INTRODUCTION:

Micro-organisms had made significant contribution to the production of foods and beverages in the last three decades. Various industries, such as food, brewing, textile pharmaceutical and confectionaries depend largely on the various products especially extra-cellular enzymes produced by these micro-organisms¹. An extra-cellular amylase, specifically raw starch digesting amylase has found important application in bioconversion of starches and starch-based substrates². Amylases are a class of enzymes (hydrolases) that are capable of digesting the glycosidic linkages found in starch or glycogen. Under aqueous conditions, amylases act on glycosidic bonds present in starch to liberate glucose, maltose, maltotriose^3-4, etc. Amylases are important enzymes employed in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents^5-6. Although amylases can be obtained from several sources, such as plants and animals, the enzymes from microbial sources generally meet industrial demand⁷. The Aspergillus species produce the extracellular amylase enzyme having significant industrial importance. Fungal amylases are used in food industry; textile and paper industries^8-9.

Microbial amylases have successfully replaced chemical hydrolysis of starch in starch processing industries. Besides their use in starch saccaharification, they also find potential application in a number of industrial processes such as in food, baking, brewing, detergent, textile and paper industries. With the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into many other fields, such as clinical, medical and analytical chemistry⁷.

MATERIALS AND METHODS:

The fungus Aspergillus oryzae MTCC 1847 was used in this study. It was maintained on Sabouraud Dextrose Agar (Hi Media, Mumbai) medium. The slants were grown at 30^oC for seven days and stored at 4^oC. The strain was sub-cultured at four week intervals.

Inoculum Preparation:

Pre-inoculum was prepared by transferring of 5 ml of sterilized distilled water to seven days old SDA slant culture and transfer the spore suspension into 250 ml Erlenmeyer flask containing 100 ml of modified Czapek Dox inoculum medium and incubated at 30^oC for seven days. 1% inoculum size is used to inoculate production media.

Substrates:

Different starch rich carbon sources such as Sweet potato, Wheat Bran and Rice Bran were used as substrate. All the substrates are obtained from the local market in Chennai. The substrates were ground into powder with a blender and passed through sieve (80/100 mesh size) to remove large size particles.

α-amylase assay:

α-Amylase activity was determined as described by Okolo et al² The reaction mixture consisted of 0.5 ml of 1% soluble starch, 0.25 ml 0.1M acetate buffer (pH 5.0), 1.5ml of distilled water, and 0.5 ml of crude enzyme extract. After 10 min of incubation at 50^oC, the liberated reducing sugars (glucose equivalents) were estimated by the dinitrosalicylic acid (DNS) method of Miller¹⁰ .The color developed was read at 540 nm using a spectrophotometer. Glucose was used as the standard. One unit (IU) of α-amylase was defined as the amount of enzyme releasing one μmol glucose equivalent per minute under the assay conditions.

Optimization of Carbon Source:

Three carbon sources, Sweet Potato, Rice Bran and Wheat Bran were added individually to 250 ml Erlenmeyer flask containing 100 ml of production medium and fermentation was carried out at 30°C. The enzyme activity was estimated daily with regular time intervals. The best result shown substrate was taken and its concentration was optimized by varying from 1% - 4%.

Optimization of pH and Temperature:

To determine the optimal temperature and pH for α-amylase production, was carried out at 30, 35, 40, 45°C and 6, 6.5, 7, 7.5 respectively. The optimal carbon source, temperature and pH were evaluated by the effects on the amylase production.

RESULTS AND DISCUSSION:

Effect of Carbon Source on Amylase Activity:

Enzyme formation is largely dependent on the selection of carbon source. Submerged Fermentation was carried out with carbon sources such as Sweet Potato, Rice Bran and Wheat Bran. After performing the above experiment, it was found that Sweet Potato (Fig.1) was better among the carbon sources; it gave higher amylase activity than others. Sweet Potato was used as a Carbon source for further studies.

Fig.1. Effect of different Carbon Sources Sweet Potato (●), Rice Bran (♦), Wheat Bran (▲) on Amylase Activity

Effect of substrate Concentration on Amylase activity:

The effect of the Carbon substrate (Sweet Potato) concentration was studied at different levels (1-4%). (Fig.2) shows that higher amylase activity was found at 2% concentration of Sweet Potato.

Fig.2. Effect of different Concentrations of sweet potato 1% (♦), 2 %(●), 3% (▲) and 4% (■)

Effect of pH on Amylase Activity:

Optimization of pH was carried out at different pH 6, 6.5, 7, 7.5 and evaluated for high amylase activity. From the (Fig.3), it was found that high enzyme activity was observed at pH 6.5.

Fig.3. Effect of pH on Amylase Activity; 6 (▲), 6.5(●), 7 (♦) and 7.5(■)

Effect of Temperature on Amylase Activity:

With optimized values of substrate concentration and pH the effect of temperature was studied at different levels (30, 35, 40, 45^oC) for the best production of amylase. After 12 days of experimentation, (Fig.4) showed that 35^oC was found to give maximum enzyme activity.

Fig.4. Effect of temperature on Amylase Activity; 30ºC (▲), 35ºC (●), 40ºC (♦) and 45ºC (■)

CONCLUSION:

The nature and amount of carbon source in culture media is important for the growth and production of extra cellular amylase in fungi. Amylase activity was high in media containing Sweet Potato as sole carbon source. It was observed that low level of carbon source is inadequate for the enzyme production and excess carbon is equally detrimental causing enzyme inhibition. Thus, 2% concentration is enough for maximum amylase activity. Further, the optimal values of pH 6.5 and Temperature 35^oC supports better amylase production.

ACKNOWLEDGEMENT:

Authors like to thank the Management and Staff of Biotechnology Department, Madha Engineering College, Kundrathur, Chennai for their support.

REFERENCES:

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5. Akpan I et al. Production of amylase by A.niger in a cheap solid medium using rice band and agricultural materials. Trop. Sci.1999b; 39: 77-79.

6. Mitchell DA et al. In: General principles of Solid State fermentation, Monogram, ed by Doelle HW, Rolz C. Publication of Oxford London, 1990.

7. Pandey A et al. Advances in microbial amylase. Biotechnol. Appl. Biochem.2000; 31: 135-152.

8. V. Ellaiah et al. Optimization of process parameters for glucoamylase production under solid state fermentation by newly isolated Aspergillus species. Process Biochem. 2002; 38: 615-620.

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10. Miller et al. Use of dinitro-salicylic acid reagent for determination of reducing sugars. Anal. Chem., 1959; 31: 426-428.

Received on 26.09.2010 Accepted on 12.10.2010

Research J. Engineering and Tech. 1(2): Oct. - Dec.2010 page 79-81