黄孢原毛平革菌锰过氧化物酶的研究
|
摘要
白腐担子菌(White-rot Basidiomycetes)分泌的胞外锰过氧化物酶(manganese peroxidase，MnP)是胞外降解木素体系的一种主要成分。来自白腐担子菌的典型种黄孢原毛平革菌(Phanerochaete Chrysosporium)的锰过氧化物酶是一种需要H_2O_2的血红素糖蛋白，其相对分子质量范围为4.5×10~4-4.7×10~4Dalton。本论文旨在确定黄孢原毛平革菌产锰过氧化物酶的最优培养基和最优发酵条件，并通过基因工程手段得到锰过氧化物酶(MnP2)基因的克隆，为进一步将其转入适宜的真核细胞表达系统进行蛋白异源表达奠定基础。其主要内容包括以下两个方面。
1．黄孢原毛平革菌产锰过氧化物酶最适条件的确定
在静置培养条件下，通过对产酶培养基的优化，得到最优组成为(L~(-1))：
葡萄糖10g，酒石酸铵2mmol，吐温80 1g，醋酸缓冲液10mmol(pH4.0)，V_(B1) 1mg，，Mn~(2+)9.9×10~(-6)g。采用该发酵限氮培养基，于34℃静置培养5d，产MnP活力达1200 U／L，比未优化提高近17倍。
进一步对产酶最适的培养条件进行优化，最终在250mL三角瓶中装液量30mL：发酵限氮培养基pH4.5；接种后，孢子终浓度为6.7×10~5个孢子／mL；温度37℃；静置培养5d；产锰过氧化物酶活力达1452U／L，比未优化条件MnP活力提高20％。
2锰过氧化物酶(MnP2)基因的克隆
以最适条件培养的菌丝为材料，采用Trizal法提取黄孢原毛平革菌5.776总RNA并以之为模板进行RT-PCR，成功扩增出预期大小约为1.3kb的特异性条带，将扩增产物提纯后与pUC19载体体外重组，转化E．coli JM109，经筛选及酶切鉴定后，获得锰过氧化物酶(MnP2)基因的克隆。通过SDS-PAGE分析表明重组质粒pUC19-mnp2／JM109比空质粒pUC19／JM109在相对分子质量4.5×10~4Dalton处多一条蛋白带，其大小与黄孢原毛平革菌锰过氧化物酶单体的大小相同，证明MnP2克隆成功。
Extracellular manganese peroxidase which is secret by white-rot Basidiomycetes is a major component of the extracellular lignin-degrading systems. Manganese peroxidase from a model strain of the white-rot Basidiomycetes-Phanerochaete Chrysosporium is a H2O2-requiring heme glycoprotein of Mr=4.5 ×104-4.7×104Dalton.The main purpose of this dissertation lies in establishing the optimum culture components and the optimum fermentation conditions that Phanerochaete Chrysosporium produce manganese peroxidase,and lies in that clone of manganese peroxidase was acquired by gene engineering means.the groundwork that manganese peroxidase albumen hetero-source expression in fitting fungal expression system was established. The mainly researching contents of the dissertation include two aspects as blow:
1 .The most agreeable conditions that Phanerochaete Chrysosporium produce manganese peroxidase was established.
Under static culture condition, the optimum culture components by optimizing culture which produce manganese peroxidase showed as blow(L-1):
Glucose 10g, ammonium tartrate 2mmol, Tween 80 1g, acetate buffer (pH4.0) lOmmol, VB1 1mg , Mn2+ 9.9×10~6 g. The culture temperature was 34℃.Under above conditions,the strain produced MnP activity up to 1200 U/L during 5 days stationary culture ,which increased almost 17 times than that of initially fermentation culture.
The most agreeable culture condition which produce manganese peroxidase was optimized farther. The volume of liquid culture is 30mL(250 mL Erlenmeyer flasks),the pH of the fementable limiting nitrogen culture is 4.5,the inoculation volume is 6.7×105 entries spores per milliliter,the culture temperature was 37 ℃ .Under above conditions,the strain produced MnP activity up to 1452U/L during 5 days stationary culture, which increased almost 20% than that of initially fermentation condition.
2. Molecular clone of cDNA encoding manganese peroxidase (MnP2). total RNA of 5.776 was withdrawed from the fungi silk which is educated in the most agreeable conditions and RT-PCR was proceeded regarding it as template, cDNA encoding specialized fragment whose size is about 1.3kb was amplified successfully just as anticipation.Amplified produut was extracellularly recombined with pUC19 vector after purification,and was transformed into E. coli JM109,then,the MnP2 gene cloning was obtained after screening and enzyme-cut identify.The analysis through SDS-PAGE showed that recombination plasmid
pUC19-mnp2/JM109 have a more albumen fragment than that of volume plasmid pUC19/JM109 and its size is about 4.5 X 104Dalton,the same as that of manganese peroxidase(MnP2) monomer come from Phanerochaete Chrysosporium which proved clone is success.
1．黄孢原毛平革菌产锰过氧化物酶最适条件的确定
在静置培养条件下，通过对产酶培养基的优化，得到最优组成为(L~(-1))：
葡萄糖10g，酒石酸铵2mmol，吐温80 1g，醋酸缓冲液10mmol(pH4.0)，V_(B1) 1mg，，Mn~(2+)9.9×10~(-6)g。采用该发酵限氮培养基，于34℃静置培养5d，产MnP活力达1200 U／L，比未优化提高近17倍。
进一步对产酶最适的培养条件进行优化，最终在250mL三角瓶中装液量30mL：发酵限氮培养基pH4.5；接种后，孢子终浓度为6.7×10~5个孢子／mL；温度37℃；静置培养5d；产锰过氧化物酶活力达1452U／L，比未优化条件MnP活力提高20％。
2锰过氧化物酶(MnP2)基因的克隆
以最适条件培养的菌丝为材料，采用Trizal法提取黄孢原毛平革菌5.776总RNA并以之为模板进行RT-PCR，成功扩增出预期大小约为1.3kb的特异性条带，将扩增产物提纯后与pUC19载体体外重组，转化E．coli JM109，经筛选及酶切鉴定后，获得锰过氧化物酶(MnP2)基因的克隆。通过SDS-PAGE分析表明重组质粒pUC19-mnp2／JM109比空质粒pUC19／JM109在相对分子质量4.5×10~4Dalton处多一条蛋白带，其大小与黄孢原毛平革菌锰过氧化物酶单体的大小相同，证明MnP2克隆成功。
Extracellular manganese peroxidase which is secret by white-rot Basidiomycetes is a major component of the extracellular lignin-degrading systems. Manganese peroxidase from a model strain of the white-rot Basidiomycetes-Phanerochaete Chrysosporium is a H2O2-requiring heme glycoprotein of Mr=4.5 ×104-4.7×104Dalton.The main purpose of this dissertation lies in establishing the optimum culture components and the optimum fermentation conditions that Phanerochaete Chrysosporium produce manganese peroxidase,and lies in that clone of manganese peroxidase was acquired by gene engineering means.the groundwork that manganese peroxidase albumen hetero-source expression in fitting fungal expression system was established. The mainly researching contents of the dissertation include two aspects as blow:
1 .The most agreeable conditions that Phanerochaete Chrysosporium produce manganese peroxidase was established.
Under static culture condition, the optimum culture components by optimizing culture which produce manganese peroxidase showed as blow(L-1):
Glucose 10g, ammonium tartrate 2mmol, Tween 80 1g, acetate buffer (pH4.0) lOmmol, VB1 1mg , Mn2+ 9.9×10~6 g. The culture temperature was 34℃.Under above conditions,the strain produced MnP activity up to 1200 U/L during 5 days stationary culture ,which increased almost 17 times than that of initially fermentation culture.
The most agreeable culture condition which produce manganese peroxidase was optimized farther. The volume of liquid culture is 30mL(250 mL Erlenmeyer flasks),the pH of the fementable limiting nitrogen culture is 4.5,the inoculation volume is 6.7×105 entries spores per milliliter,the culture temperature was 37 ℃ .Under above conditions,the strain produced MnP activity up to 1452U/L during 5 days stationary culture, which increased almost 20% than that of initially fermentation condition.
2. Molecular clone of cDNA encoding manganese peroxidase (MnP2). total RNA of 5.776 was withdrawed from the fungi silk which is educated in the most agreeable conditions and RT-PCR was proceeded regarding it as template, cDNA encoding specialized fragment whose size is about 1.3kb was amplified successfully just as anticipation.Amplified produut was extracellularly recombined with pUC19 vector after purification,and was transformed into E. coli JM109,then,the MnP2 gene cloning was obtained after screening and enzyme-cut identify.The analysis through SDS-PAGE showed that recombination plasmid
pUC19-mnp2/JM109 have a more albumen fragment than that of volume plasmid pUC19/JM109 and its size is about 4.5 X 104Dalton,the same as that of manganese peroxidase(MnP2) monomer come from Phanerochaete Chrysosporium which proved clone is success.
引文
[1] 程言君，纸浆漂白废水中有机氯化物的排放与控制[J]．中国造纸．1994，13(8)：57-61．
[2] 王彩华，文湘辉，余惠生，国际生物漂白研究及应用最新进展[J]．广东造纸．1995，3-4：8．11，32
[3] Tien M,Kirk T K.Lignin degradation enzyme from P chrysosporium .Proc.Natl.Sci.1984,81:2280-2284.
[4] 余惠生，黄秀瑜，稻草木素生物降解研究[J]．纤维素科学与技术．1993，1(1)：12-23．
[5] Ming Tien, T.Kent Kirk, Lignin-Degrading Enzyme from the Hymenomycete Phanetochaete chrysosporium Burds[J].Science. 1983, 221: 661-663.
[6] R. L. Kelley, C. Adinarayana Reddy Identification of Glucose Oxidase Activity as Primary Source of Hydrogen Peroxide Production in Ligninolytic Cultures of Phanerochaete chrysosporium, Arch Microbiol, 1986,144: 248- 253.
[7] J. K. Glenn, M. A. Morgan, M.B. Mayfield et alal. Biochem Biophysres Commum, 1983,114(3): 1077-1083.
[8] Kuwahara M, et al Purification and characterization of two lignin peroxidase isozymes produced Bjerkandera sp.strain Bos55 [J].FEBSLett, 1984,168-247.
[9] Robert Bourbonnais, Michael G. Paice. Enaymatic Delignification of Kaft Pulp Using Laccase and a Mediator [J], Tappi J,1996, 79(6): 199-204.
[10] Michel JRFC,Dass SB,Grulke EA,Reddy CA.Role of MnP and LiP of Phanetochaete chrysosporium[J]. Appl.Environ. Microbiol. 1991,57: 2368-2375.
[11] Shimada M, Higuchi T. Microbiol, Enzymatic and Biomimetic Degradation of Lignin, 1990:557-619.
[12] Ming Tien,Properties of ligninase from Phanetochaete chrysosporium and their possible application. CRC Critical Reviews in Microbiology. 1987,15(2): 141-168.
[13] 浦跃武，甄浩铭，冯书庭等．白腐菌产锰过氧化物酶条件的研究[J]．菌物系统，1998，17(3)：251-255．
[14] L.A.Andersson, V.Renganathan, A.A.Chiu et al. Spectral Characterization of Diarylpropane oxygenase, a Novel peroxidase-dependent, lignin-degrading heme enzyme[J], J. Biol. Chem. 1985, 266(10):6080-6087.
[15] Glenn, J.K., M., Gold, M.H. Purification and characterization of an extrcellul ar Mn(Ⅱ)-depedent peroxidase from the lignin-degrading Basidiomyceie
Phanerochaete chrysosporium. Arch. Biochem Biophys. 1985,242:329-341.
[16] H. P. Call, I. Mucke, 1994 NCB-Conference, Proceedings.
[17] David P.Barr, Steven D.Aust,Pollutant degradation by white rot fungi. In: Revi ews of Environmental Contamination and Texicology. Springer-Ve rlag, New York,Inc. 1994,49-72.
[18] David P.Barr, Steven D.Aust,Mechanism white rot fungi use to degrade pollut ants. Environ.Sci.Technol., 1994,28(2)78A-87A.
[19].Gellerstedt G, Zhang L-M. Nordic Pulp and Paper Research J,1991, 6(3):136- 139.
[20] P.J. Kersten, T.K.Kirk, Involvement of a new Enzyme, Glyoxal oxidasein Extracellular H_2O_2 Production by Phanerochaete chrysosporium, J.Bacterial, 1987,169(5):2195-2201.
[21] Robert Bourbonnais, Michael G. Paice. Enaymatic Delignification of Kaft Pulp Using Laccase and a Mediator [J], Tappi J,1996, 79(6): 199-204.
[22] Frederick S.Archibald.A New Assay for Lignin-type Peroxidase Employing the Dye Azure B [J]. Appl.Environ.Microbiol. 1992,58(9):3110-3116.
[23] 周金燕，张发群，桑原正章．真菌产生的锰过氧化物酶和漆酶研究Ⅱ．一株产锰过氧化物酶的担子菌—血红密孔菌K-2352[J]．微生物学通报，1994，21(3)：152-156．
[24] M.H.Gold,MnP from P.chrysosporium :Biochemical and Genetic Charaterization,International Conference on Bio-technology in Pulp and Paper Industry,447-455.
[25] Koichi Harazono,Ryuichiro Kondo,Kokki Sakai.Bleachingo of hardwood kraft pulp with MnP from Phanetochaete sordida YK-624 without addition of MnSO_4[J]. Appl. Environ.Microbiol. 1994,52(9):2456-2459.
[26] Tadakazu Hiroi,Karl-Erik Eriksson et al..Microbiological Degradation of Lignin ,Part2.Influence of Cellulose upon the Degradation of Calcium Lignosulfonate of Various Molecular Sizes by the White-rot Fungus Pleurotus ostrea us[J]. Svensk Papperstidning 1976,79(5): 162-166.
[27] Glenn JK,Morgan MA,Mayfield NB,Kuwahare M,Gold MH.Biochem Biophys Res Commun. 1983,114:1077-1083.
[28] 周金燕，张发群，桑原正章．真菌产生的锰过氧化物酶和漆酶研究Ⅰ富氮培养基筛选产酶的真菌[J]．微生物学报，1993，33(5)：387-391．
[29] Katrin; Ziegenhagen, Dirk; Hofrichter, Martin; Sorge, Silke;Gruen, Manfred (Friedrich-Schiller-Universitaet Jena; AUA Agrar- undUmweltanalytik G.m.b.H., Germany). Method for production of manganese peroxidase with
white-rot fungiScheibner, Ger. Often. DE 19741083 A1 25Mar 1999, 6 pp. (German). (Germany). CODEN: GWXXBX. CLASS:ICM: C12N009-08. ICI: C 12N009-08, C12R001-645 APPLICATION:DE 97-19741083 18 Sep 1997. DOCUMENT TYPE: Patent CA.
[30] Sundaramoorthy M, Kishi K Gold M H, et al.The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06A resolution. J Biol Chem, 1994, 269, 32759—32767.
[31] Wariishi H.Akileswaran L, Gold M H. Manganese peroxidase from basidiomycete Phanerochaete chrysosporium spectral eharacterzation of the oxidized states and the catalytic cycle. Biochemistry, 1988, 27, 5365-5370.
[32] Sergei L, Timofeevski, Nie Guojun, et al. Reading, substrate specificity of lignin peroxidase and a S168W variant of Manganese peroxidase. Arch Biochem Biophysics, 2000, 373, 147-153.
[33] Mino Y, wariishi H, Blackburn N J, et al., Spectral characterization of Manganese peroxidase,an extrocellular heme enzyme from the lignin-degrading Basidiomycete Phanerochaete chrysosporium. J Biol Chem. 1988,263,7029-7036.
[34] Mayfield M B, Godfrey B J, Gold M H, Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomcete Phanerochaete chrysosporium. Cene, 1994, 142, 231-235.
[35] Sollewijn Gelpke M D, Young H L,Gold M H. Effects of cadmium on manganese peroxidase:Competitive inhibition of Mn2+ oxidation and thermal stabilization of the enzyme. Eur J Biochem., 2000, 267, 1761-1769.
[36] Schudler DJ, Ban N, Huystee R B, et al. Structure, 1996, 4, 311-321.
[37] Harris R Z, wariishi H, Gold M H, et al The Catalytic site of manganese peroxidase. J Biol Chem, 1991, 266: 8751-8758.
[38] Role of arginine 177 in the Mn~Ⅱ binding site of manganese peroxidase:Studies with R177D, R177E, R17TN, and R177Q mutants,. Eur J Biochem, 2000, 267, 7038-7045.
[39] Whitwam R, Tien M, Heterologous expression and reconstitution of fungal Mn peroxidase. Arch Biochem Biophys, 1996, 333, 439-446.
[40] Stewart P, Whitwarn R E, Kersten P J, et al. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Asergillus oryzae. Appl Environ Microbiol, 1996, 62, 860-864.
[41] Conesa A, Jeenes D, Archer D B, Differential regulation of mnp2, a new
manganese peroxidase-encoding gene from the ligninolytic fungus Trarnetes versicolor PRL 572. Appl Envir Microbiol, 2002, 68: 2077—2080.
[42] Youngs L, Sundaramoorthy M, et al. Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl Envir Microbiol, 2002, 68, 846-851.
[43] Kishi K, Kusters-vans S M, Mayfield M B, et al. Characterzation of manganese(Ⅱ) binding site mutants of manganese peroxidase.. Biochemistry, 1994, 35, 8986—8994.
[44] Whitwan R E, Brown K R, Musick M, et al. Mutagenesis of the Mn~Ⅱ-binding site of manganese peroxidase affects xidation of Mn~Ⅱby both compound Ⅰ and compoundⅡ . Biochemistry, 1997, 36, 9766-9773.
[45] Sollewijn Gelpke M D, Moenne-Loccoz P. Gold M H, Arginine 177 is involved in Mn(Ⅱ)binding by Manganese peroxidase. Biochemstry, 1999, 38, 11481-11489.
[46] Kishi K, Hildebrand D P, Kusters—van someren M, et al. Site—directed mutations at Phe-190 of manganese peroxidase: Effects on stability, function, and coordination. Biochemistry, 1997, 36, 4268-4277.
[47] Naidu P S, Reddy C A. Nucleotide sequence of a new Iignin peroxidase gene GLG3 from the white—rot fungus. Phanerochaete chrysosporium. Nucleic Acids Res, 1990, 18(13): 7173-7179.
[48] Ballance D J. Sequences important for gene expression in filamentous fungi. Yeast. 1986, 2(1):229-236.
[49] Godfrey B J, Mayfield M B, Brown J A et al Characterization of a gene encoding a manganese peroxidase from phanerochaete chrysosporium.Gene.1990, 93(1): 119-124.
[50] Kozak M. Comparation of initiation of protein synthesis in Procaryotes, eucaryotes, and organelles. Microbiol Rev, 1983, 47(1): 1-45.
[51] Brown J A, Glenn J K. Gold M H et al. Manganese regulates tLe expression of manganese peroxidase by Phanerochaete chrysosporium.J Bacteriol, 1990, 172(6): 3125-3130.
[52] Stewart P, Kersten P, Vanden A W et al. Lignin peroxidase gene family of Phanerochaete chrysosporium:complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J Bacteriol, 1992, 174(7): 5036—5042
[53] Bonnarme P, Joffries T W. Mn() regulation of lignin peroxidases and
manganese-dependent pemxidases from lignin-degrading white rot fungi. Appl Envirom Microbiol,1990,56(1): 210-217
[54] 吴坤，张世敏，朱显峰．木质素生物降解研究进展[J]．河南农业大学学报，2000，34(4)：349-354．
[55] 张朝晖，夏黎明，柯世省，等．固定化培养黄孢原毛平革菌合成木质素过氧化物酶研究[J]．高校化学工程学报，1999，13(2)：135-140．
[56] Mohamed S A, Sami S,.Ali G. Heterologous expression of lignin peroxiedase of Phanerochaete chrysosporium in Aspergillus niger[J].Biotechnology Letters. 1999,21:849-853.
[57] Gold, M. H., H. Wariishi, and K. Valli.Extracellular peroxidases involved in lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium [J].ACS Symp.Ser, 1989,389:127-140
[58] 刘稳，李杨，高培基，等．过氧化物酶研究进展[J]．纤维素科学与技术，2000，8(2)：50-64．
[59] Yoshoiys K, Ishikaa T, Nakura Y et al. Comparative study on recombinant chloroplastic and cytosorbote peroxidase isozymes of spinach[J]. Arch Biochem Biophys, 1998,353(1):55-63
[60] Kirk, T. K.and R. L. Farrell.Enzymatic "combustion": the microbi degradation oflignin [J]. Annu Rev Microbiol,1987,41:465-505
[61] Barlev S S,Kirk T K.Effects of molocular oxygen on lignin degradation by Phanerochaete Chrysosporium[J]. Biochen Biophys Res Common, 1981,99:373-378
[62] 柯世省，夏黎明，张朝晖等．氧浓度对固定化黄孢原毛平革菌合成过氧化物酶的影响[J]．林产化学与工业，2000，20(3)：40～46．
[63] 刘伟，李若瑜，王端礼等．常见致病性丝状真菌总RNA的快速提取[J]．中国麻风皮肤病杂志，2000，16(3)：202-203．
[64] J．萨姆布鲁克，D．W．拉塞尔著．分子克隆实验指南(第三版)[美][M]．科学出版社，2002
[65] Margaret Alic,Lakshmi Akileswaran,Michael H.Gold. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete Chrysosporium [J].Biochimica et Biophysica Acta. 1997,1338:1-7.
[66] Mary B.Mayfield,Bruce J.Godfrey and Michael H.Gold.Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomycete Phanerochaete Chrysosporium[J].Gene. 1994,142:231-235.
[67] Ana C,Cees A M J,Peter J P. Studies on the production of fungal peroxidases in Aspergillus niger[J]. Applied and Environmental
Microbiology. 2000,66(7):3016-3023.
[68] Jessica M.Gettemy, Biao Ma, Margaret Alie,and Michael H.Gold.Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family[J]. Applied and Environmental Microbiology. 1998,64(2):569-57
[2] 王彩华，文湘辉，余惠生，国际生物漂白研究及应用最新进展[J]．广东造纸．1995，3-4：8．11，32
[3] Tien M,Kirk T K.Lignin degradation enzyme from P chrysosporium .Proc.Natl.Sci.1984,81:2280-2284.
[4] 余惠生，黄秀瑜，稻草木素生物降解研究[J]．纤维素科学与技术．1993，1(1)：12-23．
[5] Ming Tien, T.Kent Kirk, Lignin-Degrading Enzyme from the Hymenomycete Phanetochaete chrysosporium Burds[J].Science. 1983, 221: 661-663.
[6] R. L. Kelley, C. Adinarayana Reddy Identification of Glucose Oxidase Activity as Primary Source of Hydrogen Peroxide Production in Ligninolytic Cultures of Phanerochaete chrysosporium, Arch Microbiol, 1986,144: 248- 253.
[7] J. K. Glenn, M. A. Morgan, M.B. Mayfield et alal. Biochem Biophysres Commum, 1983,114(3): 1077-1083.
[8] Kuwahara M, et al Purification and characterization of two lignin peroxidase isozymes produced Bjerkandera sp.strain Bos55 [J].FEBSLett, 1984,168-247.
[9] Robert Bourbonnais, Michael G. Paice. Enaymatic Delignification of Kaft Pulp Using Laccase and a Mediator [J], Tappi J,1996, 79(6): 199-204.
[10] Michel JRFC,Dass SB,Grulke EA,Reddy CA.Role of MnP and LiP of Phanetochaete chrysosporium[J]. Appl.Environ. Microbiol. 1991,57: 2368-2375.
[11] Shimada M, Higuchi T. Microbiol, Enzymatic and Biomimetic Degradation of Lignin, 1990:557-619.
[12] Ming Tien,Properties of ligninase from Phanetochaete chrysosporium and their possible application. CRC Critical Reviews in Microbiology. 1987,15(2): 141-168.
[13] 浦跃武，甄浩铭，冯书庭等．白腐菌产锰过氧化物酶条件的研究[J]．菌物系统，1998，17(3)：251-255．
[14] L.A.Andersson, V.Renganathan, A.A.Chiu et al. Spectral Characterization of Diarylpropane oxygenase, a Novel peroxidase-dependent, lignin-degrading heme enzyme[J], J. Biol. Chem. 1985, 266(10):6080-6087.
[15] Glenn, J.K., M., Gold, M.H. Purification and characterization of an extrcellul ar Mn(Ⅱ)-depedent peroxidase from the lignin-degrading Basidiomyceie
Phanerochaete chrysosporium. Arch. Biochem Biophys. 1985,242:329-341.
[16] H. P. Call, I. Mucke, 1994 NCB-Conference, Proceedings.
[17] David P.Barr, Steven D.Aust,Pollutant degradation by white rot fungi. In: Revi ews of Environmental Contamination and Texicology. Springer-Ve rlag, New York,Inc. 1994,49-72.
[18] David P.Barr, Steven D.Aust,Mechanism white rot fungi use to degrade pollut ants. Environ.Sci.Technol., 1994,28(2)78A-87A.
[19].Gellerstedt G, Zhang L-M. Nordic Pulp and Paper Research J,1991, 6(3):136- 139.
[20] P.J. Kersten, T.K.Kirk, Involvement of a new Enzyme, Glyoxal oxidasein Extracellular H_2O_2 Production by Phanerochaete chrysosporium, J.Bacterial, 1987,169(5):2195-2201.
[21] Robert Bourbonnais, Michael G. Paice. Enaymatic Delignification of Kaft Pulp Using Laccase and a Mediator [J], Tappi J,1996, 79(6): 199-204.
[22] Frederick S.Archibald.A New Assay for Lignin-type Peroxidase Employing the Dye Azure B [J]. Appl.Environ.Microbiol. 1992,58(9):3110-3116.
[23] 周金燕，张发群，桑原正章．真菌产生的锰过氧化物酶和漆酶研究Ⅱ．一株产锰过氧化物酶的担子菌—血红密孔菌K-2352[J]．微生物学通报，1994，21(3)：152-156．
[24] M.H.Gold,MnP from P.chrysosporium :Biochemical and Genetic Charaterization,International Conference on Bio-technology in Pulp and Paper Industry,447-455.
[25] Koichi Harazono,Ryuichiro Kondo,Kokki Sakai.Bleachingo of hardwood kraft pulp with MnP from Phanetochaete sordida YK-624 without addition of MnSO_4[J]. Appl. Environ.Microbiol. 1994,52(9):2456-2459.
[26] Tadakazu Hiroi,Karl-Erik Eriksson et al..Microbiological Degradation of Lignin ,Part2.Influence of Cellulose upon the Degradation of Calcium Lignosulfonate of Various Molecular Sizes by the White-rot Fungus Pleurotus ostrea us[J]. Svensk Papperstidning 1976,79(5): 162-166.
[27] Glenn JK,Morgan MA,Mayfield NB,Kuwahare M,Gold MH.Biochem Biophys Res Commun. 1983,114:1077-1083.
[28] 周金燕，张发群，桑原正章．真菌产生的锰过氧化物酶和漆酶研究Ⅰ富氮培养基筛选产酶的真菌[J]．微生物学报，1993，33(5)：387-391．
[29] Katrin; Ziegenhagen, Dirk; Hofrichter, Martin; Sorge, Silke;Gruen, Manfred (Friedrich-Schiller-Universitaet Jena; AUA Agrar- undUmweltanalytik G.m.b.H., Germany). Method for production of manganese peroxidase with
white-rot fungiScheibner, Ger. Often. DE 19741083 A1 25Mar 1999, 6 pp. (German). (Germany). CODEN: GWXXBX. CLASS:ICM: C12N009-08. ICI: C 12N009-08, C12R001-645 APPLICATION:DE 97-19741083 18 Sep 1997. DOCUMENT TYPE: Patent CA.
[30] Sundaramoorthy M, Kishi K Gold M H, et al.The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06A resolution. J Biol Chem, 1994, 269, 32759—32767.
[31] Wariishi H.Akileswaran L, Gold M H. Manganese peroxidase from basidiomycete Phanerochaete chrysosporium spectral eharacterzation of the oxidized states and the catalytic cycle. Biochemistry, 1988, 27, 5365-5370.
[32] Sergei L, Timofeevski, Nie Guojun, et al. Reading, substrate specificity of lignin peroxidase and a S168W variant of Manganese peroxidase. Arch Biochem Biophysics, 2000, 373, 147-153.
[33] Mino Y, wariishi H, Blackburn N J, et al., Spectral characterization of Manganese peroxidase,an extrocellular heme enzyme from the lignin-degrading Basidiomycete Phanerochaete chrysosporium. J Biol Chem. 1988,263,7029-7036.
[34] Mayfield M B, Godfrey B J, Gold M H, Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomcete Phanerochaete chrysosporium. Cene, 1994, 142, 231-235.
[35] Sollewijn Gelpke M D, Young H L,Gold M H. Effects of cadmium on manganese peroxidase:Competitive inhibition of Mn2+ oxidation and thermal stabilization of the enzyme. Eur J Biochem., 2000, 267, 1761-1769.
[36] Schudler DJ, Ban N, Huystee R B, et al. Structure, 1996, 4, 311-321.
[37] Harris R Z, wariishi H, Gold M H, et al The Catalytic site of manganese peroxidase. J Biol Chem, 1991, 266: 8751-8758.
[38] Role of arginine 177 in the Mn~Ⅱ binding site of manganese peroxidase:Studies with R177D, R177E, R17TN, and R177Q mutants,. Eur J Biochem, 2000, 267, 7038-7045.
[39] Whitwam R, Tien M, Heterologous expression and reconstitution of fungal Mn peroxidase. Arch Biochem Biophys, 1996, 333, 439-446.
[40] Stewart P, Whitwarn R E, Kersten P J, et al. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Asergillus oryzae. Appl Environ Microbiol, 1996, 62, 860-864.
[41] Conesa A, Jeenes D, Archer D B, Differential regulation of mnp2, a new
manganese peroxidase-encoding gene from the ligninolytic fungus Trarnetes versicolor PRL 572. Appl Envir Microbiol, 2002, 68: 2077—2080.
[42] Youngs L, Sundaramoorthy M, et al. Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl Envir Microbiol, 2002, 68, 846-851.
[43] Kishi K, Kusters-vans S M, Mayfield M B, et al. Characterzation of manganese(Ⅱ) binding site mutants of manganese peroxidase.. Biochemistry, 1994, 35, 8986—8994.
[44] Whitwan R E, Brown K R, Musick M, et al. Mutagenesis of the Mn~Ⅱ-binding site of manganese peroxidase affects xidation of Mn~Ⅱby both compound Ⅰ and compoundⅡ . Biochemistry, 1997, 36, 9766-9773.
[45] Sollewijn Gelpke M D, Moenne-Loccoz P. Gold M H, Arginine 177 is involved in Mn(Ⅱ)binding by Manganese peroxidase. Biochemstry, 1999, 38, 11481-11489.
[46] Kishi K, Hildebrand D P, Kusters—van someren M, et al. Site—directed mutations at Phe-190 of manganese peroxidase: Effects on stability, function, and coordination. Biochemistry, 1997, 36, 4268-4277.
[47] Naidu P S, Reddy C A. Nucleotide sequence of a new Iignin peroxidase gene GLG3 from the white—rot fungus. Phanerochaete chrysosporium. Nucleic Acids Res, 1990, 18(13): 7173-7179.
[48] Ballance D J. Sequences important for gene expression in filamentous fungi. Yeast. 1986, 2(1):229-236.
[49] Godfrey B J, Mayfield M B, Brown J A et al Characterization of a gene encoding a manganese peroxidase from phanerochaete chrysosporium.Gene.1990, 93(1): 119-124.
[50] Kozak M. Comparation of initiation of protein synthesis in Procaryotes, eucaryotes, and organelles. Microbiol Rev, 1983, 47(1): 1-45.
[51] Brown J A, Glenn J K. Gold M H et al. Manganese regulates tLe expression of manganese peroxidase by Phanerochaete chrysosporium.J Bacteriol, 1990, 172(6): 3125-3130.
[52] Stewart P, Kersten P, Vanden A W et al. Lignin peroxidase gene family of Phanerochaete chrysosporium:complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J Bacteriol, 1992, 174(7): 5036—5042
[53] Bonnarme P, Joffries T W. Mn() regulation of lignin peroxidases and
manganese-dependent pemxidases from lignin-degrading white rot fungi. Appl Envirom Microbiol,1990,56(1): 210-217
[54] 吴坤，张世敏，朱显峰．木质素生物降解研究进展[J]．河南农业大学学报，2000，34(4)：349-354．
[55] 张朝晖，夏黎明，柯世省，等．固定化培养黄孢原毛平革菌合成木质素过氧化物酶研究[J]．高校化学工程学报，1999，13(2)：135-140．
[56] Mohamed S A, Sami S,.Ali G. Heterologous expression of lignin peroxiedase of Phanerochaete chrysosporium in Aspergillus niger[J].Biotechnology Letters. 1999,21:849-853.
[57] Gold, M. H., H. Wariishi, and K. Valli.Extracellular peroxidases involved in lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium [J].ACS Symp.Ser, 1989,389:127-140
[58] 刘稳，李杨，高培基，等．过氧化物酶研究进展[J]．纤维素科学与技术，2000，8(2)：50-64．
[59] Yoshoiys K, Ishikaa T, Nakura Y et al. Comparative study on recombinant chloroplastic and cytosorbote peroxidase isozymes of spinach[J]. Arch Biochem Biophys, 1998,353(1):55-63
[60] Kirk, T. K.and R. L. Farrell.Enzymatic "combustion": the microbi degradation oflignin [J]. Annu Rev Microbiol,1987,41:465-505
[61] Barlev S S,Kirk T K.Effects of molocular oxygen on lignin degradation by Phanerochaete Chrysosporium[J]. Biochen Biophys Res Common, 1981,99:373-378
[62] 柯世省，夏黎明，张朝晖等．氧浓度对固定化黄孢原毛平革菌合成过氧化物酶的影响[J]．林产化学与工业，2000，20(3)：40～46．
[63] 刘伟，李若瑜，王端礼等．常见致病性丝状真菌总RNA的快速提取[J]．中国麻风皮肤病杂志，2000，16(3)：202-203．
[64] J．萨姆布鲁克，D．W．拉塞尔著．分子克隆实验指南(第三版)[美][M]．科学出版社，2002
[65] Margaret Alic,Lakshmi Akileswaran,Michael H.Gold. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete Chrysosporium [J].Biochimica et Biophysica Acta. 1997,1338:1-7.
[66] Mary B.Mayfield,Bruce J.Godfrey and Michael H.Gold.Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomycete Phanerochaete Chrysosporium[J].Gene. 1994,142:231-235.
[67] Ana C,Cees A M J,Peter J P. Studies on the production of fungal peroxidases in Aspergillus niger[J]. Applied and Environmental
Microbiology. 2000,66(7):3016-3023.
[68] Jessica M.Gettemy, Biao Ma, Margaret Alie,and Michael H.Gold.Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family[J]. Applied and Environmental Microbiology. 1998,64(2):569-57