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.