Analysis of agar degradation gene and characterization of an agarase Aga0950 from a deep-sea bacterium Flammeovirga pacifica WPAGA1

Abstract:

[Objective] An agarase gene, aga0590, was found in the fine genomics mapping of a deep-sea bacterium Flammeovirga pacifica WPAGA1 using bioinformatics analysis. Recombinant Aga0950 was characterized with heterologous expression in E. coli. [Methods] Genomic sequencing of Flammeovirga pacifica WPAGA1 was performed by Illumina HiSeq2500 and agarase genes were annotated with BLAST. Subsequently, Aga0950 was obtained with heterologous expression in E. coli BL21 (DE3) and the agar-degrading activity was determined by dinitrosalicylic acid method. Enzymatic products of the agarase were analyzed by thin-layer chromatography and ion chromatography. [Results] Thirteen agarase genes with amino acid homologies from 60% to 85% were detected in the genome of Flammeovirga pacifica WPAGA1. The elected gene aga0950, which was aligned with highest identities of 67% in NCBI-nr database, possessed typical sequence characteristics of glucoside hydrolase family 16 (GH16). The specific activity of Aga0950 was 51770 U/mg, and the end-products of agar degradation by Aga0950 were neoagarotetraose (NA4) and neoagarohexaose (NA6). The optimum temperature and pH of Aga0950 was 50℃ and a range of 4.0 to 10.0, respectively. Meanwhile, rAga0950 showed outstanding temperature and pH stability. Activities of Aga0950 was enhanced by Co²⁺ and Mn²⁺ (1 mmol/L), while that was strongly inhibited by Cu²⁺. [Conclusion] Flammeovirga pacifica WPAGA1 has abundant polysaccharide-degrading genes. The agarase Aga0950 was indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo.

Key words:agarase;agaro-oligosaccharide;genome;Flammeovirga pacifica WPAGA1