Proteolytic modification of raw-starch-digesting amylase from Bacillus circulans F-2 with subtilisin: separation of the substrate-hydrolytic domain and the raw substrate-adsorbable domain.
Raw starch-digesting amylase (BF-2A, 93,000 Da) from Bacillus circulans F-2 was converted into two components during digestion with subtilisin. The two components were separated and designated BF-2A' (63 kDa) and BF-2B (30 kDa), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in Km, Vmax and in their specific activity for soluble starch hydrolysis. However, its absorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was differed greatly from that of BF-2A. The stability of the enzymes decreased below pH 5.5 and at 50 degrees C, while it was quite stable even at pH 12. On the other hand, the smaller peptide (BF-2B) could be adsorbed onto raw starch. From these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption and also contributes to the original enzyme-to-enzyme stabilization. A proposed model of the raw-starch-digesting enzyme from this strain is extensively discussed.
Kim
C H
CH
Genetic Engineering Research Institute, K.I.S.T., Yusung-ku, Taejon, South Korea.
Kwon
S T
ST
Taniguchi
H
H
Lee
D S
DS
D000681:Amylases / Q000737:chemistry* / Q000302:isolation & purification; D001407:Bacillus / Q000187:drug effects / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D013213:Starch / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry*; D013696:Temperature
D010446:Peptide Fragments; D013213:Starch; D000681:Amylases; D013381:Subtilisins