Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-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 β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-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-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 9 h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ∼10 mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu²⁺, Mn²⁺, Zn²⁺, and Al³⁺ ions. Furthermore, the K_M and k_cat values for purified Aaβ-agarase were ∼0.02 mM and ∼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 Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.

KEYWORDS:

• Agarase
• Agarivorans albus
• characterization
• Escherichia coli
• recombination
• secretory expression

Abbreviations::

• Aaβ-agarase: β-agarase from Agarivorans albus
• E. coli: Escherichia coli

Additional information

Funding

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2015R1A2A1A15053693) and by a Chung-Ang University research Grant in 2014.