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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.