Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli
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Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including micro-organisms, plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75% of its activity. The enzyme was stable at pH range of 2–10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed.
KeywordsPhytase Enzymatic region Recombinant protein expression Thermostability Activity
This study was conducted with financial assistance from Arak University of medical sciences, Iran, and we are grateful for their invaluable contribution to this study. This study was the thesis (no: 2351) by Ms. Malihe Hallaji, the master student of Medical Biotechnology at Arak university of medical sciences, Iran. Its ethical code from the ethics committee of Arak University of medical sciences, Arak, Iran is IR.ARAKMU.REC.1394.195.
All authors had equal contributions to the study.
This study was conducted with financial assistance from Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran and we are grateful for their invaluable contribution to this study.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflicts of interest.
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