Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization.
Organophosphorus hydrolase is able to hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. In this work, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Pichia pastoris at a high expression level (approximately 5.5 g/L) using 3 L high-cell-density fermentation. The expression level is higher than those produced in other expression systems. The results of the SDS-PAGE and the Western blot analyses showed a major 36 kDa polypeptide band, which was the same size as that from the original bacteria, Pseudomonas pseudoalcaligenes C2-1. The expressed enzyme was recovered from the culture supernatant and purified by a single-step purification procedure with a recovery rate of 72.78%. The main physiochemical features of the recombinant OPHC2, including its optimum temperature and pH for the reaction, its temperature and pH stability, and its sensitivity to some metal ions and chemical reagents, were also characterized. With methyl parathion as a substrate, the optimum temperature and pH for enzyme activity are 65 degrees C and pH 9.0, respectively. It also shows good thermal and pH stability.
Chu
Xiao-Yu
XY
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Wu
Ning-Feng
NF
Deng
Min-Jie
MJ
Tian
Jian
J
Yao
Bin
B
Fan
Yun-Liu
YL
D043303:Aryldialkylphosphatase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D008670:Metals; D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase