Cloning, expression and production optimization of an agarase Aga2660 from a deep sea bacterium Flammeovirga pacifica WPAGA1

Abstract:

[Background] Agaro-oligosaccharides (AOS) have become a research hotspot in cosmetics, food, medicine and other fields. Biological enzyme method is considered as an efficient method to prepare AOS. [Objective] The agarase gene aga2660 was obtained from Flammeovirga pacifica WPAGA1. The gene aga2660 was cloned and transformed to be expressed in Escherichia coli. The properties of recombinant enzyme and enzymatic hydrolysate were analyzed. [Methods] The pure expression product was obtained by clone expression and nickel column purification. Enzymatic products of the agarase were analyzed by thin-layer chromatography (TLC) and ion chromatography (IC). The optimization of enzyme production conditions was carried out in a 5 L fermentor using the strategies of exponential feeding in the feeding stage and continuous feeding of lactose in the induction stage. [Results] The elected gene aga2660 possessed typical sequence characteristics of glucoside hydrolase family 50 (GH50). The end-product of agar degradation by Aga2660 was neoagarobiose. The optimum temperature and pH of Aga2660 was 30℃ and 7.0, respectively. Meanwhile, Aga2660 showed outstanding temperature and pH stability. Activities of Aga2660 were enhanced by Mn²⁺, Ca²⁺ and Mg²⁺ (1 mmol/L). The enzyme activity reached 11.81 U/mL after fermentation optimization strategy, which was 13.2 times higher than that before optimization. [Conclusion] The agarase Aga2660 was an agarase of GH50 family and indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo. Its enzymolysis product is neoagarobiose, which lays a good foundation for large-scale preparation of AOS with single polymerization degree.

Key words:Flammeovirga pacifica WPAGA1;agarase;neoagarobiose;fermentation optimization