| Summary: | Up until now, the characterizations of GH50 agarases from <i>Vibrio</i> species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as <i>Vibrio natriegens</i> due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA–DNA hybridization. Two circular chromosomes in <i>V. natriegens</i> WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of <i>V. natriegens</i> WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in <i>E. coli</i> cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10–30 °C for 50 min. The <i>Km</i>, <i>Vmax</i>, and <i>kcat</i> values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s<sup>−1</sup>, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured <i>V. natriegens</i> WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.
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