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Engineering of a fungal β-galactosidase to remove product inhibition by galactose

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Abstract

β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp258-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K m (3.76 mM compared to 2.21 mM) and reduced V max (110.8 μmol min−1 mg−1 compared to 172.6 μmol min−1 mg−1) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

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Acknowledgements

This study was funded by grant PC/2006/25 from Enterprise Ireland Science and Technology Development Agency.

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Authors and Affiliations

  1. Microbiology, School of Natural Sciences and National Centre for Biomedical Engineering Sciences, National University of Ireland, Galway, Ireland

    Xuejun Hu & J. Gerard Wall

  2. Department of Chemical and Environmental Sciences and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland

    Xuejun Hu, Sylvain Robin, Shane O’Connell, Gary Walsh & J. Gerard Wall

  3. Medical School, Dalian Development Zone, Dalian University, Dalian, China

    Xuejun Hu

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  1. Xuejun Hu
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  2. Sylvain Robin
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  3. Shane O’Connell
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  4. Gary Walsh
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  5. J. Gerard Wall
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Correspondence to J. Gerard Wall.

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Hu, X., Robin, S., O’Connell, S. et al. Engineering of a fungal β-galactosidase to remove product inhibition by galactose. Appl Microbiol Biotechnol 87, 1773–1782 (2010). https://doi.org/10.1007/s00253-010-2662-8

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  • DOI: https://doi.org/10.1007/s00253-010-2662-8

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