High level expression of an acid-stable phytase from Citrobacter freundii in Pichia pastoris.
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²+-NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5-8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca²+, and Mg²+. These characteristics contribute to its potential application in feed industry.
Zhao
Wei
W
Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, China.
Xiong
Aisheng
A
Fu
Xiaoyan
X
Gao
Feng
F
Tian
Yongsheng
Y
Peng
Rihe
R
D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D005503:Food Additives / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D003062:Codon; D005503:Food Additives; D011994:Recombinant Proteins; D010832:6-Phytase