The 45th Academic Congress of Taiwan Association for Food Science and Technology

Expression and characterization of a β-agarase from Paenibacillus agarexedens in Escherichia coli

HJ Lin, WZ Huang, JF Lai, HZ Zeng, ZW Chen, JH Lin, JP Wang

Agar, which is composed of agarose and agaropectin, is the main component of the cell walls of some marine red algae. From the structural point of view, agar is a linear sugar chain consisting of repetitive units of 1-4-linked β-D-galactose and 1-3-linked-3,6-anhydro-α-L-galactose. Agarases are hydrolytic enzymes that degrade agar into oligosaccharides. According to the cleave pattern, agarases are classified into α-agarase (EC 3.2.1.158) and β-agarase (EC 3.2.1.81). The α-agarases cleave the α-L-(1,3) linkages of agarose to produce agarooligosaccharides, whereas the β-agarases cleave the β-D-(1,4) linkages of agarose to produce neoagarooligosaccharides. Previous studies showed that Paenibacillus agarexedens, isolated from meadow soil, is an agarolytic organism. However, the enzymatic and genetic characterization of agarases from P. agarexedens have not yet been studied. The aim of this study was to clone and express a β-agarase gene (agaB-4) from P. agarexedens in Escherichia coli, and to purify and characterize the recombinant β-agarase. The agaB-4 was predicted by using next generation sequencing approach. The open reading frame of agaB-4 consisted of 2,652 bp encoding 883 amino acids, with only 40% amino acid sequence identity with known agarases. The agaB-4 was cloned and expressed in E. coli. His-tagged recombinant AgaB-4 (rAgaB-4) could be purified from soluble fraction of E. coli cell lysate by immobilized metal-ion affinity chromatography. The rAgaB-4 showed optimum activity at pH 6.0 and 55ºC. The product of agarose hydrolysis by rAgaB-4 was confirmed to be neoagarotetraose. The results of substrate specificity showed that rAgaB-4 can degrade agar, agarose, and low melting agarose. The results from the present study indicate that rAgaB-4 has potential applications in agar degradation for production of neoagarotetraose.