Purification and characterization of -amylase produced by Penicillium citrinum HBF62

Abstract

The extracellular amylase produced by Penicillium citrinum HBF62 was purified 18 folds using starch affinity chromatography. The molecular weight of amylase was estimated to be about 65 kDa by SDS-PAGE. The temperature and pH for optimum activity of the enzyme were 55°C and 5.5, respectively. It was thermostable between 27 and 60°C and retained more than 50% activity after 24 h at these temperatures. It was found to be stable pH range between 3.5 and 8.5. Km and Vmax values for soluble starch of the enzyme were calculated to be 0.2 mg/ml and 5000 U/mg protein, respectively. The amylase exhibited broad substrate specificity because it acted on all the substrates tested. While the enzyme was activated by Mn2+, Ca2+, Co2+, Fe3+, Ba2, NH4+ and Al3+, the other ions and EDTA had no effect on its activity. The enzyme activity was inhibited in the presence of phenylmethanesulfonylfluoride (PMSF), N-bromosuccinimide (NBS) and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide methyl p-toluenesulphonate (CMC), suggesting that serine, tryptophan residues and carboxyl groups play an important role in the catalytic process. The activity of amylase was found to be tolerant up to 5 M NaCl concentration. Raw corn starch adsorption of amylase was found to be 74%. Thin layer chromatography of the starch hydrolysis products showed glucose and maltose as the main end products, indicating that the purified enzyme was α-amylase. Key words: Penicillium citrinum, α-amylase, purification, characterization.