Purification and Characterization of α-Amylase from Bacillus licheniformis CUMC305

Abstract

a-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 a-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 x 105 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. Vmax 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+, Ca2+, and Mg2+, showed stimulatory effect, whereas Hg2 , Cu2+, Ni2+, Zn2+, Ag+, Fe2+ Co +, Cd2+, A13+, and Mn2+ were inhibitory. Of the anions, azide, F-, S032-, S043-, S2032-, MoO42-, and W042showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, P-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. a-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 Cu2+ and Fe2+ but not by the addition of Ca2+ or any other divalent ions.

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54

“…Hyperthermophiles such as P. furiosus and P. woesei have also been observed to produce a-amylase at their optimum growth temperature of 100 C (Koch et al 1990;James and Simpson 1996). Neutral pH was found to be optimal for amylase production by B. thermooleovorans NP54 as also reported in B. coagulans , B. licheniformis (Krishnan and Chandra 1983) and B. brevis (Tsvetkov and Emanuilova 1989). Cultural conditions have been found to have a profound in¯uence on amylase production.…”

Production, Purification, and Characterization of Thermostableα-Amylase Produced byBacillus licheniformisIsolate AI20

“…Determination of the puri�ed -amylase enzyme using SDS-PAGE electrophoresis with silver staining as described in Section 2 is considered not only for determining the molecular mass of the puri�ed enzyme but also a method to indicate the enzyme purity. e molecular mass of the puri�ed -amylase enzyme was estimated to be about 55 kDa (Figure 3 from 42 to 150 kDa [3] and from 23 to 50 kDa [40] have been reported.…”

Enhancement of acid amylase production by an isolated Aspergillus awamori

“…Several amylase-producing bacterial, fungal and other microbial organisms have been isolated and characterized over many decades (Krishnan and Chandra 1983;Vihinen and Mantsala 1990;Freer 1993;Iefuji et al 1996;De Moraes et al 1999;Gupta et al 2003). Industrial demand for these enzymes is limited with specific applications as in the food industry, where fungal amylases are preferred over other microbial sources mainly because of their more accepted GRAS status.…”