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

• Main Authors: Zeng-Weng Chen, Hui-Jie Lin, Wen-Cheng Huang, Shih-Ling Hsuan, Jiunn-Horng Lin, Jyh-Perng Wang
• Format: Article
• Language: English
• Published: SpringerOpen 2018-03-01
• Series: AMB Express
• Subjects: β-Agarase; Paenibacillus agarexedens; Next-generation sequencing; Neoagarotetraose
• Online Access: http://link.springer.com/article/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 V max and K m 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.