Separation of functional domains for the alpha-1,4 and alpha-1,6 hydrolytic activities of a Bacillus amylopullulanase by limited proteolysis with papain.
An amylopullulanase (APase) from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, each maximally active at an alkaline pH, to generate oligosaccharides. We analyzed proteolytic fragments that were produced by exposing pure APase to various proteases, to identify its catalytic domain(s). The intact, pure 210-kDa APase was partially digested with papain for a short time, yielding simultaneously two smaller non-overlapping active fragments, designated amylose-hydrolyzing fragment (AHF114, 114 kDa) and pullulan-hydrolyzing fragment (PHF102, 102 kda). The two truncated protein fragments, each containing a single catalytic domain, were purified to homogeneity. The purified AHF114 and PHF102 had similar enzymatic properties to the amylase and pullulanase activities, respectively, of intact APase. The partial amino-terminal sequences of APase and AHF114 were both Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and that of PHF102 was Thr-Val-Pro-Leu-Ala-Leu-Val-Ser-Gly-Glu-Val-Leu-Ser-Asp-Lsy-Leu. These results were direct evidence that the alpha-1,6 and alpha-1,4 hydrolytic activities were associated with two different active sites in this novel enzyme. Our alkaline APase is obviously a "biheaded enzyme".
Ara
K
K
Tochigi Research Laboratories of Kao Corporation, Japan.
Igarashi
K
K
Hagihara
H
H
Sawada
K
K
Kobayashi
T
T
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D006026:Glycoside Hydrolases; C082507:amylopullulanase; D010206:Papain