Modifying thermostability of appA from Escherichia coli.
In order to improve the thermostability of Escherichia coli AppA phytase, Error-prone PCR was used to randomize mutagenesis appA gene, and a gene mutation library was constructed. A mutant I408L was selected from the library by the method of high-throughput screening with 4-methyl-umbelliferylphosphate (4-MUP). The appA gene of the mutant was cloned and expressed in E. coli Origami (DE3). The recombinant protein was purified by Ni-affinity chromatography, and the enzymatic features were analyzed. The results indicated that AppA phytase activities of mutant I408L and wild-type (WT) strain remained at 51.3 and 28%, respectively, after treatment at 85°C for 5 min. It means that the thermostability enhancement of AppA phytase I408L was 23.3% more as compared with WT. The K (m) of both phytase were 0.18 and 0.25 mM, respectively, which indicated that the catalyzing efficiency of I408L was improved. AppA phytase of mutant I408L showed a significant enhancement against trypsin, which was nearly three times compared with WT. In addition, AppA phytase of mutant could be activated by Mg(2+) and Mn(2+); in contrast, it could be inhibited by Ca(2+), Co(2+), Cu(2+), and K(+) in varying degrees, and the enzymatic activity was almost lost the presence of Fe(3+) and Zn(2+). It appears that screening thermotolerant phytase of E. coli by high throughput screening with a fluorescence substrate is a fast, simple, and effective method. The mutant I408L obtained in this study could be used for the large-scale commercial production of phytase.
Zhu
Weihua
W
Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, People's Republic of China.
Qiao
Dairong
D
Huang
Min
M
Yang
Ge
G
Xu
Hui
H
Cao
Yi
Y
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D016296:Mutagenesis; D010833:Phytic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010833:Phytic Acid; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli; D014357:Trypsin