995) and Nahas (1988).
Effect of inorganic nitrogen sources: Different inorganic nitrogen sources were used for extracellular lipase production by mutant strain of Rhizopus oligosporous Tuv-31. (NH4)2SO4 gave maximum production (70.97μ/g) as compared to other inorganic nitrogen sources (Table 4). It might be due to the optimum growth of mycelium when (NH4)2SO4 was used as nitrogen source.
Table 1: | Effect of different carbon sources on the production of lipase by mutant strain of Rhizopus oligosporous Tuv-31 |
Table 2: | Effect of different amounts (%) of Tween 80 on the production of lipase bymutant strain of Rhizopus oligosporous Tuv-31 |
Table 3: | Effect of different organic nitrogen sources on the production of lipase by mutant strain of Rhizopus oligosporous Tuv-31 |
According to Gao and Breuil (1995), ammonium sulphate gave the best lipase production. 0.3% concentration of (NH4)2SO4 was found to be the best for lipase production by Tuv-31.
Table 4: | Effect of different inorganic nitrogen sources on the production of lipase by mutant strain of Rhizopus oligosporous Tuv-31 |
Table 5: | Effect of different amounts of ammonium sulphate (NH4)2SO4 on the production of lipase by mutant strain of Rhizopus oligosporous Tuv-31 |
Table 6: | Effect of addition of different amounts (%) of egg yolk on the production of lipase by mutant strain of Rhizopus oligosporous Tuv-31 |
Effect of different amounts of ammonium sulphate: The data of Table 5 shows that the effect of different amounts (0.1-0.5%) of (NH4)2SO4 on the production of lipase. Maximum lipase production (74.80μ/g) was observed when 0.4% (NH4)2SO4 was added in the substrate as an additional inorganic nitrogen source. Ammonium sulphate (0.4%) may fulfill the nutritional needs of organism, presumably because the enzyme activity was associated with cell growth (Handelsman and Shoham, 1994).
Effect of addition of egg yolk: Effect of different concentration (0.5-2.0%) of egg yolk (v/w) has been used (Table 6). 1.5% (v/w) egg yolk gave the maximum production (76.69 μ/g) of lipase. As egg yolk contained 32.5% lipids, which include cholesterol as well as glycerides. Similarly an augmenting effect of fat and cholesterol on lipase production has been reported by Valero et al. (1988). They reported an increase in growth and lipase secretion in the presence of fat and sterol.