Targeted Directed Evolution of an α-L-Rhamnosidase on Hesperidin Through Six-Codon Combinatorial Mutagenesis

Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize the labor-determining screening effort remains a challenge. Herein, we propose the six-codon combinatorial mutagenesis (SCCM) strategy by using the BMT degeneracy codons encoding six amino acids with different chemical properties as the building blocks for the randomization of the amnio acid motif. SCCM requires only a small library of 646 clones for 95% coverage at the three-residue motif compared to conventional NNK degeneracy codons encoding all 20 canonical amino acids and requiring the screening of nearly 100,000 clones. SCCM generates a suitable number of mutant libraries, providing a new strategy for reducing the screening workload of saturated combination mutations in enzyme engineering. Using this approach, the α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron had been successfully engineered for improving the hydrolytic activity on natural flavonoid diglycoside hesperidin via targeted directed evolution at the motifs positioning the entrance of the substrate-binding pocket. The results indicate that the conversion rates of the four mutants on hesperidin were increased by more than 30% compared with the wild type using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of the mutant TM1-6-F5 was 1.4-fold higher than that of the wild type.