共表达谷氨酰-tRNA还原酶增强染料脱色过氧化物酶在大肠杆菌中的表达活性  

Co-expression of glutamyl tRNA reductase improves catalytic activities of a dye-decolorizing peroxidase in Escherichia coli

作　　者：顾鹏帅 潘梅[1] 丁亮亮 唐蕾[1,2] GU Pengshuai;PAN Mei;DING Liangliang;TANG Lei(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2020年第4期45-50,共6页Food and Fermentation Industries

基　　金：111引智计划(111-2-06);国家轻工技术与工程一流学科自主课题(LITE2018-27);江苏省现代工业发酵协同创新中心资助

摘　　要：染料脱色过氧化物酶(dye-decoloorizing peroxidase,DyP)属于以血红素为辅基的新型过氧化物酶类,常因缺乏辅因子而导致催化活性低。将来源于褐色嗜热裂孢菌(Thermobifida fusca)的染料脱色过氧化物酶基因(TfuDyP)与大肠杆菌谷氨酰-tRNA还原酶基因(hemA),构建重组质粒phemA-DyP,转化至E.coli BL21中进行共表达。分别以2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)和顽固性染料活性蓝19(RB19)、溴酚蓝、溴甲酚绿为底物检测TfuDyP的催化活力以及染料脱色效率。结果表明,诱导后的共表达菌株pAD胞内血红素含量为9. 8μmol/L,而单独过表达基因TfuDyP的菌株pD仅为3. 4μmol/L。TfuDyP纯酶的全波长扫描分析表明,在菌株pAD中DyP酶与血红素的结合度相比pD有较大幅度的提升。pAD菌株表达的DyP酶活力较pD菌株提高了110%,酶活力的提高使其在染料脱色应用方面也得到增强。在pAD菌株培养基中分别添加谷氨酸(Glu)、FeCl2使得胞内血红素含量、DyP酶活力和染料脱色效率比未添加时进一步提高。以上结果为TfuDyP的功能开发奠定了基础,同时也为其他血红素依赖性过氧化物酶的研发提供借鉴。Dye-decolorizing peroxidase(DyP) with heme as a prosthetic group is regarded as a novel type of peroxidase. Usually,the catalytic activity of DyP is low due to the lack of cofactor heme.In this study,the recombinant plasmid phemA-DyP with the DyP gene from Thermobifida fusca(TfuDyP) gene and glutamyl tRNA reductase gene from E. coli(hemA) was constructed and transformed into E. coli BL21 for co-expression. The catalytic ability and decolorization efficiency of TfuDyP were detected using ABTS and dyes RB19,bromophenol blue and bromocresol green as substrates respectively.The results showed that the intracellular heme content in the co-expressing strain pAD was 9. 8 μmol/L,whereas in the strain pD with over-expressed TfuDyP it was 3. 4 μmol/L. Full-wavelength scanning of purified TfuDyp showed that the binding degree of DyP to heme increased in the co-expressing strain pAD. In addition,the activity of TfuDyP from the pAD strain was 110% higher than that from the pD strain,and the increase of enzyme activity also enhanced the dye decolorization. The exogenous addition of glutamic acid(Glu) and FeCl2 to the culture medium of pAD strain increased the intracellular heme content,the TfuDyP activity and dye decolorization efficiency. The results laid the foundation for the functional development of TfuDyP,and also provided reference for the development of other hemedependent peroxidases.

关 键 词：大肠杆菌 褐色嗜热裂孢菌 血红素 染料脱色过氧化物酶 共表达 

分 类 号：Q78[生物学—分子生物学] TS201.3[轻工技术与工程—食品科学]