Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris.
Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris. The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL(-1), respectively. Both recombinant phytases exhibited high affinity for phytate but not for p-nitrophenyl phosphate. Optimal phytase activity was observed at 50 degrees C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 degrees C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.
Promdonkoy
Patcharee
P
BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand.
Tang
Kittapong
K
Sornlake
Warasirin
W
Harnpicharnchai
Piyanun
P
Kobayashi
Rutchadaporn Sriprang
RS
Ruanglek
Vasimon
V
Upathanpreecha
Tewa
T
Vesaratchavest
Mongkol
M
Eurwilaichitr
Lily
L
Tanapongpipat
Sutipa
S
D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism*; D000821:Animal Feed; D001230:Aspergillus / Q000145:classification / Q000201:enzymology / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D003313:Zea mays / Q000737:chemistry / Q000378:metabolism
D010833:Phytic Acid; D010832:6-Phytase