2.2 Expression and characterization of mutant candidates with better catalysis efficiency and thermostability
The seven mutated enzymes were obtained by site-directed mutagenesis and further expressed in Pichia pastoris. Similar to the wild type (recombinant Rha1, named as r-Rha1), the purified mutant enzymes showed a molecular mass of approximately 100 kDa (Fig. S2). Fig. 2A showed the relative hydrolytic activities of the mutants compared with the wild type r-Rha1. The mutants V302N, S303V, S356I and D525G show lower hydrolytic activities compared to r-Rha1. On the contrary, the hydrolytic activities of A355N, S356Y and D525N were noticeably enhanced by 45%, 80% and 180% than that of r-Rha1, respectively. To further investigate the effects of A355N, S356Y and D525N on the catalytic machinery of enzyme, the kinetic behaviors of A355N, S356Y and D525N onp NPR were evaluated. As shown in Table 2, bothK m and k cat values of A355N, S356Y and D525N were lower than the values of r-Rha1, whereask cat/K m values of all three mutants were increased. The mutant D525N showed the highest hydrolytic activity and catalytic efficiency among the mutants and r-Rha1. Specifically, D525N increased 2.85-fold of hydrolytic activity, and 1.54-fold of k cat/K mvalue compared to the wild type r-Rha1.
To gain a deeper insight of the mutagenesis effects of A355N, S356Y and D525N on the characteristics of enzyme, the optimum temperatures and thermostabilities were examined. Similar to wild type r-Rha1, the optimum temperatures of three mutants were 60ºC (Fig. 2A). While the mutants A355N and S356Y showed markedly weaker thermostabilities than r-Rha1 (Fig. 2C), mutant D525N exhibited slightly stronger thermostability than r-Rha1. When the thermostabilities of r-Rha1 and its mutants were tested at 60ºC, 65ºC and 70ºC, the half-lives of D525Nare shown to be 42 min, 2 min and 1 min longer (T1/2 ) than r-Rha1(Fig. 2B(b-d)), respectively. Thus, D525N was identified to have both improved thermostability as well as increased enzyme activity, which demonstrates our screening method is helpful in identifying mutants both improved enzyme activity and favorable thermostability.