Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on beta-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K-T307I were approx. 140, 190 and 200%, respectively, of wild type beta-agarase (661 U/mg) at 40A degrees C. All three mutant enzymes were stable up to 50A degrees C and E99K-T307I had the highest thermostability. The melting temperature (T (m)) of E99K-T307I, determined by CD spectra, was increased by 5.2A degrees C over that of the wild-type enzyme (54.6A degrees C). Activities of both the wild-type and E99K-T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl2. The E99K-T307I enzyme was stable at 55A degrees C with 1 mM CaCl2, reaching 260% of the activity the wild-type enzyme held at 40A degrees C without CaCl2.