Open AccessCoexpression of fungal phytase and xylanase utilizing the cis ‐acting hydrolase element in Pichia pastoris
Author(s) -
Roongsawang Niran,
Promdonkoy Peerada,
Wongwanichpokhin Minradee,
Sornlake Warasirin,
Puseenam Aekkachai,
Eurwilaichitr Lily,
Tanapongpipat Sutipa
Publication year - 2010
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2010.00669.x
Subject(s) - pichia pastoris , phytase , xylanase , biology , biochemistry , enzyme , yeast , recombinant dna , extracellular , gene
Plant‐based animal feed contains antinutritive agents, necessitating the addition of digestive enzymes in commercial feeds. Enzyme additives are costly because they are currently produced separately from recombinant sources. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. A coexpression system for the extracellular production of phytase and xylanase was established in Pichia pastoris yeast. The genes for each enzyme were fused in‐frame with the α‐factor secretion signal and linked by the 2A‐peptide‐encoding sequence. Each enzyme was expressed extracellularly as individual functional proteins. The specific activities of 2A‐expressed phytase (PhyA‐2A) and 2A‐expressed xylanase (XylB‐2A) were 9.3 and 97.3 U mg −1 , respectively. Optimal PhyA‐2A activity was observed at 55 °C and pH 5.0. PhyA‐2A also exhibited broad pH stability from 2.5 to 7.0 and retained ∼70% activity after heating at 90 °C for 5 min. Meanwhile, XylB‐2A exhibited optimal activity at 50 °C and pH 5.5 and showed pH stability from 5.0 to 8.0. It retained >50% activity after incubation at 50 °C for 10 min. These enzyme properties are similar to those of individually expressed recombinant enzymes. In vitro digestibility test showed that PhyA‐2A and XylB‐2A are as efficient as individually expressed enzymes for hydrolyzing phytate and crude fiber in feedstuff, respectively.