Role of carbohydrate binding module (CBM3c) of GH9 β-1,4 endoglucanase (Cel9W) from Hungateiclostridium thermocellum ATCC 27405 in catalysis

Abstract

The function of CBM3c in the enzymatic catalysis varies among the members of family 9 Glycoside Hydrolases (GH). A new member of family 9 GH (Cel9W) from thermophilic anaerobic bacterium, Hungateiclostridium thermocellum was explored for elucidation of the role of CBM3c in catalysis by GH9. Cel9W is a multimodular theme B1, cellulase enzyme comprising a catalytic module of family 9 glycoside hydrolase (HtGH9t) at N-terminal, a family 3c carbohydrate binding module (HtCBM3c) and a dockerin domain at the C-terminal. The ORF of Cel9W encoding full length β-1,4-glucanase, HtGH9 (containing both GH9 and CBM3c modules), the truncated GH9 catalytic module (HtGH9t) and module CBM3c (HtCBM3c) were cloned and over-expressed using E. coli BL21 cells. HtGH9 showed maximum activity at pH 6.5 and 90 °C. It displayed highest activity of 64 U/mg against lichenan followed by 44.6 U/mg (β-glucan) and 22.3 U/mg (Carboxymethyl cellulose). HtGH9 showed stability in the pH ranging from 5.0 to 9.0 and thermal stability up to 70 °C for 1.0 h. The presence of EDTA and EGTA decreased the activity of HtGH9 and also shifted the melting curve peak from 93 °C to 88 °C indicating that the enzyme inherently possesses metal ions, which play role in catalysis and structural stability. The TLC analysis of HtGH9 hydrolysed Avicel showed the presence cellotetraose indicating the processive endoglucanase activity in HtGH9. The module, HtGH9t alone exhibited very low level of activity (1.22 U/mg) against lichenan. It partially recovered (51%) the enzyme activity in presence of equimolar concentration of HtCBM3c. Non-denaturing gel electrophoresis revealed a non-covalent binding interaction between the catalytic HtGH9t module with HtCBM3c module and their physical association showed the partial recovery of its endoglucanase activity. This study confirmed that the physical association of the catalytic module HtGH9t with HtCBM3c is necessary for HtGH9 to efficiently hydrolyze the cellulosic substrates.