Expression of an endo-type β-agarase AgaDcat in Pichia pastoris and its biochemical characterization

Abstract

Neoagarooligosaccharides (NAOS), derived from the hydrolysis of agarose by β-agarases, have been found to have various biological activities. Herein, AgaDcat, originated from marine agar-degrading bacterium Zobellia galactanivorans, was for the first time successfully recombined and expressed in a eukaryotic (Pichia pastoris) system. Compared with other endo-β-agarase, AgaDcat has potential applications in producing neoagarotetraose (NA4) and neoagarohexaose (NA6) effectively because of its product specificity. The optimal temperature and pH of AgaDcat expressed in P. pastoris were 50 °C and 7.0, respectively. The maximum hydrolysis of agarose was achieved after 8h reaction at 45 °C and pH 7.0 using 750 U/g β-agarase. Through regulating hydrolysis conditions, NAOS with controllable degrees of polymerization (mainly NA4 and NA6) could be obtained. The Km value of AgaDcat expressed in P. pastoris was 1.377 mg/mL, which was significantly lower than that of AgaDcat expressed in Escherichia coli (6.358 mg/mL). Moreover, the melting temperature of AgaDcat expressed in P. pastoris (53.1 °C) was 4.5 °C higher than that of AgaDcat expressed in E. coli. AgaDcat expressed in P. pastoris exhibited higher thermal stability and substrate affinity than that expressed in E. coli, which could be ascribed to the glycosylation of proteins during translation modification period in the yeast expression system.