Purification and Characterization of α-Amylase from Bacillus licheniformis CUMC305

Abstract

α-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90�C and pH 9.0, and 91% of this activity remained at 100�C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60�C, 3 h at 70�C, and 90 min at 80�C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the α-amylase enzyme was fully stable after a 4-h incubation at 100�C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 � 10 5 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V max values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na + , Ca 2+ , and Mg 2+ , showed stimulatory effect, whereas Hg 2+ , Cu 2+ , Ni 2+ , Zn 2+ , Ag + , Fe 2+ , Co 2+ , Cd 2+ , Al 3+ , and Mn 2+ were inhibitory. Of the anions, azide, F − , SO 3 2− , SO 4 3− , S 2 O 3 2− , MoO 4 2− , and Wo 4 2− showed an excitant effect. p -Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, β-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. α-Amylase was fairly resistant to EDTA treatment at 30�C, but heating at 90�C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu 2+ and Fe 2+ but not by the addition of Ca 2+ or any other divalent ions.