Molecular Cloning, Overexpression, and Enzymatic Characterization of Glycosyl Hydrolase Family 16 β-Agarase from Marine Bacterium Saccharophagus sp. AG21 in Escherichia coli

Abstract

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

Cloning and Characterization of a Novel Agarase from a Newly Isolated Bacterium Simiduia sp. Strain TM-2 Able to Degrade Various Seaweeds

“…Interestingly, in the presence of Ca 2+ ion, the thermostability of AgaTM2 increased (Fig. 6d), suggesting that the stability of AgaTM2 may be enhanced by the binding of bivalent ions in the catalytic cleft at a site opposite the substrate binding site, as reported previously for GH26 mannanase from Bacillus subtilis, GH5 cellulase from B. subtilis 168, and rAgy1 from Saccharophagus degradans AG21 [36][37][38][39].…”

Recombinant β-agarases: insights into molecular, biochemical, and physiochemical characteristics

“…The recombinant β-agarases belong to the glycoside hydrolase (GH) family such as GH16, GH39, GH50, GH86, and GH118. Predominantly, the recombinant β-agarases were from marine microbes and their genera include Agarivorans (Liu et al 2014a, b;Lee et al 2012;Lin et al 2012), Alteromonas (Seo et al 2014;Chi et al 2014), Aquimarina (Lin et al 2017), Catenovulum (An et al 2018;Cui et al 2014;Xie et al 2013), Cellulophaga (Ramos et al 2018), Flammeovirga (Dong et al 2016;Hou et al 2015;Yang et al 2011;Chen et al 2016;Di et al 2018), Gayadomonas (Lee et al 2018;Jung et al 2017a, b), Microbulbifer (Su et al 2017), Micrococcaceae (Xu et al 2018), Pseudoalteromonas (Chi et al 2015a, b;Oh et al 2010a, b), Pseudomonas (Hsu et al 2015), Saccharophagus (Kim et al 2010(Kim et al , 2017(Kim et al , 2018Lee et al 2013), Simiduia (Tawara et al 2015), Thalassomonas (Liang et al 2014), and Vibrio (Liao et al 2011) whereas few were from soil bacteria Streptomyces (Temuujin et al 2011(Temuujin et al , 2012, Cohnella (Li et al 2015) and one exclusive source was the activated sludges of sewage plant from which Cellvibrio (Osamu et al 2012) had been identified with agarolytic property.…”

Marine enzymes and food industry: insight on existing and potential interactions

“…The β-agarase gene agy1 from Saccharophagus sp. AG21, a marine bacterium, was cloned and overexpressed as a His-tagged recombinant β-agarase in E.coli (Lee et al, 2013). The recombinant enzyme, which fit within the GH16 family, had an estimated molecular weight of 69 kDa.…”