Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics.
OBJECTIVE
Using gene cloning and overexpression to obtain a potential industrial phytase as a feed additive to upgrade the nutritional quality of phytate-rich seed-based animal feed.


RESULTS
A phyA gene from a high extracellular phytase-producing Aspergillus niger sp. was cloned and overexpressed in Pichia pastoris GS115 using the secretive expression vector pPICZalphaA. After cultivation for 4 days in buffered methanol complex medium (BMMY) containing methanol for induction, catalytically active phytase was secreted as a predominantly extracellular protein. The activity of the expressed phytase in fermented broth was 30 000-fold higher than that of native phytase with a specific activity of 503 U mg(-1). The Lineweaver-Burk plot indicated K(m) values of 0.196 mmol l(-1) for sodium phytate and 18.16 mmol l(-1) for p-nitrophenylphosphate (pNPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90 degrees C for 5 min and 65% activity after 30 min, much higher than for commercial phytase.


CONCLUSIONS
The higher activity and high thermostability of recombinant phytase enable it to withstand the temperatures of the feed pelleting process.


CONCLUSIONS
The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.
Zhao
D-M
DM
College of Life Science, Shandong Agricultural University, Shandong Taian, People's Republic of China.
Wang
M
M
Mu
X-J
XJ
Sun
M-L
ML
Wang
X-Y
XY
D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D005453:Fluorescence; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005818:Genetic Engineering; D006358:Hot Temperature; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D010832:6-Phytase