Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris.
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 degreesC and pH 5.0. PhyA-2A also exhibited broad pH stability from 2.5 to 7.0 and retained approximately 70% activity after heating at 90 degreesC for 5 min. Meanwhile, XylB-2A exhibited optimal activity at 50 degreesC and pH 5.5 and showed pH stability from 5.0 to 8.0. It retained >50% activity after incubation at 50 degreesC 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.
Roongsawang
Niran
N
Microbial Cell Factory Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong Luang, Pathumthani, Thailand. niran.roo@biotec.or.th
Promdonkoy
Peerada
P
Wongwanichpokhin
Minradee
M
Sornlake
Warasirin
W
Puseenam
Aekkachai
A
Eurwilaichitr
Lily
L
Tanapongpipat
Sutipa
S
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D021382:Protein Sorting Signals; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D013696:Temperature; D013997:Time Factors; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010832:6-Phytase; D014995:Xylosidases