Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens

Abstract

In this study, a β-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0581-8) contains supplementary material, which is available to authorized users.

Characterization and Modeling of Thermostable GH50 Agarases from Microbulbifer elongatus PORT2

“…The positive effects of the divalent cations Ca 2+ and Mg 2+ on the activities of AgaA50 and AgaC50, respectively, and the concurrent negative effect of EDTA indicate that these two GH50 agarases are metal-dependent (Klebe 2013;Pereira et al 2017). This corresponds with the features of known thermostable GH50 agarases (Li et al 2015;Chen et al 2018Chen et al , 2019. In contrast, AgaB50 was not only deactivated by EDTA, but was also negatively affected by any of the ions or other chemicals added to the reaction.…”

“…Similar to the thermostable GH50 agarases derived from thermophilic bacteria inhabiting unique niches such as AgaL4 from Microbulbifer pacificus LD25 (isolated from saltwater hot spring, activator Ca 2+ ), AgaB-4 from Paenibacillus agarexedens (soil bacterium, activator Mn 2+ and Co 2+ ), and AgaW from Cohnella sp. LGH (soil bacterium, activator Mg 2+ , Ca 2+ , Na + , and K + ) (Li et al 2015;Chen et al 2018Chen et al , 2019, they maintained more than 75% of their activities after 1-h preincubation at 50 °C.…”

“…Among the GH50 agarases, only three thermostable enzymes have been described so far, namely AgaW and AgaB-4 from the soil bacteria Cohnella sp. LGH and Paenibacillus agarexedens, respectively (Li et al 2015;Chen et al 2018), and AgaL4 from Microbulbifer pacificus LD25, a bacterium isolated from a saltwater hot spring (Chen et al 2019). All these thermostable GH50 agarases are metal-dependent and release NA2, NA4, and a mixture of NA4 and NA2, respectively, from agarose.…”

Characterization and Application of a Recombinant Exolytic GH50A β-Agarase from Cellvibrio sp. KY-GH-1 for Enzymatic Production of Neoagarobiose from Agarose

“…Several GH50 family β-agarases were previously reported to cleave agarose by either exolytic activity alone or by a combination of exolytic and endolytic activities to produce NA2, NA4, or NA2/NA4. ,− Since GH50A homologues were present in all of the agar-degrading bacteria aligned, we reasoned that GH50A β-agarase was most likely to be associated with degradation of agarose into NA2 and/or NA4 and is an essential component of the agar-degrading enzyme machinery in Cellvibrio sp. KY-GH-1.…”

“…Comparing amino acid sequence similarities among bacterial agarases, the Carbohydrate-Active Enzymes (CAZyme) database classifies agarases into different glycoside hydrolase (GH) families: GH16, GH50, GH86, and GH118 for β-agarases, − as well as GH96 and GH117 for α-agarases. , GH16, GH86, and GH118 β-agarases are endolytic and generate neoagarotetraose (NA4)/neoagarohexaose (NA6), NA6/neoagarooctaose (NA8), and NA8/neoagarodecaose (NA10). GH50 β-agarases produce neoagarobiose (NA2), NA4, or NA2/NA4 via their exolytic and endolytic activities. ,− GH96 α-agarases produce agarobiose from agarose, and GH117 α-neoagarobiose hydrolase (α-NABH) catalyzes the hydrolytic degradation of NA2 to l -AHG and d -galactose …”