title: Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801.
abstract: agWH50C, a novel β-agarase gene, was cloned from Agarivorans gilvus WH0801 by degenerate PCR and nested PCR. The gene agWH50C comprized a 2,223-bp, encoding a protein of 740 amino acids. Sequencing results demonstrated that AgWH50C shared 45 % sequence identity with a well characterized β-agarase, Aga50D, from Saccharophagus degradans 2-40. The mature agarase was expressed in Escherichia coli and purified by affinity chromatography. The optimum pH and temperature for AgWH50C activity were 6.0 and 30 °C. The K m and V max values for agarose were 12.55 mg/ml and 1.17 U/mg. Analysis of the hydrolysis products using linear ion trap mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography confirmed that the reaction product of AgWH50c was α-neoagarobiose alone. Therefore, our novel agarase has the potential for industrial applications to produce neoagarobiose as well as provides a key β-agarase for fermentation of agar biomass.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Liu
authors.forename: Xiangzhao
authors.initials: X
authors.lastname: Mao
authors.forename: Meng
authors.initials: M
authors.lastname: Yang
authors.forename: Bozhong
authors.initials: B
authors.lastname: Mu
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D034241:Alteromonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; C068544:neoagarobiose; D006026:Glycoside Hydrolases; C019624:agarase