Expression and characterization of the agarase gene aga3311 from an Antarctic bacterium

Abstract:

[Objective] The complete genome of the agarolytic bacterium Pseudoalteromonas sp. NJ21 from Antarctic sample was analyzed by bioinformatics methods and putative agarase aga3311was screened. Expression and characterization of the putative agarase aga3311 were studied.[Methods] Gene aga3311 was cloned and expressed by genetic engineering method firstly; then, the recombinant enzyme was purified by Ni-NTA chromatography and the characterization of recombinant enzyme was determined by dinitrosalicylic acid method; the hydrolysis product of recombinant enzyme Aga3311 was analyzed by thin-layer chromatography (TLC) and mass spectrometry (MS).[Results] The recombinant expression vectors (pET-30(a)+aga3311) was overexpressed in E. coli BL21(DE3) and 30% of the recombinant protein was soluble. The purified agarase (Aga3311) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 87 kDa. The optimum temperature of the recombinant agarase was 35℃, and it maintained higher activity between 30 and 45℃, but the activity declined rapidly above 50℃, typical of thermal instability enzyme. The optimum pH was 7.0, and it maintained 50% of its maximum activity between pH 4 and 10. Aga3311 was significantly activated by Fe³⁺, Be²⁺, Zn²⁺ and Ca²⁺, especially Ca²⁺ doubled the enzyme activity. The pattern of agar hydrolysis of Aga3311 is an exo-β-agarase, producing neoagarobiose (NA2) as the final main product.[Conclusion] Aga3311 is an exo-β-agarase of Glyco_hydro_42 family, producing neoagarobiose (NA2) as the final main product.