title: Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801.
abstract: AgWH50A, a novel β-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105 kDa), 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 5.97 mg/mL and Vmax 0.781 U/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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Liu
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: xzhmao@ouc.edu.cn.
authors.forename: Xiangzhao
authors.initials: X
authors.lastname: Mao
authors.affiliation: College of Marine Science, Shandong University at Weihai, Weihai 264209, China.
authors.forename: Zongjun
authors.initials: Z
authors.lastname: Du
authors.affiliation: Laboratory for Advanced Materials and Institute of Applied Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors.forename: Bozhong
authors.initials: B
authors.lastname: Mu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D034241:Alteromonadaceae / Q000737:chemistry* / Q000201:enzymology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005697:Galactosides / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry*; D016366:Open Reading Frames; D010802:Phylogeny; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012685:Sepharose / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005697:Galactosides; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C081798:neoagarotetraose; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase
keywords: Agarivorans gilvus; GH50; Neoagarotetraose; β-Agarase