CHARACTERIZATION AND CHEMICAL MODIFICATION OF GLUCOAMYLASE PRODUCED FROM INDIGENOUS FUNGAL STRAIN HUMICOLA INSOLENS
DOI:
https://doi.org/10.71146/kjmr153Keywords:
Glucoamylase, chemical modification, characterization, Humicola insolensAbstract
In the present study, glucoamylase (GA) was produced from Humicola insolens by submerged fermentation. Maximum GA production (1.002 U/mL) and protein (0.12921mg/mL) was observed under optimum conditions of temperature (45 °C), pH (5.0), substrate concentration (4 % w/v) and inoculum density (5%). The enzyme was purified using ion exchange and gel filtration column with 10 fold purity and 7 % recovery. The apparent subunit molecular weight was 57 kDa. The number of isoforms of the GA was determined by running PAGE. The optimum temperature for GA activity was 45ºC at pH 5.4. An energy of activation (Ea ) of 26.52 kJ mol-1 was required for the formation of an activated enzyme- substrate complex at 45 ºC. KD at 65 ºC= 0.005423, ∆S = 0.273 kJ mol-1 K-1 , ∆G* = 70.95 kJ mol-1,
∆H=23.88 kJ mol-1 and its half-life =36 (min) at 55ºC. Enzyme was chemically modified by using succinic anhydride and aniline for the modification of amino group and surface carboxylic group. The data revealed that the modified forms of GA had deviation from the energy of activation than the native one. The amino group modified form has large value of Ea as compared to native enzyme whereas both forms showed the same optimum pH, 6.2 at optimum temperatures and showed stability in pH range of 5.4-7.2. The pH range of modified Ani-60 and succi-130 was higher than that of native. The values of Vmax, Km and kcat (s-1) for native were 34.78 U mg-1 protein, 4.76 mg starch ml-1 and 32.75 s-1 at 45 ºC respectively. The specificity constant, kcat/Km was 6.87. For amino group modified GA, the values of Vmax, Km and kcat were 76.92 U mg- 1 protein min-1, 2.17 mg starch ml-1 and 73.74 s-1 respectively. The specificity constant, kcat/Km was 31.74.
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Copyright (c) 2024 Farhana Yasmin, Haq Nawaz Bhatti, Abdul Jabbar (Author)
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