We have previously reported a draft genome sequence for the agar degrading bacterium G. agarilyticus JEA5 [18], and were the first to describe the molecular characteristics and biochemical properties of a β-agarase (Gaa16A) isolated from G. agarilyticus JEA5 [19]. Here, we describe Gaa16B, a novel neoagarotriose-producing β-agarase. This newly identified agarase possesses the typical functional domains characteristic of β-agarases belonging to the GH16 family. Almost all GH16 β-agarases contain a GH16 domain at the N-terminus and carbohydrate binding modules in the C-terminus of the protein [20]. Gaa16B exhibits both the GH16 domain and two carbohydrate VI modules at the N- and C-termini of the protein, respectively.
The Gaa16B amino acid sequence showed the highest identity and similarity with a carbohydrate binding protein from G. polysacchariticus (WP_049721028.1), which has not yet been characterized. The NCBI database contains three genomes from the Gilvimarinus genus; however, only one agarase (Gaa16A from G. agarilyticus JEA5) has been characterized thus far [19]. Compared to the amino acid sequences of characterized agarases, Gaa16B displayed the highest similarity to an agarase from Saccharophagus degradans 2–40 (with a sequence identity and similarity of only 55.9% and 70.9%, respectively).
To determine the optimum reaction conditions for this enzyme, we investigated the effects of temperature, pH, thermostability, and metal ions on rGaa16Bc function. It showed the highest agarolytic activity at 55 °C and pH 6, although it also exhibited relatively high agarolytic activity (80% or more) between pH 5–8. This high activity in a relatively wide pH range may be an advantage for the industrial use of rGaa16Bc. Furthermore, since agar hardens at temperatures below 40 °C, it is important to have high activity above 40 °C. rGaa16B showed optimum activities at these higher temperatures, as well as under neutral ionic conditions that do not require neutralization. These properties could be particularly advantageous for industrial use.
The kinetic characterization assays revealed that the Km value of rGaa16Bc (6.4 mg/mL) is only slightly higher than that of other reported GH16 β-agarases, which is probably due to the absence of the CBMs. To assess the agarolytic activity of Gaa16B, the gaa16b gene was amplified without the carbohydrate binding region and cloned into a pMal-c2x vector. Most of the expressed protein was insoluble when the full length recombinant Gaa16B (including the carbohydrate binding region) was cloned and expressed in E. coli, and the soluble fraction also showed very low agarolytic activity (data not shown). In contrast, the recombinant rGaa16B without the CBMs showed very high activity compared to full length Gaa16B. Other studies have also reported kinetic characterizations of GH16 β-agarases lacking the CBMs and reported similar findings. A recombinant agarase containing only the GH16 catalytic region from M. thermotolerans JAMB-A94 exhibited a higher Km value than the full length fusion protein [21]. In addition, Wang et al. reported that a recombinant Aga0917 lacking a CBM from Pseudoalteromonas fuligina YTW-15-1 showed a remarkably high (39.6 mg/ml) Km value compare to other known β-agarases [22].
The TLC results showed that agarose was rapidly fragmented by rGaa16Bc. In the early stages of the reaction, rGaa16Bc hydrolyzed the agarose to generate neoagarobiose, neoagarotetraose, neoagarohexaose, and various larger oligosaccharides. The amount of neoagarosaccharides smaller than neoagarohexaose increased in a time-dependent manner. This hydrolytic pattern suggested that rGaa16Bc functions as an endo-type β-agarase. It has been reported that endo-type agarases randomly degrade agarose and rapidly lower the viscosity of agarose solution, while exo-type agarases tend to produce single major products and gradually decrease the viscosity of agarose solutions [23].
The rGaa16Bc enzyme produced mainly neoagarotetraose and neoagarobiose. Interestingly, the predicted neoagarotriose spot was observed to lie between those of neoagarobiose and neoagarotetraose, but it was not in line with the spot of the agarotriose which was used as a standard. To verify that rGaa16Bc does indeed produce neoagarotriose, the molecular mass of the hydrolytic products was measured by LC/MS. Electrospray ionization mass spectrometry of the reaction product showed peaks at 347.0 m/z [neoagarobiose + Na]+, 469.2 m/z [neoagarotriose + H]+ and 629.1 m/z [neoagarotetraose - H]−. The molecular weight of agarotriose (C18H30O15) and neoagarotriose (C18H28O14) are 486.4 and 468.4 g/mol, respectively. The sequence of agarotriose is G-A-G (G, D-galactose; A, 3-6-anhydro-a-L-galactose), whereas that of neoagarotetraose is A-G-A. This is the first report to describe a neoagarotriose-producing agarase. Some α-agarases have been reported to produce agarotriose, but to our knowledge, there are no previous studies that have noted an agarase producing neoagarotriose. The α-agarase AgaD from Thalassomonas sp. LD5 was shown to hydrolyze agarotetraose and generated agarotriose with a molecular weight of 486 g/mol and the G-A-G structural arrangement [24]. In the current study, rGaa16Bc produced neoagarotriose after hydrolyzing agarose, neoagarohexaose, and neoagarotetraose. Furthermore, it generated neoagarotriose with a molecular weight of 468 g/mol and an A-G-A structure.