Peer-Reviewed Journal Details
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Hu XJ, Robin S, O'Connell S, Walsh G, Wall JG
2010
August
Applied Microbiology And Biotechnology
Engineering of a fungal beta-galactosidase to remove product inhibition by galactose
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Optional Fields
beta-galactosidase Lactase Aspergillus niger van Tiegh Mutagenesis Product inhibition Galactose ESCHERICHIA-COLI FEEDBACK INHIBITION RANDOM MUTAGENESIS ANTIBODY FRAGMENT PICHIA-PASTORIS DOMOIC ACID L-CYSTEINE LACTOSE EXPRESSION LACTASE
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beta-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 beta-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. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-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 beta-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 mu mol min(-1) mg(-1) compared to 172.6 mu mol 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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DOI 10.1007/s00253-010-2662-8
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