多功能过氧化物酶介导Mn(Ⅲ)络合体系对酚类化合物的氧化降解  

Oxidative degradation of phenolic compounds by versatile peroxidase-mediated Mn(Ⅲ) complex system

作　　者：窦明德 姚从禹 吴敬[1,2,3] 夏伟 陈晟 DOU Mingde;YAO Congyu;WU Jing;XIA Wei;CHEN Sheng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;International Joint Laboratory on Food Safety,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [3]江南大学教育部食品安全国际合作联合实验室,江苏无锡214122

出　　处：《微生物学通报》2022年第9期3631-3643,共13页Microbiology China

基　　金：国家重点研发计划(2019YFA0706900)。

摘　　要：【背景】酚类化合物是环境中主要的水体污染物之一。多功能过氧化物酶(versatile peroxidase,VP)介导的Mn(Ⅲ)-有机酸络合体系具有较高的氧化还原电势,在酚类有机污染物降解方面具有巨大潜力。【目的】探究VP介导的Mn(Ⅲ)-有机酸络合体系降解酚类化合物的能力,为酚类化合物的降解提供新的思路和方法。【方法】研究选取了糙皮侧耳(Pleurotus eryngii)来源的多功能过氧化物酶(PeVP),采用包涵体复性的方法获得了PeVP活性蛋白,并对重组PeVP进行酶学性质研究及Mn(Ⅲ)络合体系反应条件优化,进而探究Mn(Ⅲ)络合体系对酚类污染物的氧化降解能力。【结果】确定了PeVP包涵体复性最佳条件为:pH 9.5、10%甘油、0.5 mol/L尿素、0.5 mmol/L氧化型谷胱甘肽(glutathione oxidized,GSSG)、0.1 mmol/L二硫苏糖醇(dithiothreitol,DTT)、0.1 mmol/L乙二胺四乙酸(ethylenediamine tetraacetic acid,EDTA)、5 mmol/L CaCl_(2)、5μmol/L羟高铁血红素(hematin),4℃静置透析24 h,最后5μmol/L hematin孵育12 h。通过对PeVP介导的Mn(Ⅲ)-有机酸络合体系优化,确定了最优反应条件为:75 mmol/L苹果酸缓冲液(pH 4.5)、6 mmol/L Mn^(2+)和0.2 mmol/L H_(2)O_(2)。在上述条件下,探究了络合体系对2,2-丁香醛连氮-双-3-乙基苯并噻唑啉-6-磺酸[2,2ʹ-azinobis-(3-ethylbenzthiazoline-6-sulphonate),ABTS]、2,6-二甲氧基苯酚(2,6-dimethoxyphenol,DMP)、愈创木酚和丁香醛连氮4种酚类模式底物的催化活性,发现在pH 4.5条件下,络合体系对酚类模式底物的氧化活性可达到PeVP直接氧化活性的1.5−7.5倍,并且在16 h的酶解过程中,苯酚、对苯二酚、间苯二酚和对硝基苯酚的平均降解速率分别为10.91、10.69、6.50和5.71 mg/(L·h),推测Mn(Ⅲ)-有机酸络合物对酚类底物的氧化降解是通过夺取酚类底物的电子形成酚类自由基中间体,自由基中间体经过电子重排和C−C键的断裂,最终导致酚类物质的氧化降解。【结�[Background]Phenolic compounds are among the main water pollutants in the environment.The Mn(Ⅲ)-organic acid chelate mediated by versatile peroxidase(VP)is considered to have great potential in the degradation of phenolic organic pollutants owing to the high redox potential.[Objective]To explore the degradation of phenolic compounds by VP-mediated Mn(Ⅲ)-organic acid chelate and thereby to provide a new mindset and method for the biodegradation of phenolic compounds.[Methods]The active VP from Pleurotus eryngii(PeVP)was obtained by renaturation of inclusion body in vitro.We characterized the enzymatic properties of PeVP and optimized the reaction conditions of Mn(Ⅲ)chelate to explore the ability of the complex system to degrade the phenolic compounds.[Results]The optimum conditions for renaturation of PeVP were as follows:The inclusion body was refolded in pH 9.5 solution containing 0.5 mol/L urea,0.5 mmol/L oxidized glutathione(GSSG),0.1 mmol/L dithiothreitol(DTT),0.1 mmol/L ethylenediamine tetraacetic acid(EDTA),5 mmol/L CaCl_(2),10%glycerol,and 5μmmol/L hematin at 4℃for 24 h,and then incubated with 5μmol/L hematin for 12 h.The optimal reaction conditions of PeVP-mediated Mn(Ⅲ)-organic acid chelate were 75 mmol/L malic acid buffer(pH 4.5),6 mmol/L Mn^(2+),and 0.2 mmol/L H_(2)O_(2).Under the above conditions,the catalytic activity of the chelate system on phenolic compounds 2,2ʹ-azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS),2,6-dimethoxyphenol(DMP),guaiacol,and syringaldazine was detected,and it was found that the oxidation activity of Mn(Ⅲ)chelate system at pH 4.5 was 1.5–7.5 times that of PeVP alone.Moreover,the average degradation rates of phenol,hydroquinone,resorcinol,and p-nitrophenol by Mn(Ⅲ)-malic acid chelate system were 10.91,10.69,6.50,5.71 mg/(L·h),respectively.Therefore,the Mn(Ⅲ)-organic acid complex might capture the electrons of phenolic substrate to form phenolic free radicals and lead to the fracture of benzene ring with the decrease of bond energy,thus achieving the o

关 键 词：多功能过氧化物酶 包涵体复性 Mn(Ⅲ)-苹果酸络合物 酚类污染物 氧化降解 

分 类 号：X52[环境科学与工程—环境工程]