Purification and characterization of alpha-amylase from Bacillus licheniformis CUMC305

Journal Article · Mon Aug 01 00:00:00 EDT 1983 · Appl. Environ. Microbiol.; (United States)

OSTI ID:5379453

Krishnan, T; Chandra, A K

Alpha-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 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. In the presence of substrate (soluble starch), the alpha-amylase enzyme was fully stable after a 4-hour incubation at 100 degrees 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 hours of treatment. The activation energy for this enzyme was calculated as 5.1 x 10 to the power of 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. 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+, Ca(2+), and Mg(2+), showed stimulatory effect, wheras 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/sub 3/(2-), SO/sub 4/(3-), S/sub 2/O/sub 3/(2-), MoO/sub 4/(2-), and Wo/sub 4/(2-) showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, beta-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. Alpha-amylase was fairly resistant to EDTA treatment at 30 degrees C, but heating at 90 degrees C in presence of EDTA resulted in the complete loss of enzyme activity. (Refs. 32).

• Research Organization: Microbiology Lab., Dept. of Botany, Univ. of Calcutta, Calcutta - 700 019, India
• OSTI ID: 5379453
• Journal Information: Appl. Environ. Microbiol.; (United States), Vol. 46:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GLUCOSIDASE
PURIFICATION
ACTIVATION ENERGY
AMYLASE
BACILLUS
CATALYSIS
ELECTROPHORESIS
HYDROLYSIS
PH VALUE
SUBSTRATES
BACTERIA
CHEMICAL REACTIONS
DECOMPOSITION
ENERGY
ENZYMES
GLYCOSYL HYDROLASES
HYDROLASES
LYSIS
MICROORGANISMS
O-GLYCOSYL HYDROLASES
SOLVOLYSIS
550200* - Biochemistry