Rhizopus microsporus var oligospous was procured from the institute of Microbial Technology, Chandigarh and was maintained at 4°C on potato dextrose agar (PDA). Spore suspension was made from five days old cultures that had been grown on PDA slants at 30°C. It was prepared by suspending the spores from one tube in 10 ml of sterilized distilled water containing 0.1ml Tween–80. The spore count was adjusted to10⁵ spores ml^-1 (Smith et al, 1996). One ml of inoculum was used per flask to carry out submerged fermentation.

The enzymes were produced using basal medium (Manomani et al, 1983). The basal medium contained the following ingredients (g l ^-1): (NH₄)SO₄ - 1.4, CO(NH₂)₂ - 0.3, KH₂PO₄ - 4.0, K₂HPO₄ – 0.84, _{CaCl2.2H2O – 0.3, MgSO4.7H2O – 0.3, FeSO4.7H2O – 0.005, MnSO4.H2O – 0.00156, ZnCl2 – 0.00167, Peptone 0.25, Yeast extract – 0.10, Rice Bran – 4%. All the components of the basal medium except rice bran were dissolved in distilled water. Flasks having 250 ml capacity were taken and 50 ml of this solution was poured in each flask. To each flask 2.0 g of rice bran was added (Thenawadjaja et al, 1990) and autoclaved at 1.1kg/cm² for 20 minutes. Flasks were cooled and inoculated with 1ml of the spore suspension. The inoculated flasks were incubated at 30°C on a rotary shaker cum BOD incubator revolving at 150 rpm (Seyis and Aksoz, 2005; Meenaksui et al, 2005). After 96 hours of growth, the contents of each flask were centrifuged at 4°C and this supernatant was used as the source of crude enzyme (Nehra et al, 2002).}

Protease was isolated and partially purified with isopropanol (Manomani et al, 1983). The mold filtrate (20 ml) was chilled at 4°C in a refrigerator and 25 ml of chilled isopropanol was added to it. The precipitates were separated by centrifugation. The precipitates were dissolved in 5 ml of 0.05 M citrate phosphate buffer having pH 5.2 (Rajinder, 1992) to obtain partially purified enzymes.

The test tubes containing 1ml casein solution and 1ml enzyme extract were incubated at 60°C for 10 minutes. After 10 minutes 3 ml TCA was added to each test tube to stop the reaction. The precipitates formed were centrifuged at 5000 rpm. The protein concentration in the supernatant was determined by Lowry's method (Lowry et al, 1951). Optical density was recorded at 525 nm. Protease activity was expressed as Tyrosine equivalents using the standard curve prepared for measurement of proteins under same set of conditions as described above using standard solution of Tyrosine.

Enzyme activities of the partially purified enzyme was expressed in International Units (IU). One IU was defined as one mol of tyrosine equivalents released per minute per ml under the following assay conditions using tyrosine standard curve (Silva et al, 2005). Appropriate dilution factors were used during the estimation of the enzymes.

The effect of initial culture pH on the enzyme production was investigated by adjusting the initial pH of the basal medium in the range of pH 5-8. To examine the effect of temperature on enzyme production the inoculated culture flasks were incubated at different temperature varying between 15-40°C and then the enzyme activities were determined by using the partially purified enzymes. The effect of inoculum size based on the number of spores was examined using the spore concentration of 1x10³, 1x10⁴, 1x10⁵ and 1x10⁶ spore ml^-1 of Tween-80 (Kheng and Omar, 2005) for making the spore suspension. The flasks having the sterile basal medium were inoculated with the spore suspension and were incubated at 30°C on a BOD shaker cum incubator for 96 h and then the partially purified enzymes were used for the determination of the enzyme activities.

To test the stability of the enzymes, the CMCase and xylanase were incubated at different temperatures ranging from 40-80°C for 60 min (Silva et al, 2005). The reaction was stopped in ice cold water and the remaining activity was measured under standard assay conditions.

The effect of different temperatures ranging from 15-40°C on the production of fungal enzyme protease by Rhizopus microsporus var oligosporus was studied. The basal medium was prepared, inoculated with the respective spore suspensions and was incubated at different temperatures ranging from 15°C-40°C. At 15°C, all enzyme activities were low which showed a gradual increase with the increase in temperature at 30°C. Further increase in temperature resulted in decrease in enzyme production indicating that 30°C was the optimum temperature for the maximum production of the enzymes (Table 1). Less activity of fungal enzymes at low temperature (15-25°C) and at high temperature (35-40°C) as compared to 30°C might be due to slow growth at low temperature and inactivation of the enzyme at high temperature. This observation was in accordance with the observation of Tunga et al (1999) who reported 30°C to be the optimum temperature for protease production by Rhizopus oryzae.

Figure 1

Table 1: Effect of temperature on the production of Fungal Enzymes

Medium – Basal Medium (Manomani et al, 1983)

Incubation time- 96 h

Temperature – 30± 2°C

pH - 5.5

The data represents the mean of three determinations each.

Effect of different pH values ranging from 4.0 to 9.0 on the production of fungal protease by Rhizopus microsporus var oligosporus was studied by altering the pH of the basal medium that was used for carrying out the submerged fermentation. Production of the enzyme protease increased with increase in pH value, reaching the maximum at pH 5.5, followed by a gradual decrease thereafter (Table 2). The decrease or increase in activities at different pH values than the optimum pH might be due to decreased production of the mycolytic enzymes.

Figure 2

Table 2: Effect of pH on the production of fungal enzymes

Medium – Basal Medium (Manomani et al, 1983)

Incubation time- 96 h

Temperature – 30± 2°C

pH - 5.5

The data represents the mean of three determinations each.

The effect of inoculum size based on the number of spores was examined using the spore concentration of 1x10³, 1x10⁴, 1x10⁵ and 1x10⁶ spores ml^-1 on the production of fungal protease. It was found that the increase in inoculum size resulted in rapid increase in enzyme production (Table 3) due to fast degradation of the substrate. Similar observations were reported by Raimbault and Alazard (1980) who showed that maximum enzyme production and declination was achieved much faster.

Figure 3

Table 3: Effect of inoculum size on production of fungal enzymes

Medium – Basal Medium (Manomani et al, 1983)

Incubation time- 96 h

Temperature – 30± 2°C

pH - 5.5

The data represents the mean of three determinations each.

The enzyme activity did not show a further increase at a spore count of 1x10⁶ spores ml^-1 because of the limitation of the substrate. Hence, based on this the inoculum size of 1x10⁵ was used for inoculation of basal medium.