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

Date Published: March 28, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Zeng-Weng Chen, Hui-Jie Lin, Wen-Cheng Huang, Shih-Ling Hsuan, Jiunn-Horng Lin, Jyh-Perng Wang.

http://doi.org/10.1186/s13568-018-0581-8

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.

Partial Text

Agar is a hydrophilic colloid extracted from the cell walls of red algae (Rhodophyceae), such as Gelidium spp., Gracilaria spp., and Porphyra spp. It is a heterogeneous polysaccharide which consists of agarose and porphyran (Chi et al. 2012). Agarose is a neutral polysaccharide that forms a gel; its molecular weight is approximately 120 kDa; and it consists of alternating β-d-galactose and 3,6-anhydro-α-l-galactopyranose linked by α-1,3 and β-1,4 glycosidic bonds (Armisén 1991; Yun et al. 2017). Porphyran, the non-gelling fraction, is a linear sulfated galactan; its composition is similar to that of agarose, except that some 3,6-anhydro-α-l-galactose are replaced with α-L-galactose-6-sulfate (Knutsen et al. 1994; Chi et al. 2012).

P. agarexedens is an agar-degrading bacterium that was isolated from meadow soil by Miehlmanni in 1972 (Uetanabaro et al. 2003). The culture conditions, physiological characteristics, and genetic characteristics of this bacterium have been studied. Agarase production by this bacterium has been proven by the appearance of depressions around bacterial colonies when cultured in solid culture medium containing agar. However, the type of agarase produced by this bacterium and the cellular localization, gene and enzyme characteristics, and applications of agarase have yet to be analyzed. In this study, the agarase gene of this bacterium was explored through whole-genome sequencing and bioinformatics analysis. Subsequently, cloning and expression of agaB-4 were performed to reveal the characteristics of the recombinant enzyme.

 

Source:

http://doi.org/10.1186/s13568-018-0581-8

 

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