Intact-Cell Detection of Haloacid Dehalogenase Activity Based on
Fluorescence Spectroscopy
CHEN Peng1, 2, LU Feng2, ZHAO Yun-li1*
1. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
2. School of Pharmacy, Naval Medical University, Shanghai 200433, China
Abstract：Haloacid dehalogenase can degrade halocarboxylic acids that are harmful to the environment. In addition, their stereoselectivity can produce pure chiral halocarboxylic acid. Therefore, haloacid dehalogenase has potential applications in environmental protection and chemical synthesis. It is of great significance to establish a method to detect the activity of haloacid dehalogenases. Haloacid dehalogenase DehE has good catalytic performance and can catalyze the dehalogenation of 2-chloropropionic acid to produce protons in the weak alkaline range (pH 8.00~9.50), changing the pH of the reaction system with weak buffering capacity. The HP of the solution greatly affects the fluorescence intensity of some substances. Based on this, the selected fluorescence pH indicator established an intact-cell detection method for haloacid dehalogenase activity. The results showed that the fluorescence pH indicator 2-naphthol-6,8-disulfonate could sensitively reflect the pH change in the weak alkaline range. The established method has the best detection effect under the condition of 20 mmol·L-1 HEPES, pH 8.50, 45 ℃ and 45 mmol·L-1 2-chloropropionic acid, avoiding the appearance of false positive results. A 50% fluorescence change was observed at 15 min with OD600 2.0 of the tested bacterial suspension. HPLC verified the specificity of method. The method can rapidly, and sensitively detect haloacid dehalogenase activity at the intact-cell level. In addition, when the activity of haloacid dehalogenases in the microbe is comparable to that of DehE, the detection limit of the established method for the microbe is OD600 0.3 of the bacterial suspension. In summary, this method has the potential for in situ qualitative screening and analysis of microorganisms containing haloacid dehalogenases.
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