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Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics
Author(s) -
Zhao D.M.,
Wang M.,
Mu X.J.,
Sun M.L.,
Wang X.Y.
Publication year - 2007
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2007.02216.x
Subject(s) - pichia pastoris , phytase , aspergillus niger , cloning (programming) , molecular cloning , pichia , microbiology and biotechnology , biology , recombinant dna , saccharomyces cerevisiae , gene , chemistry , biochemistry , enzyme , complementary dna , computer science , programming language
Aims: 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. Methods and 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 pPICZαA. 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 ( p NPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90°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. Significance and Impact of the Study: The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.