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 heme-dependent peroxidases.
GU Pengshuai
,
PAN Mei
,
DING Liangliang
,
TANG Lei
. Co-expression of glutamyl tRNA reductase improves catalytic activities of a dye-decolorizing peroxidase in Escherichia coli[J]. Food and Fermentation Industries, 2020
, 46(4)
: 45
-50
.
DOI: 10.13995/j.cnki.11-1802/ts.022459
[1] PASSARDI F,COSIO C,PENEL C,et al.Peroxidases have more functions than a Swiss army knife[J].Plant Cell Reports,2005,24(5):255-265.
[2] DAVIES M J,HAWKINS C L,PATTSON D I,et al.Mammalian heme peroxidases:from molecular mechanisms to health implications[J].Antioxidants & Redox Signaling,2008,10(7):1 199-1 234.
[3] 汪立群,CHANG A K,袁文杰,等.一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定[J].生物工程学报,2013,29(6):772-784.
[4] COLPA D I,FRAAIJE M W,VANBLOOIS E.DyP-type peroxidases:A promising and versatile class of enzymes[J].Journal of Industrial Microbiology & Biotechnology,2014,41(1):1-7.
[5] RAMZI A B,HYEON J E,HAN S O.Improved catalytic activities of a dye-decolorizing peroxidase (DyP) by overexpression of ALA and heme biosynthesis genes in Escherichia coli[J].Process Biochemistry,2015,50(8):1 272-1 276.
[6] SANTOS A,MENDES S,BRISSOS V,et al.New dye-decolorizing peroxidases from Bacillus subtilis,and Pseudomonas putida,MET94:Towards biotechnological applications[J]. Applied Microbiology & Biotechnology, 2013,98(5):2 053-2 065.
[7] COUTO S R,HERRERA J L T.Industrial and biotechnological applications of laccases:A review[J].Biotechnology Advances,2006,24(5):500-513.
[8] GRAVES P E,HENDERSON D P,HORSTMAN M J,et al.Enhancing stability and expression of recombinant human hemoglobin in E. coli:Progress in the development of a recombinant HBOC source[J].Biochimica et Biophysica Acta,2008,1 784(10):1 471-1 479.
[9] 李芳芳.大肠杆菌血红素合成途径的改造与调控对5-氨基乙酰丙酸积累和菌体代谢的影响[D].济南:山东大学,2014.
[10] 陈丹园,沈云杰,杨燕,等.关键酶基因的过表达与环境因素对大肠杆菌血红素合成的调控[J].食品与发酵工业,2018,44(11):7-14.
[11] PRANAWIDJAJA S,CHOI S,LAY B W,et al.Analysis of heme biosynthetic pathways in a recombinant 3 Escherichia coli[J].Journal of Microbiology Biotechnology,2014,25(6):880-886.
[12] RAMZIA B,HYEON J E,KIM S W,et al.5-aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway[J].Enzyme and Microbial Technology,2015,81:1-7.
[13] OGOLA H J O,KAMIIKE T,HASHIMOTO N,et al.Molecular characterization of a novel peroxidase from the Ccyanobacterium Anabaena sp.Strain PCC 7120[J].Applied and Environmental Microbiology,2009,75(23):7 509-7 518.
[14] VANBLOOIS E,PAZMIÑUDET,WINTER R T,et al.A robust and extracellular heme-containing peroxidase from Thermobifida fuscaas prototype of a bacterial peroxidase superfamily[J].Applied Microbiology and Biotechnology,2010,86(5):1 419-1 430.
[15] ROBERTS J N,SINGH R,GRIGG J C,et al.Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1[J]. Biochemistry,2011,50(23):5 108-5 919.
[16] 林俊芳,刘志明,陈晓阳等.真菌漆酶的酶活测定方法评价[J].生物加工过程,2009,7(4):1-8.
[17] DICKO M H,GRUPPEN H,HILHORST R,et al.Biochemical characterization of the major sorghum grain peroxidase[J].The FEBS Journal,2006,273(10):2 293-2 307.
[18] CHANWUN T,MUHAMAD N,CHIRAPONGSATONKUL N,et al. Hevea brasiliensis cell suspension peroxidase:Purification,characterization and application for dye decolorization[J].AMB Express,2013,3(1):14.
[19] ZUBIETA C,KRISHNA S S,KAPOOR M,et al.Crystal structures of two novel dye-decolorizing peroxidases reveal a β-barrel fold with a conserved heme-binding motif[J].Proteins Structure Function and Bioinformatics,2007,69(2):223-233.
[20] COLPA D I,FRAAIJE M W.High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX[J].Journal of Molecular Catalysis B Enzymatic,2016,134:372-377.
[21] 朱竹兵,孙亚武,唐蕾.褐色嗜热裂孢菌脱色过氧化物酶的表达及发酵条件优化[J].食品与发酵工业, 2019,45(13):23-30.
