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

Abstract

A recombinant β-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 β-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 °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), β-glucan (36.2 U/mg) and hydroxy ethyl cellulose (17.9 U/mg). Catalytic module, AtGH9C showed insignificant 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 °C for 90 min with unfolding transition midpoint (T_m) of 65 °C. The generation of cellotetraose and other higher oligosaccharides by AtGH9C-CBM3A-CBM3B confirmed it as an endo-β-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 %, respectively. 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.

Keywords: Carbohydrate binding module; Catalysis; Endoglucanase; Glycoside hydrolase; Oligosaccharides.