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Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3

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Abstract

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

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Abbreviations

GH:

Glycoside hydrolases

Rf:

Ruminococcus flavefaciens

TLC:

Thin-layer chromatography

IMAC:

Immobilized metal ion affinity chromatography

ESI-MS:

Electron spray ionization mass spectrometry

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Acknowledgements

This research work was supported by NERBPMC Twinning project from DBT (No. BT/PR24786/NER/95/853/2017), Ministry of Science and Technology, Govt. of India to AG. The authors are thankful to Central Instrumentation Facility, IIT Guwahati for providing ESI-MS facility. The authors are thankful to Vikky Rajulapati for his help in ESI-MS analysis.

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Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Dishant Goyal, Krishan Kumar, Abhijeet Thakur & Arun Goyal

  2. CIISA – Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal

    Maria S. J. Centeno, Pedro Bule & Carlos M. G. A. Fontes

  3. NZYTech – Genes & Enzymes, Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E - R/C, 1649-038, Lisbon, Portugal

    Virgínia M. R. Pires & Carlos M. G. A. Fontes

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  1. Dishant Goyal
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Goyal, D., Kumar, K., Centeno, M.S.J. et al. Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3. Mol Biotechnol 61, 826–835 (2019). https://doi.org/10.1007/s12033-019-00205-2

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