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Applied Microbiology and Biotechnology

, Volume 98, Issue 18, pp 7825–7836 | Cite as

An intermolecular disulfide bond is required for thermostability and thermoactivity of β-glycosidase from Thermococcus kodakarensis KOD1

  • Kuo-Yuan HwaEmail author
  • Boopathi Subramani
  • San-Tai Shen
  • Yu-May Lee
Biotechnologically relevant enzymes and proteins

Abstract

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. β-glycosidase, Tkβgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkβgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the k cat/K m of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkβgly enzyme.

Keywords

β-glycosidase Thermostability Thermoactivity Cysteine residues Disulfide bond 

Notes

Acknowledgments

This study was supported by the grants from National Taipei University of Technology (NTUT-98-140-01, NTUT-99-140-01 and NTUT-100-140-01) to Kuo-Yuan Hwa and Academia Sinica, Taiwan. We thank Dr. Sathyadevi (National Chiao Tung University, Taiwan, ROC) for her assistance in fluorescence spectroscopy. We warmly acknowledge the reviewers for their valuable comments for improving the manuscript.

Supplementary material

253_2014_5731_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1118 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Kuo-Yuan Hwa
    • 1
    • 2
    • 3
    Email author
  • Boopathi Subramani
    • 1
  • San-Tai Shen
    • 4
  • Yu-May Lee
    • 4
  1. 1.Department of Molecular Science and EngineeringNational Taipei University of TechnologyTaipeiTaiwan
  2. 2.Institute of Organic and Polymeric MaterialsNational Taipei University of TechnologyTaipeiTaiwan
  3. 3.Centre for Biomedical IndustryNational Taipei University of TechnologyTaipeiTaiwan
  4. 4.Institute of Biological Chemistry, Academia SinicaTaipeiTaiwan

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