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 hy drolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, each being 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 sm aller non-overlapping active fragments, designated amylose-hydrolyzing fragment (AHF114, 114 kDa) and pullulan-hydrolzing fragment (PHF102, 102 kDa). The two truncated protein fragments, each containing a singl e catalytic domain, were purified to homogeneity. The purified AHF114 and PHF102 had similar enzymatic properties to the amylase and pullula nase activities, respectively, of intact APase. The partial amino-term inal sequences of APase and AHF114 were both hr-Gly-Asp-Lys-Arg-Ile-Gl u-Phe-Ser-Tyr-Glu-Arg-Pro and that of PHF102 was u-Ala-Leu-Val-Ser-Gly -Glu-Val-Leu-Ser-Asp-Lys-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 i s obviously a ''biheaded enzyme''.