Cloning, expression, and characterization of three agarases in Vibrio natriegens WPAGA4

Abstract:

[Objective] To clone and express three β-agarase genes agaW3418, agaW3419, and agaW3472 in Vibrio natriegens WPAGA4 isolated from the deep-sea sediment and study the enzymatic properties of the proteins. [Methods] The three agarase genes were expressed in Escherichia coli BL21(DE3) cells, and the 3,5-dinitrosalicylic acid (DNS) method was employed to measure the activities of the recombinant agarases. [Results] The three agarases, AgaW3418, AgaW3419, and AgaW3472, belonged to the GH50 family, with the highest activities at 50, 60, and 30℃ and pH 6.0, 7.0, and 7.0, respectively. AgaW3472 showed the highest activity and maintained good stability at 20℃. The highest production of AgaW3472 was detected in the super optimal broth (SOB) medium with 1% (W/V) lactose as the carbon source, 20 mmol/L MgCl₂, and 0.1 mmol/L isopropyl-β-d-thiogalactoside (IPTG) at the induction temperature of 37℃. [Conclusion] The agarase AgaW3472 has high enzyme activity, low temperature adaptability, and good stability, serving as a potential bio-tool for the development of agar degradation-related industries. The three recombinant agarases provided a foundation for understanding the agarose metabolism in V. natriegens WPAGA4 and the role of this strain in the marine carbon cycling.

Key words:agarose;agarose oligosaccharides;Vibrio;cold adaptation