褐色嗜热裂孢菌脱色过氧化物酶的表达及发酵条件优化
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摘要
为了提高来源于褐色嗜热裂孢菌(Thermobifida fusca)的脱色过氧化物酶(TfuDyP)对蒽醌染料降解能力,将含有目的基因的重组质粒p ET-28a(+)-TfuDyP,转化至E. coli BL21进行异源表达,并对重组TfuDyP的发酵条件进行优化,分析酶活与血红素饱和度之间的关系。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测到分子质量为46 k Da的重组TfuDyP蛋白条带。TfuDyP的最佳诱导条件为:诱导剂(IPTG)浓度0. 3 mmol/L,诱导时间14 h,诱导温度30℃,在此条件下,TfuDyP比酶活达到27. 9 U/g。氯高铁血红素、5-氨基乙酰丙酸(5-ALA)、谷氨酸(Glu)、FeCl_2、MnCl_2均可提高重组TfuDyP的催化活性,酶活的提高与血红素饱和度之间存在一定的正相关性。实验结果可为利用外源添加物提高血红素的饱和度,应用于染料脱色过氧化物酶的工业发酵提供理论依据。
This study aimed to improve the catalytic ability of Thermobifida fusca dye-decolorizing peroxidase( TfuDyP) to degrade anthraquinone dyes. The recombinant plasmid pET-28 a( +)-TfuDyP was transformed into Escherichia coli BL 21 for heterologous expression,and the fermentation conditions of recombinant Tfu DyP was optimized. Additionally,the relationship between enzyme activity and heme saturation was analyzed. SDS-PAGE showed that the molecular weight of recombinant Tfu DyP was 46 k Da. Moreover,induction with 0. 3 mmol/L IPTG at 30 ℃for 14 h was determined to be the optimal fermentation condition. Under this condition,the specific activity of TfuDyP was 27. 9 U/g. Furthermore,hemin,5-aminolevulinic acid( 5-ALA),glutamic acid( Glu),FeCl_2 and MnCl_2 promoted the enzyme activity. There was a positive correlation between the increase in enzyme activity and heme saturation. Therefore,this study provides a theoretical basis for improving heme saturation by adding exogenous promoters and applying this in industrial fermentation of dye-decolorizing peroxidase.
This study aimed to improve the catalytic ability of Thermobifida fusca dye-decolorizing peroxidase( TfuDyP) to degrade anthraquinone dyes. The recombinant plasmid pET-28 a( +)-TfuDyP was transformed into Escherichia coli BL 21 for heterologous expression,and the fermentation conditions of recombinant Tfu DyP was optimized. Additionally,the relationship between enzyme activity and heme saturation was analyzed. SDS-PAGE showed that the molecular weight of recombinant Tfu DyP was 46 k Da. Moreover,induction with 0. 3 mmol/L IPTG at 30 ℃for 14 h was determined to be the optimal fermentation condition. Under this condition,the specific activity of TfuDyP was 27. 9 U/g. Furthermore,hemin,5-aminolevulinic acid( 5-ALA),glutamic acid( Glu),FeCl_2 and MnCl_2 promoted the enzyme activity. There was a positive correlation between the increase in enzyme activity and heme saturation. Therefore,this study provides a theoretical basis for improving heme saturation by adding exogenous promoters and applying this in industrial fermentation of dye-decolorizing peroxidase.
引文
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[22]MORAWSKI B,LIN Z,CIRINO P,et al.Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris[J].Protein Engineering,2000,13(5):377-384.
[23]张俊丽.代谢工程改造大肠杆菌血红素合成途径生产5-氨基乙酰丙酸[D].无锡:江南大学,2016.
[24]VARNADO C L,GOODWIN D C.System for the expression of recombinant hemoproteins in Escherichia coli[J].Protein Expression and Purification,2004,35(1):76-83.
[25]KERY V,ELLEDER D,KRAUS J.δ-Aminolevulinate increases heme saturation and yield of human cystathionineβ-synthase expressed in Escherichia coli[J].Archives of Biochemistry and Biophysics,1995,316(1):24-29.
[26]KRAINER F W,CAPONE S,JAGER M,et al.Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris[J].Microbial Cell Factories,2015,14:4.
[2]SUGANO Y,ISHII Y,SHODA M.Role of H164 in a unique dye-decolorizing heme peroxidase Dy P[J].Biochemical and Biophysical Research Communications,2004,322(1):126-132.
[3]DUNFORD H B.Heme Peroxidases[M].Wiley-Ych,1999.
[4]SUGANO Y,MURAMATSU R,ICHIVANAGI A,et al.Dy P,a unique dye-decolorizing peroxidase,represents a novel heme peroxidase family ASP171 replaces the distal histidine of classical peroxidases[J].Journal of Biological Chemistry,2007,282(50):36 652-36 658.
[5]SATO T,HARA S,MATSUI T,et al.A unique dye-decolorizing peroxidase,Dy P,from Thanatephorus cucumeris Dec 1:heterologous expression,crystallization and preliminary X-ray analysis[J].Acta Crystallographica Section D:Biological Crystallography,2004,60(1):149-152.
[6]ZUBIETA C,KRISHNA S S,KAPOOR M,et al.Crystal structures of two novel dye-decolorizing peroxidases reveal aβ-barrel fold with a conserved heme-binding motif[J].Proteins:Structure,Function,and Bioinformatics,2007,69(2):223-233.
[7]FARAC0 V,PISCITELLI A,SANNIA G,et al.Identification of a new member of the dye-decolorizing peroxidase family from Pleurotus ostreatus[J].World Journal of Microbiology and Biotechnology,2007,23(6):889-893.
[8]LAUBER C,SCHWARZ T,NGUYEN Q K,et al.Identification,heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus[J].AMBExpress,2017,7(1):164.
[9]LIERS C,BOBETH C,PECVNA M,et al.Dy P-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes[J].Applied Microbiology and Biotechnology,2010,85(6):1 869-1 879.
[10]DOERGED R,DIVI R L,CHURCHWELL M I.Identification of the colored guaiacol oxidation product produced by peroxidases[J].Analytical Biochemistry,1997,250(1):10-17.
[11]RAHMANPOUR R,REA D,JAMSHIDI S,et al.Structure of Thermobifida fusca Dy P-type peroxidase and activity towards kraft lignin and lignin model compounds[J].Archives of Biochemistry and Biophysics,2016,594:54-60.
[12]LONCAR N,COLPA D I,FRAAIJE M W.Exploring the biocatalytic potential of a Dy P-type peroxidase by profiling the substrate acceptance of Thermobifida fusca Dy P peroxidase[J].Tetrahedron,2016,72(46):7 276-7 281.
[13]YOSHIDA T,SUGANO Y.A structural and functional perspective of Dy P-type peroxidase family[J].Archives of Biochemistry and Biophysics,2015,574:49-55.
[14]AHMAD M,ROBERTS J N,HARDIMAN E M,et al.I-dentification of Dyp B from Rhodococcus jostii RHA1 as a lignin peroxidase[J].Biochemistry,2011,50(23):5 096-5 107.
[15]MENDES S,CATARINO T,SILVEIRA C,et al.The catalytic mechanism of A-type dye-decolourising peroxidase Bs Dy P:neither aspartate nor arginine is individually essential for peroxidase activity[J].Catalysis Science&Technology,2015,5(12):5 196-5 207.
[16]HENDRIKS I A,VERTEGAAL A C O.A comprehensive compilation of SUMO proteomics[J].Nature Reviews Molecular Cell Biology,2016,17(9):581.
[17]COLPA D I,FRAAIJE M W.High overexpression of dye decolorizing peroxidase Tfu Dy P leads to the incorporation of heme Precursor protoporphyrin IX[J].Journal of Molecular Catalysis B:Enzymatic,2016,134:372-377.
[18]贺娟妮.彩绘蛋白胶结材料MALDI-TOF-MS分析及表征研究[D].西安:西北大学,2015.
[19]余波,程安春,汪铭书.大肠杆菌中重组蛋白可溶性表达的研究进展及展望[J].黑龙江畜牧兽医,2008(10):19-21.
[20]CONESA A,VAN DEN HONDEL C A,PUNT P J.Studies on the production of fungal peroxidases in Aspergillus niger[J].Applied and Environmental Microbiology,2000,66(7):3 016-3 023.
[21]SEGURA M M,LEVIN G,MRANDA M V,et al.Highlevel expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture[J].Process Biochemistry,2005,40(2):795-800.
[22]MORAWSKI B,LIN Z,CIRINO P,et al.Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris[J].Protein Engineering,2000,13(5):377-384.
[23]张俊丽.代谢工程改造大肠杆菌血红素合成途径生产5-氨基乙酰丙酸[D].无锡:江南大学,2016.
[24]VARNADO C L,GOODWIN D C.System for the expression of recombinant hemoproteins in Escherichia coli[J].Protein Expression and Purification,2004,35(1):76-83.
[25]KERY V,ELLEDER D,KRAUS J.δ-Aminolevulinate increases heme saturation and yield of human cystathionineβ-synthase expressed in Escherichia coli[J].Archives of Biochemistry and Biophysics,1995,316(1):24-29.
[26]KRAINER F W,CAPONE S,JAGER M,et al.Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris[J].Microbial Cell Factories,2015,14:4.