Abstract
•A highly thermostable and alkaline lichenase was purified.•The purified lichenase was stable over a broad range of temperature and pH.•A genomic library was screened to sort out lichenase-active clones.•Lichenase gene showed 98% homology with Bacillus subtilis 168 lichenase.•3D structure suggests that Val69 stabilizes a Ca2+ binding site resulting in a higher stability.
A highly thermostable and alkaline lichenase was isolated from the newly isolated strain Bacillus UEB-S. Single step purification was achieved by heating the enzyme extract for 30min at 90°C. The enzyme was a monomeric protein with a molecular weight of 28kDa. The optimal temperature and pH for UEB-S lichenases activity were 60°C and 6.0, respectively. More remarkably, the purified lichenase was stable over a broad range of temperature and pH. It retained more than 60% of its activity after incubation at 90°C for 30min. Substrate specificity studies revealed that the enzyme is a true lichenase. A genomic library was screened. It allows the identification of a gene that encodes a putative lichenase showing 98% identity with the lichenase from Bacillus subtilis 168. Sequence comparison revealed that the two enzymes differed by two mutations at positions 69 and 83, where Val69 and Ser83 are replaced by Met and Ala amino acids, respectively. Therefore, a theoretical structural model was built using the lichenase from B. subtilis 168 Pdb code (3o5sA) structure as template. Comparison of the two 3D structures suggested that Val69 stabilizes a calcium-binding site and could be involved in the higher stability of the enzyme.