Abstract: Based on the codon preference of Escherichia coli,the gene of AgaO from Flammeovirga sp. MY04 was optimized and artificially synthesized. The new gene was cloned into the expression vector pET-30a(+)and transformed into E. coli BL21(DE3). Under the optimized expression condition,the recombinant protein rEAgaO was purified and its enzymatic properties were analyzed. The results showed that the recombinant agarase rEAgaO had the highest expression levels when induced by 0.1 mmol/L IPTG at 16 ℃ for 20 h. SDS-PAGE analysis indicated that more than 95% of the recombinant proteins were expressed in water-soluble form with a yield of approximately 1432.7 mg/L,which was increased by 10.9 folds compared with that of the original gene before optimization. The recombinant enzyme rEAgaO showed its optimal agarose-degrading activity at 45 ℃ and pH7.0 respectively. After an incubation below 40 ℃ for 2 h,rEAgaO retained more than 90% activity. It was stable in environments ranging from pH6.0 to 10.0 and retained more than 69.5% of its residual activity after incubation at 4 ℃ for 2 h. Moreover,the final degradation product of agarose by rEAgaO was identified as neoagarobiose by TLC analysis. Although codon optimization did not change the amino acid sequence of protein,it could significantly improve the yield and enzyme activity of water-soluble recombinant protein,which would benefit the production and application of related enzymes.