Effects of carbohydrate binding module of pullulanase type I on the raw starch rearrangement by enhancing the hydrolysis activity

Starch, a versatile ingredient used in various industries, often requires modification for improvements in its physicochemical and functional properties for utilization. Although carbohydrate-binding modules (CBMs) are non-catalytic domains that recognize and bind to the substrate without any enzymatic activity, CBMs are commonly found in the starch-modifying enzyme, pullulanase (PulA). Herein, we identified two PulAs from Deinococcus radiodurans and Deinococcus wulumuqiensis that differed in activity only by CBM composition. Structural comparison analysis showed unique substrate-binding coordination of CBM_b1. The analysis with a deletion mutant (ΔCBM_b1-DrPulA), and a chimeric mutant (CBM_b1-DwPulA) revealed that CBM_b1 can improve amylopectin but not pullulan hydrolysis. Moreover, CBM_b1 introduction increased the hydrolysis activity of raw waxy corn starch by 60% which leads to the formation of distinct α-glucan microparticles (GMP). GMP produced using CBM_b1-DwPulA exhibited B-type crystallinity, similar to that produced by DwPulA. However, a relatively high proportion of short-length maltooligosaccharides was observed in the GMP produced by CBM_b1-DwPulA compared with that produced by DwPulA. This contributed to the formation of smaller particles, high colloidal dispersion stability, and increased the absolute value of the zeta potential. Altogether, our findings showed that Deinococcus sp.-derived CBMs can be used to customize GMPs by changing the debranching pattern.