Importance of Trp139 in the product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04.

The maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites - 5 and - 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.