Intercalation of the disulfide bond between the A2 and A4 loop of Cellobiohydrolase (Cel7A) of Aspergillus fumigatus enhances catalytic activity and thermostability

Disulfide bond is important for maintaining the structural conformation and stability of the protein. Introducing new disulfide bond is a promising strategy for rational protein design. In this report, disulfide bond engineering has been applied to improve the stability of an industrially important enzyme, Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of A fumigatus origin. Disulfide by Design 2.0 (DbD2), an online tool, was used for the detection of the mutation sites and created four mutations (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) both inside and outside of the peripheral loops but, not in the catalytic region. The disulfide bond formed between the A2 and A4 loop of DSB3 showed higher thermostability (70% activity at 700C), higher substrate affinity (Km= 0.081mM) and higher catalytic activity (Kcat =9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild type AfCel7A (50% activity at 700C; Km= 0.128mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min -1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3:13.6 [A]; Wt: 5.3 [A]) at the product exit site; giving flexibility in the entrance region and mobility of the substrate. It may facilitate substrate entry into the catalytic tunnel and releases the product faster than the wild type. Whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop. ImportanceCellulases are important for their role in the production of bioethanol, the cleanest renewable replacement of fossil fuels. Engineering of the cellulases is a chalange to increase their catalytic activity and thermostability for production of cheap ethanol. In this report we have introduced disulfide bond and successfully increased the both thermostabilty and catalytic activity of AfCel7A.