Enzyme immobilization on α-1,3-glucan: development of flow reactor with fusion protein of α-1,3-glucan binding domains and histamine dehydrogenase

alpha-1,3-Glucanase Agl-KA from Bacillus circulans KA-304 consists of a discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), a threonine-proline- rich-linker (TP linker), a discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. The binding of DS1, CBM6, and DS2 to alpha-1,3-glucan can be improved in the presence of two of these three domains. In this study, DS1, CBM6, and TP linker were genetically fused to histamine dehydrogenase (HmDH) from Nocardioides simplex NBRC 12069. The fusion enzyme, AGBDs-HmDH, was expressed in Escherichia coli Rosetta 2 (DE3) and purified from the cell-free extract. AGBDs-HmDH bound to 1% micro-particle of alpha-1,3-glucan (diameter: less than 1 mu m) and 7.5% coarse- particle of alpha-1,3-glucan (less than 200 mu m) at about 97 % and 70% of the initial amounts of the enzyme, respectively. A reactor for flow injection analysis filled with AGBDs-HmDH immobilized on the coarse-particle of alpha-1,3-glucan was successfully applied to determine histamine. A linear calibration curve was observed in the range for about 0.1 to 3.0 mM histamine. These findings suggest that the combination of alpha-1,3-glucan and alpha-1,3-glucan binding domains is a candidate for novel enzyme immobilization.