title: Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein.
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 PfMBP-BCX165, 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.
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
authors.forename: Vandan
authors.initials: V
authors.lastname: Shah
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
authors.forename: Brennal
authors.initials: B
authors.lastname: Pierre
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
authors.forename: Tamari
authors.initials: T
authors.lastname: Kirtadze
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
authors.forename: Seung
authors.initials: S
authors.lastname: Shin
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D020027:Pyrococcus furiosus / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases
keywords: domain insertion; enzyme stabilization; protein engineering; protein stability; thermophilic protein