Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzyme's substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made. Here we show that two asparagine residues (N194 and N197) positioned in the loop covering the glucopyranose subsite −4 (recently denoted B2 loop) of the thermostable Cel7A from Rasamsonia emersonii had profound effects on both substrate interactions and catalytic efficacy. At room temperature the double mutant N194A/N197A showed strongly reduced substrate affinity with a water-cellulose partitioning coefficient threefold lower than the wild type. Yet, this variant was catalytically efficient with a maximal turnover about twice as high as the wild type. Analogous but smaller changes were found for the single mutants. Analysis of these changes in affinity and kinetics as a function of temperature, led to the conclusion that replacement of N194 and particularly N197 with alanine leads to faster enzyme-substrate dissociation. Conversely, these residues appeared to have little or no effect on the rate of association. We suggest that the controlled adjustment of the enzyme-substrate dissociation prompts faster cellulolytic enzymes.