Highly efficient raw starch degradation by a novel dual-function maltogenic debranching enzyme of the intermediate GH13_20 subfamily from Deinococcus radiodurans

The neopullulanase subfamily comprises diverse enzymes involved in starch modification. In Deinococcus radiodurans , we newly identified a maltogenic debranching amylase (DrMD) that lacks the N-terminal domain and uniquely exhibits both pullulanase (EC 3.2.1.41) and maltogenic amylase (EC 3.2.1.133, EC 3.2.1.54) activities at a single active site. Notably, DrMD produced a higher amount of reducing sugars from various types of gelatinized raw starch, showing a 4-fold increase in efficiency over type I pullulanase. The HPSEC-RI analysis indicated that DrMD hydrolyzes the branch point of the large amylopectin fraction, followed by the cleavage of the amylose structure. With side chain length-dependent debranching activity and maltogenic amylase-like function, DrMD was effective in producing glucose (G1) and maltooligosaccharides ranging from G2 to G5. As a result, 86.97% of the raw starch fraction was degraded into low-molecular-weight glucans (<1 × 10⁴ Da). These findings highlight efficiency of DrMD in starch modification and its potential as a versatile enzyme for industrial starch processing.