Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris

Arch Biochem Biophys. 1999 Apr 1;364(1):83-90. doi: 10.1006/abbi.1999.1115.

Abstract

Economical and thermostable phytase enzymes are needed to release phytate-phosphorus in plant foods for human and animal nutrition and to reduce phosphorus pollution of animal waste. Our objectives were to determine if a methylotrophic yeast, Pichia pastoris, was able to express a phytase gene (phyA) from Aspergillus niger efficiently and if suppression of glycosylation by tunicamycin affected its functional expression. The gene (1.4 kb) was inserted into an expression vector pPICZalphaA with a signal peptide alpha-factor, under the control of AOX1 promoter. The resulting plasmid was transformed into two P. pastoris strains: KM71 (methanol utilization slow) and X33 (wild-type). Both host strains produced high levels of active phytase (25-65 units/ml of medium) that were largely secreted into the medium. The expressed enzyme was cross-reacted with the polyclonal antibody raised against the wild-type enzyme and showed two pH optima, 2.5 and 5.5, and an optimal temperature at 60 degrees C. Compared with the phyA phytase overexpressed by A. niger, this phytase had identical capacity in hydrolyzing phytate-phosphorus from soybean meal and slightly better thermostability. Deglycosylation of the secreted phytase resulted in reduction in the size from 95 to 55 kDa and in thermostability by 34%. Tunicamycin (20 microg/ml of medium) resulted in significant reductions of both intracellular and extracellular phytase activity expression. Because there was no accumulation of intracellular phytase protein, the impairment did not seem to occur at the level of translocation of phytase. In conclusion, glycosylation was vital to the biosynthesis of the phyA phytase in P. pastoris and the thermostability of the expressed enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 6-Phytase / genetics*
  • 6-Phytase / metabolism*
  • 6-Phytase / physiology
  • Aspergillus niger / enzymology*
  • Aspergillus niger / genetics
  • Blotting, Western
  • Enzyme Stability
  • Glycosylation
  • Hexosaminidases / pharmacology
  • Hydrogen-Ion Concentration
  • Phosphates / metabolism
  • Phytic Acid / metabolism
  • Pichia / drug effects
  • Pichia / enzymology*
  • Pichia / genetics
  • Temperature
  • Time Factors
  • Tunicamycin / pharmacology

Substances

  • Phosphates
  • Tunicamycin
  • Phytic Acid
  • 6-Phytase
  • Hexosaminidases