Micro organisms are the important component of soil. Soil bacteria and fungi mediate soil processes such as decomposition, Nutrient mobilization and mineralization. Storage release of nutrients and water, Nitrogen fixation and denitrification in the frame of agriculture. The micro flora is the great significance because it has both beneficial and detrimental influence upon man's ability to feed himself (Gaur, 1992; Motsara et al., 1995).
Micro organisms represent an excellent source of proteases owing to their broad biochemical diversity and susceptibility to genetic manipulation (Rao et.al., 1998). Various species of Bacillus, Streptomyces and Aspergillus have been extensively studied organisms for the production of alkaline proteases (Kumar et.al., 1999 and keay, 1971).Microbial proteases are among the most important hydrolytic enzymes and have been studied extensively since the advant of enzymology (Gupta et al., 2002). These proteases represent one of the three largest groups of industrial enzymes and account for approximately 60% of the total enzyme sales in the world (Rao et al., 1998).
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Lipase is the first enzyme to be discovered by Claude Bernard in 1856 and since then researchers have obtained an increased interest in discovering potential enzyme producing microorganisms and identifying in the unique enzyme properties (Rajan, 2004). Today lipases stand amongst the most important biocatalysts carrying out novel reactions in both aqueous and non-aqueous medium. The lipases are being exploited due to their low cost of extraction, thermal and PH stability, substrate specificity and activity in organic solvent. The cheap producers of commercial lipase are Aspergillus niger, Candida cylindracea, Humicola lanuginose, Mucor michei, Rhizopus arrhizus, Rhizopus, oryzae, Rhizopus delimer, Rhizopus japonious, and Rhizopus invius.
Amylase represents a group of enzyme of great importance to food industry and other needs of life. They were also one of the first to be produced industrially by micro organisms (Reed, 1975). It is an enzyme that breaks starch down into sugar. It is produced in human saliva, where it begins the chemical process of digestion. Diastase, Amylase was the first enzyme to be discovered and isolated (Anselme and payen, 1833).Amylase are enzymes with hydrolyze starch molecules to give dextrin and progressively smaller polymers composed of glucose units [Windish, 1965].Starch is an important renewable biological resource. Bacterial α- amylases have several applications in food industry and potentially useful in pharmaceutical and fine chemical industries if enzymes with suitable properties can be found [Roychoudhury et.al 1975, Hewitt and Solomons, Hiller et. al., 1998, Lin et.al, 1998].The starch possessing industry requries the use of amylolytic enzymes at high temperature [Uguru et.al., 1997, Bolton et.al., 1997].
Collection of the soil samples:
The soil samples were collected in sterile bags. The soils were loosened and samples were taken from the region of courtrallam. 10 different soil samples were collected.
Glass wares and Culture media were sterilized by 15lbs pressure and 1210c for 15 minutes in autoclave. Pipettes were sterilized at 1800c for 2 hours in hot air oven. Inoculation work was carried out under Aseptic conditions in laminar air flow chamber and the plates were incubated at suitable temperature in the incubator.
Isolation of the organisms
Isolation of organisms from the collected soil samples were done by serial dilution technique. 1gm of soil sample was suspended in 9ml of sterile distilled water and 10-fold dilution were prepared up to 10-6 1ml of diluted suspension was inoculated into each sterile Petri plate containing 20ml of Nutrient agar. The plates were rotated for uniform distribution and were incubated at room temperature for 24 hours. The colonies developed were pure cultured and maintained in Nutrient agar slants. 20 isolates were obtained and they were designed by the numbers 1-20.
Proteases Enzyme Assay:
Protease production media were prepared. The culture was inoculated and inoculated at 370c for 24 hours. After inoculation the protease production culture media with culture were centrifuged at 10,000 rpm for 10 minutes to obtain the crude enzyme which used for enzyme activity
Casein is used as a substrate to determine the proteolytic activity for the sample. To determine the presence of protease casein solution with 0.5ml of crude sample solution and 1.25ml of tris buffer (PH7.2) and incubated for 30 minutes. After incubation 3ml of 5% trichloro acetic acid was added and incubated at 40c for 10 minutes. There the mixture was centrifuged at 5000 rpm for 15 minutes and the supernatant was collected. Take 0.5ml of supernatant. To that add 2.5ml of 0.5M sodium carbonate and incubate for 20 minutes. There add 0.5ml of Folin ciocalteus reagent. The color developed was read at 660nm in a colorimeter. A blank was prepared with out enzyme sample in the same manner.
Lipase Enzyme Assay:
Always on Time
Marked to Standard
The lipase production medium was prepared (Bora et al 2007). The medium was inoculated with 2ml of overnight culture and incubated in mechanical shaker at 370c. After 24 hours incubation the culture was centrifuged and the cell free culture supernatant fluid was used as enzyme source.
The production method (PH7.5) containing 50ml of olive oil emulsions composed of 25ml of olive oil and 75ml of 2% polyvinyl alcohol solution 4ml of 0.2M tris buffer, 1ml of 10MM calcium chloride and 1ml of enzyme solution (Watnable et al., 1977). The control containing boiled inactivated enzyme (at 1000c for 5 min was treated similarly. After incubation enzyme activity was blocked by 20ml of Acetone ethanol (1:1) mixture and liberated free fatty acid was titrated against 0.02N sodium hydroxide using phenolphthalein as indicator, 1unit of lipase was defined as the amount of enzyme, which liberated 1molecule of fatty acid per minute (1ml of 0.02 N sodium hydroxide is equivalent to 100 molecules of fatty acid liberated per minute).
Amylase Enzyme Assay:
Amylase production media was prepared. The culture was inoculated and incubated at 300c for 40 hours. After incubation the Amylase production culture media with culture were centrifuged at 4000 rpm for 15 minutes. The resultant supernatant was used as enzyme source.
Assay system for Amylase Activity was carried out by measuring the amount of reducing sugar according to the DNSA method (Micro et al., 1976). The substrate was 1% soluble starch dissolved in phosphate buffer (PH7).0.1ml of the test solution was added to 1ml of the substrate. After incubation for 10 minutes at 370c the reaction mixture was stopped by adding 2ml of DNSA reagent (Micro et al., 1979). The reaction mixture was heated at 1000c for 10 minutes and cooled. Then 17ml of water was added to the solution stand for 15 minutes at room temperature. The OD was measured of 530nm. 1unit of Amylase was defined as the amount of enzyme which liberated 1µm per molecule of maltose per minute in this assay system (Murao et al., 1979 and Barley 1988).
Result and Discussion:
Isolation of Micro organisms
From the collected 10 different soil samples 20 different isolates were isolated and they are sub cultured in separate tubes for further use and they are numbered from 1-20
The protease assay was performed to determine the protease activity in the sample organisms. The results were presented in Fig: 1. It shows that the maximum amount of protein was seen in isolate 15 and next to it isolate isolates 10 and 11shows the same amount (i.e.) 0.08 mg/ml and also isolates 6, 18 and 19 shows 0.05mg/ml and remaining isolates shows varies amount and only trace amount (i.e.) 0.01mg/ml was present in sample 8 and 12.
The Lipase assay was done to determine the activity of Lipase for the different isolates and the activity was expressed in U/ml. the maximum lipase activity was seen in isolate 3 and its activity about 1140U/ml and the least activity was seen in isolate 19 and its activity is 520 U/ml. The results is presented in Fig: 2.
The Amylase assay is carried out to determine the amylase activity of different isolates. The result is presented in Fig: 3 the maximum activity was seen in isolate 5 and the activity was 0.42 mg/ml minimum activity was seen in isolate 10 and 13 and the activity was 0.01mg/ml. the same amount of activity was seen in isolates 16 and 17 its activity was about 0.05mg/ml and in isolates 6,7,12 and 18 and its activity was about 0.04 mg/ml.