Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): Molecular size, glycosylation pattern, and engineering of proteolytic resistance

Citation
M. Wyss et al., Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): Molecular size, glycosylation pattern, and engineering of proteolytic resistance, APPL ENVIR, 65(2), 1999, pp. 359-366
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,Microbiology
Journal title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN journal
0099-2240 → ACNP
Volume
65
Issue
2
Year of publication
1999
Pages
359 - 366
Database
ISI
SICI code
0099-2240(199902)65:2<359:BCOFP(>2.0.ZU;2-8
Abstract
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found natura lly in plants and microorganisms, particularly fungi. Interest in these enz ymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce envir onmental pollution due to excess phosphate excretion in areas where there i s intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emer icella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Esc herichia col. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of th e fungal phytases proved to be highly variable, differing for individual ph ytases, for a given phytase produced in different expression systems, and f or individual batches of a given phytase produced in a particular expressio n system. Whereas the extents of glycosylation were moderate when the funga l phytases were expressed in filamentous fungi, they were excessive when th e phytases were expressed in yeasts. However, the different extents of glyc osylation had no effect on the specific activity, the thermostability, or t he refolding properties of individual phytases. When expressed in A, niger, several fungal phytases were susceptible to limited proteolysis by proteas es present in the culture supernatant. N-terminal sequencing of the fragmen ts revealed that cleavage invariably occurred at exposed loops on the surfa ce of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidula ns phytases at the cleavage sites yielded mutants that were considerably mo re resistant to proteolytic attack. Therefore, engineering of exposed surfa ce loops may be a strategy for improving phytase stability during feed proc essing and in the digestive tract.