Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

Background: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis.
Results: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s ^-1 and 568.93 L mmol ^-1 s ^-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s ^-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol ^-1 s ^-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference.
Conclusions: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.
(© 2020 Society of Chemical Industry.)