Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei.
The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.
Pérez-Pomares
F
F
División de Bioquímica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain.
Bautista
V
V
Ferrer
J
J
Pire
C
C
Marhuenda-Egea
F C
FC
Bonete
M J
MJ
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D003470:Culture Media; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D019623:Haloferax mediterranei / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D013058:Mass Spectrometry; D008970:Molecular Weight; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003470:Culture Media; D015636:Magnesium Chloride; D012965:Sodium Chloride; D004492:Edetic Acid; D000516:alpha-Amylases; D002122:Calcium Chloride