Identification, characterization, and overexpression of a phytase with potential industrial interest.
A high phytase-producing strain of Aspergillus niger N-3 was identified by screening 104 microbial strains. The gene for A. niger N-3 was cloned and expressed in Pichia pastoris. The coding region without the introns and putative signal sequence was comprised of 1347 nucleotides. It encoded a polypeptide of 448 amino acids, exhibiting high amino acid sequence homologies (94.87%) with the typical phytase of A. niger NRRL 3135. The molecular mass of the recombinant phytase as determined by SDS-PAGE was 60-70 kDa, with maximum activity at approximately 55 degrees C (after incubation at 10 min). The phytase retained about 45% of its enzymatic activity under heat treatment at 90 degrees C for 5 min. It showed a greater affinity for sodium phytate than for p-nitrophenyl phosphate. Dual optima pH (2.0 and 5.5) was gained. The activity at pH 2.0 was about 30% higher than at pH 5.5, which was more suitable to the circumstance of the stomachs of monogastric animals. The extent of glycosylation influenced the characterization of phytase. The deglycosylated phytase showed pH optima at 3.5 and 5.5, and the molecular mass had dropped to 50 kDa.
Shi
Xiao-Wei
XW
College of Life Science, Shandong Agricultural University, Shandong Tai'an, People's Republic of China.
Sun
Ming-Lv
ML
Zhou
Bo
B
Wang
Xiao-Yun
XY
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007438:Introns; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D021382:Protein Sorting Signals; C008644:nitrophenylphosphate; D010833:Phytic Acid; D010832:6-Phytase