Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus.
An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.
Yoon
Seong-Ae
SA
Center for Agricultural Biomaterials, Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
Ryu
Soo-In
SI
Lee
Soo-Bok
SB
Moon
Tae-Wha
TW
D000681:Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000687:Amylopectin; D000688:Amylose / Q000378:metabolism; D004795:Enzyme Stability; D005110:Extracellular Space / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology*; D012623:Seawater / Q000382:microbiology*; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D009844:Oligosaccharides; C021705:maltooligosaccharides; D013213:Starch; D000688:Amylose; D000687:Amylopectin; D014166:Transferases; D000681:Amylases