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Purification and Characterization of a Recombinant β-d-xylosidase from Thermobifida fusca TM51

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Abstract

The subject of our investigations was a recombinant β-d-xylosidase (TfBXyl43) from Thermobifida fusca TM51 which was expressed in E. coli BL21DE3 and was purified to apparent homogeneity. The SDS-PAGE investigations demonstrated that the molecular weight of the monomer unit is 62.5 kDa but the native-PAGE studies indicated that the mass of the enzyme is 240–250 kDa which proves the presence of a characteristic homo oligomer quaternary structure in solution phase. Optimal parameters of the enzyme activity were at pH 6.0 and 50 °C. The enzyme showed little stability under pH 4.5 and above 60 °C. The substrate specificity investigations indicated that the TfBXyl43 is an exo-glycosidase, hydrolyzing only xylobiose and –triose from the nonreducing end. Besides the enzyme shows very high specificity on the glycon part of the substrate, since it can only hydrolyze β-d-xylopyranoside derivatives. The importance of hydrophobic interactions in the binding of the substrates are supported that the enzyme can hydrolize about four times more efficiently the artificial p-nitrophenyl-β-d-xylopyranoside substrate compared to the natural one, xylobiose. Furthermore we could detect transxylosidase activity both in the case of xylobiose and p-nitrophenyl-β-d-xylopyranoside donors which is the first example among the inverting β-d-xylosidases from T. fusca.

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Acknowledgments

The authors wish to thank József Kukolya for the technical helping in the cloning process, to Teréz Barna for methodical advices, to Gyöngyi Gyémánt for the contribution in the MALDI-TOF measurments, to Gyula Batta for the support in 1H-NMR investigations and to Rita Somosi for her technical assistance.

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Authors and Affiliations

  1. Department of Genetics and Applied Microbiology, Faculty of Sciences, University of Debrecen, P.O. Box 56, H-4010, Debrecen, Hungary

    Csaba Attila Fekete & László Kiss

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  1. Csaba Attila Fekete
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  2. László Kiss
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Correspondence to Csaba Attila Fekete.

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Fekete, C.A., Kiss, L. Purification and Characterization of a Recombinant β-d-xylosidase from Thermobifida fusca TM51. Protein J 31, 641–650 (2012). https://doi.org/10.1007/s10930-012-9440-7

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