Starch metabolism in Pseudomonas stutzeri. I. Studies on maltotetraose-forming amylase

The extracellular maltotetraose-forming amylase of Pseudomonas stutzeri was purified to homogeneity by a combination of affinity and hydroxyapatite chromatography. Sodium dodecyl sulfate-gel electrophoresis indicated that the oligomeric enzyme contains two different subunits with molecular weights of 48 000 and 58 000. Cross-linking studies using dimethyl suberimidate have demonstrated that the native enzyme consists of dimers. Seven isozymes of the amylase have been identified after polyacrylamide gel electrophoresis and amylose-digestion zymograms. The amylase of Ps. stutzeri is known to produce maltotetraose from linear and branched alpha-glucans by an exomechanism. The relatively high conversion rate of starch (75% hydrolysis), and the hydrolysis of cross-linked blue starch by this amylase indicate that the enzyme can cleave its substrates also by an endomechanism. Further strong evidence for an endomechanism was obtained from the action of the amylase on maltotetraose units which are located within the pullulan molecule. Dextran, pullulan, and maltotetraose are compeititve inhibitors. EDTA caused reversible inactivation. Amylase activity could be restored by addition of Ca2+. Heavy metals are inhibitory.