Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein

Vandan Shah, Brennal Pierre, Tamari Kirtadze, Seung Shin, Jin Ryoun Kim

Research output: Contribution to journalArticle

Abstract

High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBPBCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.

Original languageEnglish (US)
Pages (from-to)281-290
Number of pages10
JournalProtein Engineering, Design and Selection
Volume30
Issue number4
DOIs
StatePublished - Apr 1 2017

Keywords

  • Domain insertion
  • Enzyme stabilization
  • Protein engineering
  • Protein stability
  • Thermophilic protein

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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