Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Abstract

Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a Km of 3.78 mg mL−1 for agarose and a Vmax of 1.14 × 104 U mg−1. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.

Agarolytic bacteriumPersicobactersp. CCB-QB2 exhibited a diauxic growth involving galactose utilization pathway

“…Based on amino acid sequence similarities, putative genes assigned β-agarases function have been classified into four families: GH16, GH50, GH86, and GH118 (Cui et al, 2014;Martin, Portetelle, Michel, & Vandenbol, 2014). Four agarases (PdAgaA, PdAgaB, PdAgaC, and PdAgaD) were identified in the QB2 genome using the Rapid Annotation using Subsystem Technology (RAST) annotation server while two agarases were found from Persicobacter sp.…”

“…Many agarolytic bacteria have been identified from seawater, marine algae, and marine sediments, including Agarivorans (Fu, Lin, & Kim, 2008;Lin et al, 2012), Alteromonas (Potin et al, 1993), Catenovulum (Cui, Dong, Shi, Zhao, & Zhang, 2014), Cytophaga (Duckworth & Turvey, 1969), Flammeovirga (Hou, Chen, Chan, & Zeng, 2015;Yang et al, 2011), Microbulbifer (Jonnadula & Ghadi, 2011;Miyazaki et al, 2008;Ohta et al, 2004), Microscilla (Naganuma, Coury, Polne-Fuller, Gibor, & Horikoshi, 1993;Zhong et al, 2001), Paenibacillus (Mei et al, 2014), Pseudoalteromonas (Chi, Park, Kang, & Hong, 2014;Lu et al, 2009;Oh et al, 2010;Vera, Alvarez, Murano, Slebe, & Leon, 1998;Xavier Chiura & Kita-Tsukamoto, 2000), Pseudomonas (Belas, Bartlett, & Silverman, 1988;Morrice, McLean, Williamson, & Long, 1983), Pseudozobellia (Nedashkovskaya et al, 2009), Saccharophagus (Ekborg et al, 2006), Vibrio (Sugano et al, 1993;Zhang & Sun, 2007), and Zobellia (Jam et al, 2005). These agarolytic bacteria metabolize agar primarily with β-agarase.…”

A Novel Neoagarotriose-Producing Agarase from the Marine Bacterium Gilvimarinus Agarilyticus JEA5

“…The β-agarases are classi ed into four families on the basis of their amino acid sequence homology: glycoside hydrolase 16 (GH16), GH50, GH86, and GH118 [4]. To date, many β-agarases have been found from many different genera including Agarivorans [5], Alteromonas [6], Bacillus [7], Catenovulum [8], Pseudoalteromonas [9], Pseudomonas [10], Vibrio [11], Streptomcyes [12], Saccharophagus [13] and Gilvimarinus [14].…”

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

“…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].…”