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Microbial amylases are as important in industrial processes as are proteases. Among the microbes, fungi are gaining repute for the production of amylases. Keeping this in view, the present study was carried out to isolate, identify, characterize and explore the biotechnological applications of indigenous fungal strains. The study began by reviving fungal cultures from the stock collection in our lab and six more fungi were further isolated from the contaminated starch-agar plates. The isolates identified on the basis of cultural and morphological characteristics belonged to genus Aspergillus, Penicillium and Rhizopus. Preliminary screening was performed on starch-agar plate method with minor modification. Amylase production from the fungal isolates was also carried out under submerged fermentation conditions using mineral-salt media supplemented with starch and amylase production was quantitatively evaluated. Based on the results for quantitative production of amylases, 4 fungal isolates showing high IU/ml of amylase productivity were selected for further studies. The amylases from these isolates were characterized on the basis of activities at high temperatures and 2 fungal strains A. tubingensis SY 1 and A. niger MS 101 showing activities at 60oC and 64oC, respectively, were selected. Afterwards, the conditions for the optimum production of amylases from A. tubingensis SY 1 and A. niger MS 101 were worked out. The fungal strains showed optimum amylase production at 30oC with an initial pH of 5.9. Among the carbon sources; starch, glucose and maltose displayed higher amylase production along with the organic nitrogen source peptone. Amylase production was also optimized using a Plackett-Burman statistical design, and the results revealed peptone as the superior factor responsible for higher amylase titers. The optimum pH for amylase activity was determined along with the determination of optimum substrate concentration, the effect of various metal-ions and enzyme modulators. The pH 5.6 was optimum for amylase activity from both the fungal strains, while starch concentration of 0.5% was found to be optimum for the enzyme-substrate reaction to be carried out. Mn2+, K+ and NH4+ ions enhanced amylase activities while urea crystals and EDTA slightly inhibited the amylase activities of both fungal strains. Studies on solid-state fermentation (SSF) and submerged fermentation (SmF) for amylase production was also performed using variety of natural substrates including 2 halophytic plants and the results were compared. Whenever studies were compared with crude natural carbon substrates, whether under solid-state or submerged fermentation conditions together with the quantitative determination of amylase, the concentration of other enzymes, like xylanase, pectinase and cellulose enzyme system (β-glucosidase, endoglucanase, filter paper assay) were also determined. Potato-peels were found to be the most suitable substrate for amylase production by both fungal strains under SmF and SSF conditions. The Tm of amylase from the strain MS101 of A. niger was 65oC and from A. tubingensis SY 1 was 67oC, while Ea values were 73.64 KJ/mol and 46.07 KJ/mol for A. niger MS101 and A. tubingensis SY 1 amylases, respectively. Because of higher Tm values and low energies of activation (Ea) the industrial potential of amylases was determined. For this purpose, the starch-sized fabric was treated with fungal amylases at different temperatures for different time intervals to determine the d-sizing efficiency of amylases. The fabric after de-sizing by A. niger MS 101 amylase resulted in a TEGEWA rating of 8, while by A. tubingensis SY 1 amylase a TEGEWA rating of 9 was observed at 54oC in 12 hr. The results are promising for the use of these amylases in de-sizing. Co-culture studies for bioethanol production under SmF and SSF conditions were carried out using potato-peels under SmF and SSF, when the fermentation medium was simultaneously inoculated with the fungal and yeast strains, ~4 g/Kg and 6 g/Kg ethanol was produced in 120 hr. of incubation at 30oC. The yeast Pichia kudriavzevii SY 11 was also able to produce almost similar amount of ethanol under SmF of potatopeels. Indicating no contribution of fungal amylase to bioethanol. However, when coculture studies were carried out on purified starch 7- to 12- fold more ethanol production was noted (12 and 28 g/Kg) compared to potato-peel (1 and 4 g/Kg). Amylases were subjected to purification using different techniques: affinity and gelfiltration chromatography. No fruitful results were obtained by affinity chromatography while by using gel-filtration technique; a band of ~116 kDa was observed for A. tubingensis SY 1 amylase.
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