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 micromol min(-1) mg(-1) compared to 172.6 micromol 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.