Food and Nutritional Sciences

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Browsing Food and Nutritional Sciences by Author "Abdisa Melese (PhD)"

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    Isolation and Characterization of Thermostable Α-Amylase From Afar Regional State of Ethiopia
    (Addis Ababa University, 2013-06-02) Duga Abdi; Abdisa Melese (PhD); Retta Nigussie (Professor)
    Thermophilic microorganisms could be isolated from harsh environments where the environmental conditions are not favorable for most microorganisms. Enzymes isolated from these microbes could have higher activity and stability. Thermostable enzymes can be extracted from thermophilic microbes and be employed for industrial purposes. The isolation and characterization of these enzymes are crucial steps in biotechnology and enzyme sciences. In this study, five (5) water samples and five (5) soil samples from Afar, a regional state in Ethiopia. Isolates in all the samples were grown at 70 0C to screen for thermophilic microorganisms. Among these thermophiles, thirty five (35) colonies from each sample which showed a positive amylolytic response were selected and the ratio of the clear halo created to the colony size (HCR) was compared. Based on the HCR, isolates from ALS and MS were selected for further characterization. The optimum fermentation temperature was found to be 65 0C for both isolates with a fermentation period of 48 hours under the normal pH of the basal medium. The pH optima for the production of the enzymes were pH 6 for MS and pH 8 for ALS. The enzymes isolated from both isolates were found to be very active at high temperature. Enzyme from ALS had a maximum activity at 90 0C while the enzyme from MS had its maximum activity at 85 0C. pH optimum for ALS was 8 and it was functional in a pH range of 6- 11. The enzyme from MS had maximum activity at pH value of 6 and it had amylolytic activity in a pH range of 4-9. Both enzymes showed good stability for pH and temperature. The enzyme from ALS retained 62% of its original activity (2.3817 ± 0.11 U/ml) after 40 minutes of incubation at 900C however, maintained 6% of its activity at 100 0C. The enzyme was deactivated after 30 minutes (retaining only 5% at 105 0C of its original activity). This enzyme was fairly stable for 30 minutes maintaining more than 50 % of its activity at pH values of 6-9, with no activity at pH values less than 6. The ion Zn2+ and higher concentrations (10 mM) of Mg2+ strongly inhibited enzyme iv activity while 5mM concentration of Ca2+ and Ba2+ induced higher catalysis on the enzyme from ALS. The enzyme from MS retained 65% of its original activity (2.5782 ± 0.56 U/ml) after 40 minutes of incubation at 85 0C however, maintained 4% of its activity at 100 0C. The enzyme was fairly stable for 40 minutes maintaining more than 50 % of its activity at pH values of 5-7, with no activity at pH values higher than 9. It had 69 % of its activity at pH value of 6 after 40 minutes of reaction time and maintained 53 % of its original activity at pH value of 4 after 30 minutes of reaction time. The ion Zn2+ and higher concentrations (10 mM) of Mg2+ strongly inhibited enzyme activity while 5mM concentration of Ca2+ and Ba2+ induced higher catalysis. Considering the requirement of starch liquefaction process of an enzyme with a low pH optimum, the enzyme isolated from MS may offer an advantage since it functions well in acidic pH value of up to 4. Even though the enzyme activities of ALS and MS improved with the addition of CaCl2, they had good amylolytic activities in the absence of Ca2+ which is a requirement in large-scale starch hydrolysis processes. Therefore, the enzymes from ALS and MS can be employed for starch hydrolysis in large-scale processes.