Studies on amylase synthesis by thermophilic bacillus sp. isolated from refuse dump and its action on starchy waste materials

Omoboye, Olumide Owolabi (2014)

xix,109

Thesis

This study isolated and characterized α-amylase-producing thermophilic bacteria from a refuse dumpsite as well as characterized the partially purified α-amylase produced by the isolated bacteria. This was with a view to obtaining a thermostable α-amylase-producing bacteria capable of digesting raw starches. Four soil samples were collected at different dumpsites in Ile-Ife, Osun State, Nigeria. Serially diluted inocula were plated on nutrient agar in order to isolate bacteria for their α-amylase activity. The isolated bacteria were identified by morphological and biochemical characterization while the bacteria of interest was identified by molecular analysis using 16S rRNA gene sequencing. The bacterium with the highest α-amylase activity was selected for enzyme production, purification and characterization. The optimal conditions for α-amylase secretion by the bacterium were determined by varying the pH, temperature, percentage soluble starch, nitrogen sources and carbon sources. Sources of raw starches were also varied. The enzyme was partially purified by ion exchange chromatography on CM Sepharose CL-6B. The molecular weight of the enzyme was determined using gel filtration on Sephadex G-100. Kinetic parameters (Km and Vmax) of the purified enzyme, effects of temperature, pH, metal ions and ethylenediaminetetra acetic acid (EDTA) were studied. The isolated and identified bacteria were Bacillus alvei (40%) Bacillus licheniformis (40%) and Bacillus brevis (20%) while Bacillus licheniformis RD24 was identified by 16S rRNA gene sequencing. The peak of amylase productivity was at 20 h of incubation (925 µg/ml/min). The optimum pH and temperature for the production of Bacillus licheniformis RD24 α-amylase were 7 (with 150 ± 1.33 µg/ml/min) and 45 oC (with 58 ± 1.66 µg/ml/min enzyme activity) respectively. One percent (1%) starch composition of the enzyme production medium gave highest enzyme activity of 102 ± 5.3 µg/ml/min. Peptone gave an enzyme activity of 165 ± 8.97 µg/ml/min and yeast extract gave 52.26 ± 2.86 µg/ml/min. Starch gave the highest activity of 33 ± 4.98 Units/ml followed by lactose (32 ± 2.99 Units/ml) and melibiose (27 ± 2.99 µg/ml/min). Of the raw starches, cassava flour gave the highest specific activity of 72 ± 0.07 Units/mg protein, while sorghum starch gave the lowest specific activity 5 ± 1.52 Units/mg protein. The specific activity of the partially purified Bacillus licheniformis RD24 α-amylase for starch was 1.634 Units/mg protein with a purification fold of 4.76. The partially purified Bacillus licheniformis RD24 α-amylase had a molecular weight of 50 kDa. The Vmax and Km of the partially purified Bacillus licheniformis RD24 α-amylase with soluble starch as substrate were 4654 ± 108 Units/mg protein and 79.11 ± 1.84 mg/ml respectively. The optimum pH and temperature of partially purified Bacillus licheniformis RD24 α-amylase were 8.0 and 70 oC respectively. Sodium ion had stimulatory effect on the enzyme while Mg2+, Ca2+ and Al3+ inhibited the enzyme. EDTA inhibited the enzyme at all concentrations. The study concluded that α-amylase synthesized by Bacillus licheniformis RD24 had a unique characteristic of thermostability and ability to withstand alkaline pH.

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