Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization.
Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58 degrees C to 53 degrees C for bridge number 1, 37 degrees C for bridge number 3 and 4, and 42 degrees C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K (m) was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.
Mullaney
Edward J
EJ
United States Department of Agriculture, Southern Regional Research Center, ARS, New Orleans, LA 70124, USA. ed.mullaney@ars.usda.gov
Locovare
Heather
H
Sethumadhavan
Kandan
K
Boone
Stephanie
S
Lei
Xin Gen
XG
Ullah
Abul H J
AH
D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics* / Q000378:metabolism
D004220:Disulfides; D005656:Fungal Proteins; D010832:6-Phytase