- Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01T
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Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60°C and had a Km of 3.78 mg mL-1 for agarose and a Vmax of 1.14 × 104 U mg-1. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50°C and retained 13% activity after incubation at 80°C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.
- Cui, Fangyuan,Dong, Sujie,Shi, Xiaochong,Zhao, Xia,Zhang, Xiao-Hua
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p. 2731 - 2747
(2014/06/10)
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- A simple method of preparing diverse neoagaro-oligosaccharides with β-agarase
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In order to prepare pure and well-defined oligosaccharides from agarose in a rapid and simple manner, an enzymatic degradation method was developed, which includes degradation with either recombinant β-agarase (EC 3.2.1.81) AgaA or AgaB and gel permeation chromatography. Agarose was degraded with AgaA at the optimized conditions, yielding 47% and 45% of neoagarotetraose and neoagarohexaose, respectively. These neoagaro-oligosaccharides were conveniently separated by consecutive column chromatography on Bio-Gel P2 or P6 and were identified by FACE. The structure of these neoagaro-oligosaccharides was confirmed by MALDI-TOF MS and 13C NMR spectroscopy.
- Li, Jingbao,Han, Feng,Lu, Xinzhi,Fu, Xiaoyan,Ma, Cuiping,Chu, Yan,Yu, Wengong
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p. 1030 - 1033
(2008/02/03)
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- 13C-N.M.R.-SPECTROSCOPIC INVESTIGATION OF AGAROSE OLIGOMRS
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A complete, unambiguous assignment of all of the 13C-n.m.r.-spectral signals of agarose oligomers produced by enzymic hydrolysis has been achieved.The 1J 13C-H coupling constants are reported, and the chemical shifts and coupling constants of both the agarose polymer and oligomers are compared.
- Rochas, Cyrille,Lahaye, Marc,Yaphe, Wilfred,Viet, Minh Tan Phan
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p. 199 - 208
(2007/10/02)
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