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Modifying Thermostability of appA from Escherichia coli

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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 Mg2+ and Mn2+; in contrast, it could be inhibited by Ca2+, Co2+, Cu2+, and K+ in varying degrees, and the enzymatic activity was almost lost the presence of Fe3+ and Zn2+. 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.

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Acknowledgments

This study was supported by The National Natural Science Foundation of China (30871321, 30771312, and 30971817), The National Special Basic Research Projects of China (SB2007FY400-4), and The National Basic Research Program of China (2009CB125910).

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Authors and Affiliations

  1. Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, 610064, Sichuan, People’s Republic of China

    Weihua Zhu, Dairong Qiao, Min Huang, Ge Yang, Hui Xu & Yi Cao

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  1. Weihua Zhu
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  2. Dairong Qiao
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  3. Min Huang
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  4. Ge Yang
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  5. Hui Xu
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  6. Yi Cao
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Correspondence to Yi Cao.

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Zhu, W., Qiao, D., Huang, M. et al. Modifying Thermostability of appA from Escherichia coli . Curr Microbiol 61, 267–273 (2010). https://doi.org/10.1007/s00284-010-9606-5

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  • DOI: https://doi.org/10.1007/s00284-010-9606-5

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