Carbohydrate binding module (CBM) as a kind of non-catalytic domain has significant effects on the substrate binding and catalytic properties of glycoside hydrolases. CBM68 of an Anoxybacillus sp. pullulanase (PulA) was identified as a new type of CBM in our previous studies. Then, four key substrate binding amino acid residues (Y14, V91, G92, and R96) were obtained by alanine substitutions in this work. Through kinetic analysis of the mutants, V91A and G92A showed significant reduction both in K-m values and k(cat) values against pullulan. To further identify the changes of substrate affinities of V91A and G92A, devitalized mutants V91A-D413N and G92A-D413N were under measuring by surface plasmon resonance (SPR). Compared with that of PulA-D413N, the substrate affinities of V91A-D413N and G92A-D413N were improved by 1.6-fold and 2.2-fold, respectively. However, as to the product (maltotriose) binding force tested by the isothermal titration calorimetry (ITC), G92A showed higher binding force than that of V91A and PulA by 4.2-fold and 6.2-fold, respectively. That may cause G92A showing significantly lower catalytic efficiency than V91A and PulA. Moreover, four different kinds of amino acids (leucine, serine, glutamic acid and arginine) substitutions for V91 and G92 showed various changes both on the kinetic parameters and enzymatic properties, which demonstrated that V91 and G92 were the critical binding residues in the CBM68. The results of this study made contributed to the rational design for improving the catalytic efficiency of PulA. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.