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Factors Responsible for Production of Amylases from Aspergillus Fumigatus Fresenius

MUNIRA AKHTER
Department of Microbiology, University of Chittagong, Chittagong- 4331, Bangladesh

M. N. ANWAR
Department of Microbiology, University of Chittagong, Chittagong- 4331, Bangladesh

MD. TOWHID HOSSAIN
Department of Microbiology, University of Chittagong, Chittagong- 4331, Bangladesh

Abstract/ Executive Summary:

Aspergillus fumigatus Fresenius showed maximum amylase production at 27⁰C with an initial culture pH of the medium 6.0 after 72 h. of incubation. One per cent soluble starch and 0.15% ammonium nitrate in the medium supported the highest amylase activity. During enzyme-substrate reaction maximum enzyme activity was observed at 50⁰C and pH 4.0 with 2% starch.

Keywords: Factors, Amylases, Aspergillus fumigatus

Location: Department of Microbiology, University of Chittagong, Bangladesh.

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Fortified food

Objectives:

1. The present work has been under taken to investigate the influence of some factors on the production and activity of amylases from the fungal isolate Aspergillus fumigatus.

Methodology:

Organism - Aspergillus fumigatus Fresenicus was obtained from the Department of Microbiology, University of Chittagong, Bangladesh. Culture Medium - Aspergillus fumigatus was cultured in defined salt medium containing 0.5% KH₂PO₄, 0.67% K₂HPO₄, 0.1% (NH₄)₂SO₄, 0.01% MgSO₄.7H₂O and 4% soluble starch. Assay of Enzyme Activity - Assay system for amylase activity was carried out by measuring the amount of reducing sugar following Nelson’s modification of Somogyi method. The reaction mixture contained 5 ml of 1% soluble starch prepared in 0.2 M citrate phosphate buffer (pH 6.0), 1 ml of 0.2 M citrate phosphate buffer and 1 ml of enzyme solution. The reaction mixture was incubated at 37?C for 1 h. Then 1 ml of alkaline copper reagent was added to 1 ml of reaction mixture, and the mixture was boiled for 20 min, cooled and 1 ml of arsenomolybdate colour reagent was added. After 10 min the reaction mixture was diluted with 22 ml of distilled water and the absorbance was measured at 550 nm. Enzyme activity was expressed as the amount of glucose released per ml of enzyme extract per unit time. [One unit (U) enzyme activity is the amount of enzyme required to release 1μmol glucose equivalent in 1 min under the assay condition]. Protein Estimation - Protein was determined. Biomass yield - Biomass was determined by dry weight basis (mg/g substrate). Saccharification - For saccharification the enzyme preparation (crude) was adjusted to pH 4.0 and sodium azide (0.2%) was added to inhibit the microbial growth. Hydrolysis was carried out under stationary condition in 25 ml screw cap test tubes at 50?C with substrate (starch) concentration of 7.5% (w/v) for 24 h. and 48 h. of intervals. The sugar content in the hydrolysate was measured by Nelson’s modification. Effect of Culture Conditions on Amylase Production - The fungal isolate was grown at various pH (4, 5, 6, 7, 8 and 8.5) and temperature (10ºC, 27ºC, 37ºC and 45ºC) for various incubation periods (48, 72, 96, 120 and 144 h.) using different nitrogen sources (NH₄NO₃, NH₄Cl, (NH₄)₂HPO₄, glutamine, glycine and peptone) for the optimization of growth conditions and to investigate the effects of these factors on amylase production. To determine optimum percent of carbon and nitrogen sources, the study was also carried out with 1 to 5% carbon and 0.05 to 0.30% nitrogen sources keeping other experimental conditions at optimum. Effect of Incubation Time on Amylase Activity - To determine the effect of incubation time on enzyme activity the enzyme substrate mixture was incubated at different incubation time (15, 30, 45, 60, 75 and 90 min). Effect of pH and Temperature on Amylase Activity - The effect of pH on amylase activity was determined by incubating the reaction mixture at pH 3.0 to 8.0 using citrate phosphate buffer (adjusted with 1% Na₂HPO₄.7H₂O solution). The optimum temperature for enzyme activity was also determined by assaying the enzyme activity at various temperatures from 35 to 55ºC.

Source of Information: The Chittagong Univ. J. B. Sci.,Vol. 3(1 &2):pp.11-20 , 2008

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present observation, high temperature (50⁰C) and low pH (4.0) for maximum enzyme activity indicate a good commercial probability of the amylase enzymes produced by the fungal isolate Aspergillus fumigatus.

Knowledge Management: Journal