Molecular Characterization of a Novel 1,3-α-3,6-Anhydro-L-Galactosidase, Ahg943, with Cold- and High-Salt-Tolerance from Gayadomonas joobiniege G7

Abstract

Agarose is a major constituent of agar, the main component of red algal biomass. Agar is a heterogeneous hydrophilic colloidal polysaccharide composed of alternately linked 3-O-linked β-D-galactopyranose (G) and 4-O-linked α-L-galactopyranose (L) [1, 2]. Agarose is a main component of agar, where L is substituted with 3,6-anhydro-L-galactose (L-AHG), forming a linear polymer with an average molecular mass of 120,000 Da [3]. Agar has been used as a dietary food and gelling agent for a long time in many Asian countries and has been generally recognized as safe by the United States Food and Drug Administration. Owing to the unique properties of thermal hysteresis in the sol-to-gel transition and its structural stability, agar has been widely used in bacterial plate culture as well as in electrophoretic and chromatographic supporting materials [4]. Recently, marine algal biomass has been highlighted in many studies because it is a valuable and sustainable resource that can substitute fossil-based chemical feed stocks, including petroleum [5]. In addition, agar can be used as a supporting material for enzyme or bacterial immobilization to enhance the stability of the system, which allows long-term operation [6, 7]. Agar can also be used to manufacture biodegradable polymers, such as bioplastics [8], and can be used in wet-fiber [9], eco-friendly biocleaning processes [10] as well as in medical treatments, such as microencapsulation [11], drug delivery [12], and bone generation [13]. In this sense, agar is expected to be widely used in the food and chemical industry as well as in the medical field. Agar can be degraded by chemical treatments or enzymatic hydrolysis for industrial applications. Because the 1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In parti...

NADP+-Dependent Dehydrogenase SCO3486 and Cycloisomerase SCO3480: Key Enzymes for 3,6-Anhydro-L-Galactose Catabolism inStreptomyces coelicolorA3(2)

“…The dried sample was extracted with methanol and analyzed using liquid chromatography/time-of-flight (LC-TOF) mass spectrometry (JMS-T100LP 4G, JEOL Ltd., Japan). Mass spectra in the m/z range 100–250 were obtained using electrospray ionization (ESI) in the positive-ion mode, under the conditions described [ 5 ].…”

“…Moreover, as the various biological activities of agar-derived oligosaccharides and agar decomposition products are discovered, the industrial use of agar is expected to expand [ 4 ]. Agarose, in nature, is mainly hydrolyzed by microbial beta-agarases into neoagarobiose (3,6-anhydro-L-galactosyl-α-1,3-D-galactose), which is further hydrolyzed by neoagarooligosaccharide (NAOS) hydrolase into monomers D‐galactose (D‐Gal) and 3,6‐anhydro‐L‐galactose (L-AHG) [ 5 ]. D‐Gal is a valuable chemical feedstock that can be converted into bioenergy or other useful chemicals [ 4 ].…”