荧光光谱法的卤酸脱卤酶活性全细胞检测

Intact-Cell Detection of Haloacid Dehalogenase Activity Based on Fluorescence Spectroscopy

卤酸脱卤酶能够降解对环境具有危害性的卤代羧酸类化合物,并且其具有立体选择性,可以拆分出纯手性的卤代羧酸,在环境保护和化学合成领域具有潜在的应用价值。建立卤酸脱卤酶活性的分析方法具有重要意义。催化性能较好的卤酸脱卤酶DehE在弱碱性范围内(pH 8.00~9.50)能催化2-氯丙酸发生脱卤反应产生氢离子,从而改变弱缓冲能力反应体系的pH,而某些物质的荧光强度受溶液pH影响较大。进行了荧光-pH指示剂的筛选,并以此建立了基于荧光光谱法的卤酸脱卤酶活性全细胞检测方法。结果表明:筛选出的荧光-pH指示剂2-萘酚-6,8-二磺酸二钾能够灵敏地反映弱碱性范围内(pH 8.00~9.50)的pH变化。所建方法在20 mmol·L^(-1)的4-羟乙基哌嗪乙磺酸、pH 8.50、45℃、45 mmol·L^(-1)的2-氯丙酸条件下检测效果最好,并且能避免假阳性结果的出现。当被检测的菌液OD600=2.0时,15 min即可出现50%的荧光变化。采用HPLC验证了方法的专属性。该方法能够快速、简单、灵敏地在全细胞水平上检测卤酸脱卤酶活性。当微生物含有卤酸脱卤酶的活性与DehE相当时,所建立的方法对该微生物检测限为菌液OD600=0.3,具备对含有卤酸脱卤酶的微生物原位筛查分析潜力。

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 OD6002.0 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 OD6000.3 of the bacterial suspension.In summary,this method has the potential for in situ qualitative screening and analysis of microorganisms containing haloacid dehalogenases.