Purification and characterization of an alkaline amylopullulanase with both alpha-1,4 and alpha-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378.
The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the alpha-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the alpha-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and alpha-amylase activities were pH 9.5 and 50 degrees C and pH 8.5 and 50 degrees C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, alpha-cyclodextrin and beta-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of alpha-1,4-linked substrates and one for the cleavage of alpha-1,6-linked substrates.
Ara
K
K
Tochigi Research Laboratories of Kao Corporation, Japan.
Saeki
K
K
Igarashi
K
K
Takaiwa
M
M
Uemura
T
T
Hagihara
H
H
Kawai
S
S
Ito
S
S
D000595:Amino Acid Sequence; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002236:Carbohydrate Conformation; D003505:Cyclodextrins / Q000494:pharmacology; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism
D001426:Bacterial Proteins; D003505:Cyclodextrins; D005936:Glucans; D010446:Peptide Fragments; D014312:Trisaccharides; C008317:maltotriose; C009109:pullulan; D006003:Glycogen; D000688:Amylose; D000687:Amylopectin; D006026:Glycoside Hydrolases; C082507:amylopullulanase; D008628:Mercury