Directed Evolution Of A Novel β-agarase

We have found a novel extracellularβ-agarase AgaB from Marine Pseudoalteromonas sp. CY24 recently. It has no significant sequence similarity with any known protein, including currently known glycoside hydrolases. The novel enzyme degrades agarose to generate neoagarooctaose and neoagarodecaose as main products. It apperars to have a large substrate binding cleft that accommodates twelve sugar units. And this novelβ-agarase hydrolyzes glycosidic bond with inversion of anomeric configuration in contrast to other known agarases which are retaining type. However, the poor thermostability (when reaction temperature is above 35°C, its catalytic activity will lose quickly) inhibited the wide use of AgaB in industry. In former research, we have constructed a thermostable mutant, called M446, but its catalytic activity decreases more than 20% compared to the wild tye. The aim of this paper is to improve the catalytic activity of M446 using directed evolution, while its thermostability and other characters are well maintained.The two key steps of directed evolution are the generation of molecular diversity and the building of high throughput screening method. Error prone PCR and chemical mutation are used to generate gene diversity and a constitutive expression vector pBS-ks(sv)is used to creat the mutants library. Chemical reagent EMS usually generate G mutation while error prone PCR usually generate A and T mutation. The screening strategy used in this study is based on clear zones around colonies, which is formed by extracellular agarase hydrolyzing agar. Larger clear zone represents higher catalytic activity, thermostability or increased quantity of enzyme. Therefore, after incubation at 37°C overnight, clones which show larger clear zone dimension/colony dimension than M446 were verified further by measuring the enzymes'residual activity using DNS method. Finally we got a mutant strain named M117, which had three folds catalytic activity of M446 but retained the other characters of M446. DNA sequence analysis reveales that M446 containes two amino acid transitions (R9K and I111V) and two synonymous mutations. Since the former site (R9K) is located in the signal peptide,which is cut down during transmembrane, the main reason that results in the improvement of catalytic activity should be attributed to the 111th substitution.To verify the mechanism of M117, the kinetic parameters were tested. The results showed that the Km of M117 decreases more than 14 folds compared to M446, while the Kcat has no obvious change. This indicated that the increasement in substrate binding ability conduced to the higher catalytic activity of M117. We presumed that the 111th site might be around the substrate binding site or be part of it. Both of Val and Ile are hydrophobic amino acids, but the former one has shorter side ligand and might has smaller spatial hindrance of substrate binding. On the other hand, due to the similarity of the two amino acids, this substitution doesn't led to other character changes.To summarize, we have used directed evolution to create an AgaB mutant with increased catalytic activity and thermostability. The higher specific activity of this AgaB mutant, in conjunction with its high thermostability and product specificity, will allow it to have potential applications in such areas as food, cosmetic, and medical industries. Moreover, the success of the directed evolution method used in this paper will open a new phase of research on the structure-function relationship ofβ-agarases.