Role of carbohydrate binding modules, CBM3A and CBM3B in stability and catalysis by a 8-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405

A recombinant 8-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC27405 was explored for biochemical properties and the role of its associated CBMs in catalysis. The gene expressing full-length multi-modular 8-1,4-endoglucanase (AtGH9C-CBM3A-CBM3B) and its truncated derivatives (AtGH9C-CBM3A, AtGH9C, CBM3A and CBM3B) were independently cloned and expressed in Escherichia coli BL21(DE3) cells and purified. AtGH9C-CBM3A-CBM3B showed maximal activity at 55 degrees C and pH 7.5. AtGH9C-CBM3A-CBM3B exhibited highest activity against carboxy methyl cellulose (58.8 U/mg) followed by lichenan (44.5 U/ mg), 8-glucan (36.2 U/mg) and hydroxy ethyl cellulose (17.9 U/mg). Catalytic module, AtGH9C showed insig-nificant activity against the substrates, signifying the essential requirement of CBMs in catalysis. AtGH9C-CBM3A-CBM3B displayed stability in pH range, 6.0-9.0 and thermostability up to 60 degrees C for 90 min with unfolding transition midpoint (Tm) of 65 degrees C. The generation of cellotetraose and other higher oligosaccharides by AtGH9C-CBM3A-CBM3B confirmed it as an endo-8-1,4-glucanase. AtGH9C activity was partially recovered by the addition of equimolar concentration of CBM3A, CBM3B or CBM3A + CBM3B by 47 %, 13 % or 50 %, respec-tively. Moreover, the associated CBMs imparted thermostability to the catalytic module, AtGH9C. These results showed that the physical association of AtGH9C with its associated CBMs and the cross-talk between CBMs are necessary for AtGH9C-CBM3A-CBM3B in effective cellulose catalysis.

Automated summary

This study investigated the biochemical properties of a recombinant 8-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B, and the role of its associated CBMs in catalysis. The enzyme showed maximum activity at 55°C and pH 7.5, and exhibited high activity against carboxy methyl cellulose, lichenan, 8-glucan, and hydroxy ethyl cellulose. The catalytic module, AtGH9C, showed insignificant activity, emphasizing the essential requirement of CBMs in catalysis. The associated CBMs also contributed to thermostability. The results highlight the importance of the physical association and cross-talk between AtGH9C and its associated CBMs in efficient cellulose catalysis.