Improvement Thermal Stability of D-Lactate Dehydrogenase by Hydrophobin-1 and in Silico Prediction of Protein-Protein Interactions.

Hydrophobins are small surface-active proteins. They can connect to hydrophobic or hydrophilic regions and oligomerize in solution to form massive construction. In nature, these proteins are produced by filamentous fungi at different stages of growth. So far, researchers have used them in various fields of biotechnology. In this study, recombinant hydrophobin-1 (rHFB1, 7.5 kDa) was used to stabilize recombinant D-lactate dehydrogenase (rD-LDH, 35 kDa). rD-LDH is a sensitive enzyme deactivated and oxidized by external agents such as O2 and lights. So, its stabilization with rHFB1 can be the best index to demonstrate the positive effect of rHFB1 on preserving and improving enzyme's activity. The unique ability of rHFB1 for interacting with hydrophobic regions of rD-LDH was predicted by protein-protein docking study with ClusPro and PIC servers and confirmed by fluorescence experiments, and Colorless Native-PAGE. Measurement of thermodynamic parameters allows for authenticating the role of rHFB1 as a thermal stabilizer in the protein-protein complex (rD-LDH@rHFB1). Interaction between rHFB1 and rD-LDH improved half-life of enzyme 2.25-fold at 40 °C. Investigation of the kinetic parameters proved that the presence of rHFB1 along with the rD-LDH enhancement strongly the affinity of the enzyme for pyruvate. Furthermore, an increase of Kcat/Km for complex displayed the effect of rHFB1 for improving the enzyme's catalytic efficiency.