In-silico structural analysis affecting thermostability in recombinant psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12

Abstract

Cold-adapted enzymes are significant with structure flexibility and high catalytic activity at low temperature. High structural flexibility could be due to combination of several features such as weak intramolecular bonds, decreased compactness of hydrophobic core and reduced number of proline and arginine residues. However, to compensate the structural flexibility, cold-adapted enzymes are also thermolabile which causes them to be easily inactivated at elevated temperature. Therefore, it would be more interesting and beneficial if more stable cold-adapted enzymes are produced to fulfill the industrial needs. In this study, a novel cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was rationally designed to improve their thermostability thus make them more resistant to increased temperature. Four CHI II mutants were designed through rational design named as A157Q, I134P, mutant Loop and Y257R by manipulating the structural hydrophobicity, introduction of proline in the loop regions, introduction of arginine salt bridges and loop shortening. Mutant Loop was designed by removing 9 residues in loop regions thus makes loop involved became shorter. Stability of all mutants was first predicted through a computational approach where all structures were subjected to 10 ns molecular dynamics simulation at three temperatures; 273 K, 288 K and at 300 K. Based on the simulation, it was found that mutants I134P, mutant Loop and Y257R exhibited structural stability at 300 K. This conclusion was made based on low and stable root-mean square deviation (RMSD) value at 300 K in comparison to RMSD values at 288 K and 273 K. Low RMSD values indicated mutant structure experienced low structural deviation throughout the simulation. Besides, this observation is correlated with reduction of structure compactness (radius of gyration), reduced solvent accessible surface area and increased numbers of hydrogen and salt bridges. However, mutant A157Q experienced structure destabilization at 300 K. Substitution of helix-preferred residue, alanine with a thermolabile residue, glutamine had caused A157Q structure become loosely packed at 300 K indicating a thermal denaturation. To support the theoretical model, CHI II and all mutants were then cloned into Pichia pastoris expression vector pPICZαC and expressed in P. pastoris (GS115).