Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach

Abstract Pullulanase is a well-known debranching enzyme that can specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability catalytic efficiency under industrial conditions. In the present study, four residues (A365, V401, H499, T504) lining pocket of Anoxybacillus sp. AR-29 pullulanase (PulAR) were selected for site-directed mutagenesis (SDM) by using structure-guided consensus approach. Five beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, PulAR-A365V/V401C/T504V/H499A) created, which showed enhanced thermostability, pH stability, efficiency. Among them, quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- 9.6-fold higher toward pullulan at 60 ℃, 6.0 5.0, respectively. addition, its thermostabilities ℃ 65 improved 2.6- 3.1-fold, respectively, compared to those wild-type (WT). Meanwhile, stabilities 4.5 5.0 1.6- 1.8-fold than WT, summary, performance PulAR was significantly The resultant demonstrated potential applications industry. Graphical