Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801

•We cloned and characterised a novel β-agarase from Agarivorans gilvus WH0801.•Compared with other known β-agarases, the identity of amino acid sequence was 53%.•The agarase was purified and characterised.•An exo-glycoside agarase which produced neoagarotetraose was first revealed.•This study laid a foundation for the preparation of neoagarotetraose in future. AgWH50A, a novel β-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105kDa), including a 21-amino acid signal peptide. AgWH50A shares the highest amino acid sequence homology with AgaD02 from Agarivorans sp. QM38 (53%). The recombinant agarase gene was expressed in Escherichia coli and purified by affinity chromatography. Maximum enzymatic activity (Km 5.97mg/mL and Vmax 0.781U/mg) was observed at pH 6.0 and 30°C. Using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography, we analysed the hydrolysis products and concluded that AgWH50A is a neoagarotetraose-forming β-agarase, which can cleave agarose into neoagarotetraose. This novel agarase has potential applications in the industrial production of neoagarotetraose and provides a new agarose hydrolysis model for future research.