The cold-adapted pullulanase Pul13A is an industrial useful amylolytic enzyme, but its low solubility is the major bottleneck to produce the protein in recombinant form. In a previous approach, a complex and time-consuming purification strategy including a step-wise dialysis procedure using decreasing concentrations of urea to renature the insoluble protein from inclusion bodies had been established. In this study, a truncation strategy was developed to facilitate the purification and handling of the type-I pullulanase. Pul13A has a size of 155-kDa with a multidomain architecture that is composed of the following predicted modules: CBM41/E-set/Amy-Pul/DUF3372/E-set/E-set/E-set, with CBM and E-set domains being putative carbohydrate-binding modules, Amy-Pul is the catalytic region and DUF is a domain of unknown function. Consecutive N- and C-terminal deletions of domains were applied to construct minimized enzyme variants retaining pullulanase activity and exhibiting improved renaturation efficiencies. A total of seven truncation constructs were generated and tested, which still led to the production of inclusion bodies. However, the parallel deletion of the exterior CBM41 and E-set domain enabled the direct refolding of active enzymes during one-step dialysis in urea-free buffer. Catalytic properties of truncation construct Pul13A-N1/C1 were not impaired indicating that this enzyme variant may be superior for industrial applications over the full-length pullulanase.
Keywords: Cold-adaptation; One-step renaturation; Purification efficiency; Truncations; Type-I pullulanase.