Engineering of a fungal beta-galactosidase to remove product inhibition by galactose.
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.
Hu
Xuejun
X
Microbiology, School of Natural Sciences and National Centre for Biomedical Engineering Sciences, National University of Ireland, Galway, Ireland.
Robin
Sylvain
S
O'Connell
Shane
S
Walsh
Gary
G
Wall
J Gerard
JG
D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism*
D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D005690:Galactose