Purification and characterization of an antimicrobial protein from &lt;i&gt;Gastrodia elata&lt;/i&gt; Blume tubers

Chen Chen,Youmei Xu,Sanqiao Wu,Xian Jing,Xinxin Li,Jing Li,Xiang Liu,Xiaoying Zhang

doi:10.4314/tjpr.v17i9.5

Purification and characterization of an antimicrobial protein from <i>Gastrodia elata</i> Blume tubers

Chen Chen, Youmei Xu + Show 6 more

Open Access

https://doi.org/10.4314/tjpr.v17i9.5

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Abstract

Purpose: To purify and characterize a novel antimicrobial protein from the Gastrodia elata Blume (Bl.) plant, which has long been used in herbal medicine. Methods: The procedure for isolation and purification of Gastrodia elata protein (GEP) involved phosphate buffer extraction, ammonium sulfate precipitation, ion-exchange chromatography, and gelfiltration chromatography. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis was employed to detect the apparent molecular mass and determine homogeneity, while paper disc diffusion was used to measure the antibacterial activity of GEP. A hemolytic assay was performed on rabbit red blood cells. The effect of pH, salt concentration, and temperature on the antibacterial activity of GEP was evaluated by minimum inhibitory concentration assay. Results: GEP was a 14-kDa monomer and displayed antimicrobial activity against Staphylococcus aureus and Candida albicans , with 8.0-mm and 9.4-mm zones of inhibition, respectively, but no antibacterial activity was observed against Escherichia coli . GEP had little hemolytic activity on red blood cells even at a concentrations of up to 200 mg/ml. GEP was thermally stable at temperatures below 70 °C for 30 min, and displayed higher antibacterial activity in the pH range 5.0 to 7.0. Conclusion: GEP protein is relatively thermostable and possesses antimicrobial activity. The results suggest that GEP protein has potential agricultural and industrial applications, such as in transgenic plants. Keywords: Antimicrobial protein, Gastrodia elata , Protein characterization

Highlights

Plants exhibit a variety of defenses to combat bacteria in the environment
The protein was purified from Gastrodia elata Bl. tubers using ammonium sulfate precipitation, ionexchange chromatography using DEAE-52 columns, and gel-filtration chromatography using Sephadex G-50 columns (Table 1)
Our research showed that the Gastrodia elata protein (GEP) protein retains significant antibacterial activity at pH values up to 10.0, and displays good thermal stability up to 60°C

Summary

INTRODUCTION

Plants exhibit a variety of defenses to combat bacteria in the environment. Despite not having an integrated immune system, plants produce proteins and peptides with antibacterial activity to combat pathogenic invasion [1]. -©---2-0--1--8---T--h-e---a--u-t-h--o-r-s--.--T-h--i-s--w--o--r-k--i-s--l-i-c-e--n-s--e--d--u--n--d-e--r--t-h-e---C--r-e--a-t-i-v-e---C--To-r-m-o-mp---oJ-n-P-s-h-A-a-t-rt-mr-i-b-Ru--te-io-s-,n--S-4e-.-p0--te-In-m-t-eb--re-n-ra--2t-i0o--1n--8a-;l--L1--i7c--(e-9n-)-s:--e1--7--1-7 flowers, seeds, roots, and stems of plants [2,3] These proteins and peptides display antibacterial activity against phytopathogens, as well as bacteria that are pathogenic to humans. Fresh Gastrodia elata Bl. tubers were purchased from Lueyang County, Shaanxi province, China, and stored at –20 °C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis was used to detect the apparent molecular mass and homogeneity of the purified antibacterial proteins. MIC was used to determine antibacterial activity at different pH values after incubating at room temperature for 30 min. The minimum inhibitory concentration (MIC) of the antibacterial protein-of-interest was detected using microdilution methods [10] with an initial strain concentration of 5 × 105 CFU/mL.

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CONCLUSION