Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus.
A medium  was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.
D000681:Amylases / Q000302:isolation & purification / Q000378:metabolism*; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001439:Bacteroides / Q000201:enzymology*; D002241:Carbohydrates / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D013213:Starch / Q000378:metabolism; D013402:Sugar Alcohols / Q000494:pharmacology; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D002241:Carbohydrates; D013402:Sugar Alcohols; D003035:Cobalt; D013213:Starch; D000688:Amylose; D000687:Amylopectin; D000681:Amylases; D008628:Mercury; D008274:Magnesium; D015032:Zinc