Cloning, Expression, and Biochemical Characterization of a Novel Acidic GH16 β-Agarase, AgaJ11, from Gayadomonas joobiniege G7

Abstract

A novel β-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 β-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 °C, respectively. Notably, AgaJ11 is an acidic β-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 K and V 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 β-agarase that hydrolyzes agarose into neoagarohexaose, neoagarotetraose, and neoagarobiose. Therefore, this study shows that AgaJ11 from G. joobiniege G7 is a novel GH16 β-agarase with an acidic enzymatic feature that may be useful for industrial applications.

Biochemical Characterization of a Novel GH86 ��-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7

“…A Novel Agarase (AgaJ5) of G. joobiniege G7 There are 12 putative agarase genes in G. joobiniege, a marine bacterium with strong agar-degrading ability [30]. Among them, we have characterized two novel β-agarases, AgaJ9 and AgaJ11 [3,27]. AgaJ9 has cold-adapted agarase activity, and AgaJ11 is an acidic β-agarase.…”

“…After evaporation of the methanol, the molecular mass of the extracted oligosaccharides was determined using a matrixassisted laser desorption ionization time-of-flight mass spectrometer (Autoflex III, Germany). 2-(4'-Hydroxybenzeneazo)benzoic acid was used as the matrix [27].…”

“…Alteromonas [11], Pseudomonas [12], Gayadomonas [3], Vibrio [13,14], Pseudoalteromonas [15], Zobellia [16], Catenovulum [17], Agarivorans [18], Cohnella [19], Bacillus [20], Thalassomonas [21], Streptomyces [22,23], Stenotrophomonas [24], Simiduia [25], and Flammeovirga [26]. They have been classified into a number of glycoside hydrolase (GH) families, including GH16 [27], GH39 [3], GH50 [26], GH86 [28], and GH118 [18]. Various GH16 β-agarases have been extensively studied by biochemical analyses [1,11,15,27].…”

“…They have been classified into a number of glycoside hydrolase (GH) families, including GH16 [27], GH39 [3], GH50 [26], GH86 [28], and GH118 [18]. Various GH16 β-agarases have been extensively studied by biochemical analyses [1,11,15,27]. In other GH families, such as GH86, few β-agarases have been biochemically characterized [28,29].…”

“…The β-agarase AgaJ9, a member of GH39, is a cold-tolerant enzyme having more than 80% of its activity at 5°C [3]. Another GH16 β-agarase, AgaJ11, is an acidic β-agarase that has agarase activity only at the acidic pH range of 4-5 [27].…”