Purification and some properties of a novel maltohexaose-producing exo-amylase from Aerobacter aerogenes.
Maltohexaose producing amylase (EC 3.2.1.-) is the fourth known exo-amylase, the three previously known being glucoamylase, beta-amylase and Pseudomonas stutzeri maltotetraose producing amylase. The enzyme after release from Aerobacter aerogenes cells by 0.1% sodium lauryl sulfate extraction was purified by ammonium sulfate precipitation, DEAE-Sephadex column chromatography and Sephadex G-100 gel filtration to 80-fold of the original sodium lauryl sulfate extract activity, It gave a single band on disc electrophoresis, and the molecular weight by gel filtration was 54 000. This amylase showed maximal activity at 50 degrees C and pH 6.80. The pH stability range was relatively wide, the enzyme retaining more than 90% of its initial activity in the range of 6.50-9.0. 80% of the activity was retained after 15 min at 50 degrees C. This enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on alpha- or beta-cyclodextrin, pullulan or maltohexaitol. Also the enzyme acted on beta-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The unusual reaction of this enzyme on beta-limit dextrin is discussed from the standpoint of the stereochemistry of 1,4-alpha- and 1,6-alpha-glucosidic bonds. This is the anomalous amylase for which it is recognized that 1,6-alpha-glucosidic linkages in the substrates can mimic the effect of 1,4-alpha-bonds, as previously observed in pseudo-priming reactions of E. coli phosphorylase.
D000681:Amylases / Q000302:isolation & purification / Q000378:metabolism*; D000687:Amylopectin / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003912:Dextrins / Q000378:metabolism; D004754:Enterobacter / Q000201:enzymology*; D004755:Enterobacteriaceae / Q000201:enzymology*; D006003:Glycogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D008956:Models, Chemical; D009844:Oligosaccharides / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D003912:Dextrins; D007461:Iodoacetates; D009844:Oligosaccharides; D006003:Glycogen; D000687:Amylopectin; D000681:Amylases; D002118:Calcium