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Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801

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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.

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References

  • Araki C (1959) Seaweed polysaccharides. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interest. Pergamon Press, London, pp 15–30

    Google Scholar 

  • Du ZJ, Lv GQ, Alejandro PR, Miao TT, Xu QQ, Chen GJ (2011) Agarivoransgilvus sp. nov. isolated from seaweed. Int J Syst Evol Microbiol 61:493–496

    Article  CAS  Google Scholar 

  • Duckworth M, Yaphe W (1972) The relationship between structures and biological properties of agars. In: Nisizawa K (ed) Proceedings of the 7th international seaweed symposium. Halstead Press, New York, pp 15–22

    Google Scholar 

  • Fu XT, Kim SM (2010) Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8:200–218

    Article  CAS  Google Scholar 

  • Hehemann JH, Kelly AG, Pudlo NA, Martens EC, Boraston AB (2012) Bacteria of the human gut microbiomecatabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes. Proc Natl Acad Sci USA 109:19786–19791

    Article  CAS  Google Scholar 

  • Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877

    Article  CAS  Google Scholar 

  • Kim HT, Lee S, Lee D, Kim HS, Bang WG, Kim KH, Choi IG (2010) Overexpression and molecular characterization of Aga50D from Saccharophagusdegradans 2–40: an exo-type β-agarase producing neoagarobiose. Appl Microbiol Biotechnol 86:227–234

    Article  CAS  Google Scholar 

  • Kim HT, Lee S, Kim KH, Choi IG (2012) The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production. Bioresour Technol 107:301–306

    Article  CAS  Google Scholar 

  • Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci Biotechnol Biochem 61:162–163

    Article  CAS  Google Scholar 

  • Lee DG, Park GT, Kim NY, Lee EJ, Jang MK, Shin YG, Park GS, Kim TM, Lee JH, Lee JH, Kim SJ, Lee SH (2006) Cloning, expression, andcharacterization of a glycosidehydrolase family 50 β-agarase from a marine Agarivorans isolate. Biotechnol Lett 28:1925–1932

    Article  CAS  Google Scholar 

  • Liao L, Xu XW, Jiang XW, Cao Y, Yi N, Huo YY, Wu YH, Zhu XF, Zhang XQ, Wu M (2011) Cloning, expression, and characterization of a new β-agarase from Vibrio sp. Strain CN41. Appl Environ Microbiol 77:7077

    Article  CAS  Google Scholar 

  • Lin B, Lu G, Zheng Y, Xie W, Li S, Hu Z (2012) Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105. World J Microbiol Biotechnol 28:1691–1697

    Article  CAS  Google Scholar 

  • Long M, Yu Z, Xu X (2010) A novel β-agarase with high pH stability from marine Agarivorans sp. LQ48. Mar Biotechnol 12:62–69

    Article  CAS  Google Scholar 

  • Ma C, Lu X, Shi C, Li J, Gu Y, Ma Y, Chu Y, Han F, Gong Q, Yu W (2007) Molecular cloning and characterization of a novel β-agarase, AgaB, from marine Pseudoalteromonas sp. CY24. J Biol Chem 282:3747–3754

    Article  CAS  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Oppenheimer CH, ZoBell CE (1952) The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J Mar Res 11:10–18

    Google Scholar 

  • Rochas C, Lahaye M, Yaphe W (1986) 13C-NMR-spectroscopic investigation of agarose oligomers. Carbohydr Res 148:199–207

    Article  CAS  Google Scholar 

  • Roh H, Yun EJ, Lee S, Ko HJ, Kim S, Kim BY, Song H, Lim K, Kim KH, Choi IG (2012) Genome sequence of Vibrio sp. strain EJY3, an agarolytic marine bacterium metabolizing 3,6-anhydro-l-galactose as a sole carbon source. J Bacteriol 194:2773–2774

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Segel IH (1976) Enzyme kinetics. In: Biochemical calculations. How to solve mathematical problems in general biochemistry, 2nd edn. Wiley, New York, pp 214–229

  • Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

    Article  CAS  Google Scholar 

  • Sugano Y, Matsumoto T, Kodama H, Noma M (1993) Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Appl Environ Microbiol 59:3746–3750

    Google Scholar 

  • Temuujin U, Chi WJ, Chang YK, Hong SK (2011) Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type beta-agarase-producing neoagarobiose. J Bacteriol 194:142–149

    Article  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  Google Scholar 

  • Wang J, Jiang X, Mou H, Guan H (2004) Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium. J Appl Phycol 16:333–340

    Article  CAS  Google Scholar 

  • Xie W, Lin B, Zhou Z, Lu G, Lun J, Xia C, Li S, Hu Z (2012) Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3. Appl Microbiol Biotechnol 97:4907–4915

    Article  Google Scholar 

Download references

Acknowledgments

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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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Nan Liu & Dongzhi Wei

  2. College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China

    Nan Liu, Xiangzhao Mao & Meng Yang

  3. Laboratory for Advanced Materials, Institute of Applied Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Bozhong Mu

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  1. Nan Liu
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  2. Xiangzhao Mao
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  3. Meng Yang
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  4. Bozhong Mu
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  5. Dongzhi Wei
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Correspondence to Xiangzhao Mao or Dongzhi Wei.

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Liu, N., Mao, X., Yang, M. et al. Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801. World J Microbiol Biotechnol 30, 1691–1698 (2014). https://doi.org/10.1007/s11274-013-1591-y

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  • DOI: https://doi.org/10.1007/s11274-013-1591-y

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