Secretory expression and enzymatic characterization of recombinant Agarivorans albus -agarase in Escherichia coli

Abstract

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding -agarase from Agarivorans albus (Aa-agarase) in Escherichia coli. The synthetic -agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aa-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aa-agarase in Luria-Bertani broth containing 0.6mM isopropyl -D-1-thiogalactopyranoside for 9h at 37 degrees C. The expressed recombinant Aa-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding approximate to 10mg/L Aa-agarase. The purified recombinant Aa-agarase exhibited optimal activity at pH 7 and 40 degrees C, and its activity was strongly inhibited by Cu2+, Mn2+, Zn2+, and Al3+ ions. Furthermore, the K-M and k(cat) values for purified Aa-agarase were approximate to 0.02mM and approximate to 45/s, respectively. These kinetic values were up to approximately 15-100-fold lower than the K-M values reported for other agarases and approximately 7-30-fold higher than the k(cat)/K-M values reported for other agarases, indicating that recombinant Aa-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aa-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.