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

Content Provider	Springer Nature Link
Author	Hu, Xuejun Robin, Sylvain O’Connell, Shane Walsh, Gary Wall, J. Gerard
Copyright Year	2010
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.
Starting Page	1773
Ending Page	1782
Page Count	10
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	87
Issue Number	5
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2010-05-23
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	β-galactosidase Lactase Aspergillus niger van Tiegh Mutagenesis Product inhibition Galactose Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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