Production Of Protease By Submerged Fermentation Using Rhizopus Microsporus Var Oligospous
L Sarao, M Arora, V Sehgal, S Bhatia
Keywords
protease, rhizopus microsporus var oligospous, submerged fermentation
Citation
L Sarao, M Arora, V Sehgal, S Bhatia. Production Of Protease By Submerged Fermentation Using Rhizopus Microsporus Var Oligospous. The Internet Journal of Microbiology. 2009 Volume 9 Number 1.
Abstract
Production of protease via submerged fermentation using
Introduction
Enzymes are proteins composed of 20 amino acids. They are produced by cellular anabolism, the naturally occurring biological process of making more complex molecules from simpler ones. Enzymes increase the rate of biochemical reaction and decrease the time for those reactions to reach equilibrium. They are not consumed in the chemical reaction, and thus their action is catalytic (Nielson, 1991). Enzymes used in food processing are produced by bacteria, fungi, higher plants and animals. Most of the organisms that produce enzymes are fungi. Mycolytic enzymes are primarily originated as a desire of microbiologists to understand the chemical nature and ultra structure of fungal cell wall. A number of enzymes can be classified under mycolytic enzymes viz; protease, cellulase, xylanase etc.
Protease enzyme conducts proteolysis by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain. Proteases are the most valuable commercial enzymes and account for 60% of the total enzyme market (Rao
Materials and methods
Microorganism and inoculun preparation
Shake flask cultivation media
The enzymes were produced using basal medium (Manomani
Partial purification of fungal protease
Protease was isolated and partially purified with isopropanol (Manomani
Protease activity
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
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
Factors affecting enzyme production
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.
Testing of enzyme stability
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
Results and discussions
Effect of temperature
The effect of different temperatures ranging from 15-40°C on the production of fungal enzyme protease by
Medium – Basal Medium (Manomani
Incubation time- 96 h
Temperature – 30± 2°C
pH - 5.5
The data represents the mean of three determinations each.
Effect of pH
Effect of different pH values ranging from 4.0 to 9.0 on the production of fungal protease by
Medium – Basal Medium (Manomani
Incubation time- 96 h
Temperature – 30± 2°C
pH - 5.5
The data represents the mean of three determinations each.
Effect of inoculum size (spore density) on the production of fungal enzymes
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.
Medium – Basal Medium (Manomani
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.