Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801
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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.
Keywordsβ-Agarase Neoagarobiose GH50 Agarivorans gilvus
This work was supported by the National Science Foundation of China (31271923), the China Postdoctoral Science Foundation (2013T60687) and the Fundamental Research Funds for the Central Universities (201262021). The authors would like to thank Enago (www.enago.cn) for the English language review.
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