Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR

In our previous work, a recombinant aflatoxin-degrading enzyme derived from Myxococcus fulvus (MADE) was reported. However, the low thermal stability of the enzyme had limitations for its use in industrial applications. In this study, we obtained an improved variant of recombinant MADE (rMADE) with enhanced thermostability and catalytic activity using error-prone PCR. Firstly, we constructed a mutant library containing over 5000 individual mutants. Three mutants with T-50 values higher than the wild-type rMADE by 16.5 degrees C (rMADE-1124), 6.5 degrees C (rMADE-1795), and 9.8 degrees C (rMADE-2848) were screened by a high-throughput screening method. Additionally, the catalytic activity of rMADE-1795 and rMADE-2848 was improved by 81.5% and 67.7%, respectively, compared to the wild-type. Moreover, structural analysis revealed that replacement of acidic amino acids with basic amino acids by a mutation (D114H) in rMADE-2848 increased the polar interactions with surrounding residues and resulted in a threefold increase in the t(1/2) value of the enzyme and made it more thermaltolerate.

Automated summary

An improved variant of recombinant aflatoxin-degrading enzyme (rMADE) with enhanced thermostability and catalytic activity was obtained using error-prone PCR. A mutant library containing over 5000 individual mutants was constructed, and three mutants with higher T-50 values compared to the wild-type rMADE were screened. The catalytic activity of two mutants, rMADE-1795 and rMADE-2848, was significantly improved compared to the wild-type.