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β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme

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Abstract

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s−1 mM−1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

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Acknowledgments

This research was partially supported from the Directorate General of Higher Education, Ministry of Education and Culture, the Republic of Indonesia through Hibah Tim Pascasarjana Program to NNTP (2011–2012), Sandwich Course Program 2011 to AAID, as well as Hibah Doktor Program to AAID. We thank Mr. Kimiya Mizutani for his assistance in the preparation of enzyme purification, Mr. Didik Huswo Utomo for molecular art work as well as Mr. Tubagus Andhika Nugraha for critical reading of this manuscript.

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Authors and Affiliations

  1. Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jember, Jalan Kalimantan 37, Jember, 68121, Indonesia

    Anak Agung Istri Ratnadewi

  2. Proteomic Laboratory of Institute of Tropical Diseases, Universitas Airlangga, Kampus C Mulyorejo, Surabaya, 60115, Indonesia

    Muchzainal Fanani, Eddy Bagus Wasito & Ni Nyoman Tri Puspaningsih

  3. Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung, 40132, Indonesia

    Sari Dewi Kurniasih & Zeily Nurachman

  4. Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, 514-8507, Japan

    Makiko Sakka & Kazuo Sakka

  5. Department of Chemistry, Faculty of Sciences and Technology, Universitas Airlangga, Kampus C Mulyorejo, Surabaya, 60115, Indonesia

    Ni Nyoman Tri Puspaningsih

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  1. Anak Agung Istri Ratnadewi
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  2. Muchzainal Fanani
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Correspondence to Zeily Nurachman or Ni Nyoman Tri Puspaningsih.

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Ratnadewi, A.A.I., Fanani, M., Kurniasih, S.D. et al. β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme. Appl Biochem Biotechnol 170, 1950–1964 (2013). https://doi.org/10.1007/s12010-013-0329-5

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  • DOI: https://doi.org/10.1007/s12010-013-0329-5

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