Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD 434

Abstract

The raw starch-digesting alpha amylase of Bacillus sp. IMD 434 was purified to homogeneity and displayed substantial hydrolysis of raw starch but did not adsorb onto the insoluble substrates, corn, rice, wheat or potato starch, at any of the pH values examined. The degree of hydrolysis ranged from 10% hydrolysis of potato starch to 32% hydrolysis of corn starch after 24 h. α- and β-Cyclodextrins (CDs) inhibited raw starch digestion but did not affect hydrolysis of soluble starch. In the presence of 10 mM α-CD or β-CD, hydrolysis of raw corn starch by the amylase decreased by 88 and 97%, respectively. The enzyme did adsorb onto α-CD Sepharose 6B, suggesting that an affinity site may be present on this non-raw starch-adsorbing amylase. After incubation with Pronase E, alpha amylase (434a M r 69,200) was hydrolysed into two components, a large enzymatically active component, EA 434b ( M r 56,200) and a small inactive peptide, IA 434c ( M r 13,000). EA 434b, although active on soluble starch, was incapable of hydrolysing raw starch, unable to adsorb onto raw starch and lost its ability to adsorb onto α-CD Sepharose 6B. Conversely, IA 434c was inactive on soluble and raw starch, did not adsorb onto raw starch but did adsorb onto α-CD Sepharose 6B and a range of linear maltooligosaccharide Sepharose 6B matrices. Thus, the alpha amylase simultaneously lost the ability to hydrolyse raw starch and adsorb onto α-CD Sepharose 6B when IA 434c was removed by proteolysis. The ability of the raw starch-digesting alpha amylase to adsorb onto α-CD Sepharose 6B was then exploited successfully in the development of a one-step purification for the amylase using CD affinity chromatography.