Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Abstract

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and (13)C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.

Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH

“…The similar recognition mode was believed to be present in the hydrolysis of agarose as well. This hypothesis was further confirmed by 13 C NMR analysis of the anomeric configuration of their agarolytic products (Chi et al 2014;Park et al 2015). In present study, we also utilized p-nitrophenyl-α-Dgalactopyranoside and p-nitrophenyl-β-D-galactopyranoside to determine the mode of action of AgaW firstly.…”

“…Two GH16 β-agarases AgaG1 from Alteromonas sp. GNUM-1 (Chi et al 2014) The recombinant AgaW hydrolyzed neoagarohexaose into neoagarobiose and neoagarotetraose as two end products. c Changes in the viscosity of the recombinant AgaW reaction mixture.…”

“…The agarolytic activity was measured by the 3,5-dinitrosalicylic acid (DNS) method as previously reported (Chi et al 2014;Temuujin et al 2011), with slight modification. Briefly, 10 μl of the enzyme solution was mixed with 390 μl of 10 mM potassium phosphate buffer (pH 7.0) plus 0.5 % agarose and incubated at 50°C for 10 min.…”

Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides

“…Due to the broad substrate ranges of CAZyme families such as GH16, some homologs may however target other polysaccharides. For instance, A. stellipolaris, A. addita and strains Mac1/Mac2 encode a second GH16 with 99% sequence similarity to a β-agarase [104], probably opening further 'substrate niches' during degradation of algal biomass.…”

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

“…The kinetic parameters of K m and V max for different agarases are various. Most reported agarases exhibited a K m between 1 and 50 mg·mL −1 , and a V max between 10 and 1000 μM·min −1 ·mg −1 [27,30,31,32,33,34,35]. AgaML displayed a relatively higher specific activity than most of the reported agarases.…”