PMID:20213104
| Citation |
Zhu, W, Qiao, D, Huang, M, Yang, G, Xu, H and Cao, Y (2010) Modifying thermostability of appA from Escherichia coli. Curr. Microbiol. 61:267-73 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1007/s00284-010-9606-5 |
| Keywords |
6-Phytase/chemistry; 6-Phytase/genetics; 6-Phytase/metabolism; Acid Phosphatase/chemistry; Acid Phosphatase/genetics; Acid Phosphatase/metabolism; Cloning, Molecular; Directed Molecular Evolution; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Hot Temperature; Hydrolysis; Mutagenesis; Phytic Acid/metabolism; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Trypsin/metabolism |
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