Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that -3, -2, -1, +1 and + 2 subsites have a major role while, -4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of -1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that -1 and -2 subsite mutants had 27- and 30-fold higher binding affinity (K-D)( )for carob galactomannan when compared with catalytic mutant. The -1 subsite mutant also had highest K-D values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher K-D) and ivory nut mannan (up to 4.3-fold higher K-D) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, -3 mutant displayed 10-fold higher K-D for both carob galactomannan and glucomannan while, -4 mutant did not show any noticeable change in K-D values when compared to catalytic mutant. (C) 2019 Elsevier Inc. All rights reserved.