Purification and characterization of extracellular alpha-amylase and glucoamylase from the yeast Candida antarctica CBS 6678.
An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and affinity chromatography on acarbose--AH-Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (Szero20,w), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48,500, 4.7 S, 10.1, 1.74 cm2 mg-1, 4.2 and 57 degrees C, respectively, for glucoamylase and as 50,000, 4.9 S, 10.3, 1.53 cm2 mg-1, 4.2 and 62 degrees C, respectively, for alpha-amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. alpha-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both alpha-amylase (Ki less than 1 microM) and glucoamylase (Ki less than 0.1 microM), being more effective than Bay e 4609 (Ki less than 10 microM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki less than 0.1 microM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki less than 1 mM), maltitol and amino alcohols (Ki less than 10 mM). Unlike alpha-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical with the active site.
D000327:Adsorption; D002175:Candida / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D005959:Glucosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D013213:Starch; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D013213:Starch; D005959:Glucosidases; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase