Applied Biochemistry and Biotechnology

, Volume 184, Issue 4, pp 1232–1246 | Cite as

Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH

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Abstract

High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.

Keywords

Protein engineering Thermal stabilization Insertional fusion Bacillus circulans xylanase Maltodextrin-binding protein from Pyrococcus furiosus 

Notes

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12010_2017_2619_MOESM1_ESM.docx (1.8 mb)
ESM 1 (DOCX 1890 kb)

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Othmer-Jacobs Department of Chemical and Biomolecular EngineeringNew York UniversityBrooklynUSA

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