Effects of mutations of non-catalytic aromatic residues on substrate specificity of Bacillus licheniformis endocellulase cel12A.

Cel12A , a gene that encodes an endocellulase of Bacillus licheniformis , was cloned and overexpressed in E. coli DE3. The rBLCel (wild-type recombinant cellulase) enzyme was assayed and characterized for its physical properties, including its optimum pH (9.0), temperature (50 °C), thermostability (175 h) and kinetic parameters ( K m 16.38 ± 0.550 mg ml −1 ; k cat 134.91 ± 0.377 min −1 and k cat /K m 8.23 ± 0.026 ml min −1  mg −1 ). To determine the role of conserved amino acids in the substrate binding pocket, five single mutations and one double mutation were studied for their effects on substrate specificity. All six purified engineered enzymes were assayed and compared to rBLCel regarding their catalytic activity with a specific substrate (CMC). All of the recombinant enzymes (wild type and engineered) were also assayed with non-specific substrates, such as xylan, avicel and filter paper (FP). Out of five point-mutant enzymes, three mutants (W197A, W98A and Y89A) showed reduced specific activity compared to rBLCel. However, the W139A and W53A mutants exhibited a 67% and 4% relative increase in their specific activity with a specific substrate (CMC), respectively. After replacing aromatic residues, the affinity towards the CMC increased in the case of the W139A and W53A mutants, indicating that the aromatic residues play a key role in substrate affinity and binding. One of the significant results that we obtained was that the increase in activity was additive in the double mutant enzyme, W139A_W53A, which showed 1216% higher activity than rBLCel on filter paper (FP). The activity of all of the mutated enzymes with non-specific substrates indicates that the substrate binding pocket is altered and has become flexible toward other substrates after mutation. Moreover, given the importance of the two enzymes in degrading lignocellulose, one bifunctional enzyme endowed with the two catalytic activities may be of great advantage. Despite the broad spectrum of cellulases being isolated, no single enzyme is completely suitable as it is, for the hydrolysis of lignocellulose. These enzymes have been used due to their biotechnological applications in several industries, including bio pulping wood, treatment of animal feed to increase digestibility, agro-processing, juice processing and baking. [ABSTRACT FROM AUTHOR]