Applied Microbiology and Biotechnology

, Volume 98, Issue 15, pp 6667–6677 | Cite as

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

  • Yukari Ohta
  • Yuji Hatada
  • Yuko Hidaka
  • Yasuhiro Shimane
  • Keiko Usui
  • Tetsuya Ito
  • Koki Fujita
  • Gaku Yokoi
  • Marina Mori
  • Shona Sato
  • Takatsugu Miyazaki
  • Atsushi Nishikawa
  • Takashi Tonozuka
Biotechnologically relevant enzymes and proteins

Abstract

A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4G-β-d-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

Keywords

Thermostability Random mutagenesis β-Fructofuranosidase Lactosucrose GH68 

Supplementary material

253_2014_5645_MOESM1_ESM.pdf (4.3 mb)
ESM 1(PDF 4411 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yukari Ohta
    • 1
  • Yuji Hatada
    • 1
  • Yuko Hidaka
    • 1
  • Yasuhiro Shimane
    • 1
  • Keiko Usui
    • 1
  • Tetsuya Ito
    • 2
  • Koki Fujita
    • 2
  • Gaku Yokoi
    • 3
  • Marina Mori
    • 3
  • Shona Sato
    • 3
  • Takatsugu Miyazaki
    • 3
  • Atsushi Nishikawa
    • 3
  • Takashi Tonozuka
    • 3
  1. 1.Japan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan
  2. 2.Ensuiko Sugar Refining Co., Ltd.YokohamaJapan
  3. 3.Department of Applied Biological ScienceTokyo University of Agriculture and TechnologyTokyoJapan

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