Cloning Expression and Biochemical Characterization of a Novel Acidic GH 16 b Agarase AgaJ 11 from Gayadomonas joobiniege G 7
A novel b agarase AgaJ11 belonging to the glycoside hydrolase (GH) 16 family was identified from an agar degrading bacterium Gayadomonas joobiniege G7. AgaJ11 was composed of 317 amino acids (35 kDa) including a 26 amino acid signal peptide, and had the highest similarity (44 identity) to a putative b agarase from an agarolytic marine bacterium Agarivorans albus MKT 106. The agarase activity of purified AgaJ11 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ11 activity were determined to be 4.5 and 40 degC, respectively. Notably, AgaJ11 is an acidic b agarase that was active only at a narrow pH range from 4 to 5, and less than 30 of its enzymatic activity was retained at other pH conditions. The Km and Vmax of AgaJ11 for agarose were 21.42 mg/ml and 25 U/mg, respectively. AgaJ11 did not require metal ions for its activity, but severe inhibition by several metal ions was observed. Thin layer chromatography and agarose liquefying analyses revealed that AgaJ11 is an endo type b agarase that hydrolyzes agarose into neoagarohexaose, neoagarotetraose, and neoagarobiose. Therefore, this study shows that AgaJ11 from G. joobiniege G7 is a novel GH16 b agarase with an acidic enzymatic feature that may be useful for industrial applications.