固定化黄孢原毛平革菌生产木素过氧化物酶及其对染料脱色降解的研究
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摘要
本文研究了多孔材料—聚氨脂泡沫固定化黄孢原毛平革菌合成木素过氧化物酶的产酶条件。在此基础上，研究了酶系的分离、固定化条件及固定化酶对染料的脱色降解。
经研究发现：使用聚氨脂泡沫固定黄孢原毛平革菌生产木素过氧化物酶的效果很好。本文研究了五种不同类型的聚氨酯泡沫的固定化效果，经发酵试验证明聚氨脂载体3固定化菌丝的产酶较高，且发酵液澄清，对后处理有利。聚氨脂泡沫价格便宜，是比较好的固定化载体。
用正交实验法对三种影响产酶较大的因素(载体量、酒石酸铵和转速)进行了优化，得到优化后的培养条件：载体量4g／L，酒石酸铵量2g／L和转速190r／min。优化后木素过氧化物酶活力可达200U／L，通过实验证明接种量对产酶影响不大。
固定化菌丝重复间歇培养生产木素过氧化物酶的试验表明：重复加入与原始培养基组分相同的培养基、碳源减半或氮源减半的培养基时，均可以较早的产酶。固定化菌丝重复产酶，可以省去菌种的生长阶段，并且重复过程中碳氮源减半也节省了成本。
用硫酸铵沉淀法对木素过氧化物酶进行了初步分离与纯化，结果表明：45％浓度硫酸铵对沉淀木素过氧化物酶的效果最好，沉淀后酶的回收率最高。用正交实验确立了木素过氧化物酶固定化的最佳条件：海藻酸钠浓度为3％，戊二醛浓度为0.4％，氯化钙浓度为0.2mol／L，在此条件下木素过氧化物能够被很好的固定。固定化后，酶的稳定性增强，对温度和pH的抗性增加。
以红玉为对象研究了游离酶对其的脱色条件：游离酶脱色低浓度红玉时效果较好，而对高浓度的红玉脱色率较差，在脱色过程中过氧化氢的最适浓度为100μmol／L，最适pH值为4.0，最适温度为35℃。
在研究固定化酶对红玉脱色时发现：固定化酶对红玉的脱色速率比游离酶要差，并且达到平衡的时间要较长。但是固定化酶可以重复使用7次，在重复使用时发现固定化酶对染料的脱色效率下降，这主要是在脱色过程中有过氧化氢的参与，而过氧化氢对酶液有失活作用，导致在重复使用过程中酶液的不断失活而导致对红玉的脱色效率下降。
In this thesis ,we first study lignin peroxidase production by Phanerochaete chrysosporiun on porous polyurethane foams . On the basis of this , we studied crude separation and purification of lignin peroxidase , immobilization of the lignin peroxidase and the degradation of the dyes by the immobilized lignin peroxidase.
From our investigation , we found that effection of lignin peroxidase production by the Phanerochaete chrysosporiun immobilized porous polyurethane foams are good . Among the five kind of on porous polyurethane are tested , the third is the most effective in the fermentation experiment.The mycelium immobilized stablely in the foam of the following and fermentation liquor was clear, which is beneficial on the post-treatment, Polyurethane foam is a suitable carrier for its cheap price and effectiveness.
We optimize three parameters which are the most effective in the lignin peroxidase production through orthogonal method : the amount of the carrier , the amount of ammonium tartrate and the the rotation. The optimal value of the three parameters are 4g/L, 2g/L and 190r/min respectively. Under which the production was increased and the activities of the lignin peroxidase can reach 200U/L Additionally , inoculation amount test show that it influence little in the fermentation.
Repeated batch fermentation of the immobilized Phanerochaete chrysosporiun indicated that we can get the lignin peroxidase if we adding initial fermentation medium or adding medium which is lower in glucose or ammonium tartrate, We can reused the immobilized mycelium and bypass the incubation period after immobilization. Furthermore , reducing glucose or ammonium tartrate is an economic way in reducing production cost.
We did some crude separation and purification of the lignin peroxidase.In the research we found that the concentration of 45% ammonium
sulfate is the best when precipitation of the lignin peroxidase, the acquirement rate of which is 86% , We optimize three parameters that are the most effective in the activity of immobilized lignin peroxidase production through orthogonal method : The optimal value of the three parameters are 3% sodium alginate, 0.4% glutaraldehyde and 0.2mol/L Calcium Chloridere respectively, under which the spectrums of the optional pH and optional temperature were expand and had a better stability.
When the four kind of dyes were degraded directively by the enzyme solution. They all had different extents of the degradation . In the test of using immobilized lignin peroxidase degrade dyes , We chose disperse Rubine as the object and investigated the potential of our immobilization system. In the comparison of the decolorization results of the three concentrations of the disperse Rubine we tested , we could conclude that the decolorization rate of the higher concentration are lower than those of the lower concentration..The result indicated that the immobilized lignin peroxidase could be reused at least of 7 times. In the test of the decolorization we found the colorization rate is declined gradully which due to the reducement of the concentration of H2O2.
经研究发现：使用聚氨脂泡沫固定黄孢原毛平革菌生产木素过氧化物酶的效果很好。本文研究了五种不同类型的聚氨酯泡沫的固定化效果，经发酵试验证明聚氨脂载体3固定化菌丝的产酶较高，且发酵液澄清，对后处理有利。聚氨脂泡沫价格便宜，是比较好的固定化载体。
用正交实验法对三种影响产酶较大的因素(载体量、酒石酸铵和转速)进行了优化，得到优化后的培养条件：载体量4g／L，酒石酸铵量2g／L和转速190r／min。优化后木素过氧化物酶活力可达200U／L，通过实验证明接种量对产酶影响不大。
固定化菌丝重复间歇培养生产木素过氧化物酶的试验表明：重复加入与原始培养基组分相同的培养基、碳源减半或氮源减半的培养基时，均可以较早的产酶。固定化菌丝重复产酶，可以省去菌种的生长阶段，并且重复过程中碳氮源减半也节省了成本。
用硫酸铵沉淀法对木素过氧化物酶进行了初步分离与纯化，结果表明：45％浓度硫酸铵对沉淀木素过氧化物酶的效果最好，沉淀后酶的回收率最高。用正交实验确立了木素过氧化物酶固定化的最佳条件：海藻酸钠浓度为3％，戊二醛浓度为0.4％，氯化钙浓度为0.2mol／L，在此条件下木素过氧化物能够被很好的固定。固定化后，酶的稳定性增强，对温度和pH的抗性增加。
以红玉为对象研究了游离酶对其的脱色条件：游离酶脱色低浓度红玉时效果较好，而对高浓度的红玉脱色率较差，在脱色过程中过氧化氢的最适浓度为100μmol／L，最适pH值为4.0，最适温度为35℃。
在研究固定化酶对红玉脱色时发现：固定化酶对红玉的脱色速率比游离酶要差，并且达到平衡的时间要较长。但是固定化酶可以重复使用7次，在重复使用时发现固定化酶对染料的脱色效率下降，这主要是在脱色过程中有过氧化氢的参与，而过氧化氢对酶液有失活作用，导致在重复使用过程中酶液的不断失活而导致对红玉的脱色效率下降。
In this thesis ,we first study lignin peroxidase production by Phanerochaete chrysosporiun on porous polyurethane foams . On the basis of this , we studied crude separation and purification of lignin peroxidase , immobilization of the lignin peroxidase and the degradation of the dyes by the immobilized lignin peroxidase.
From our investigation , we found that effection of lignin peroxidase production by the Phanerochaete chrysosporiun immobilized porous polyurethane foams are good . Among the five kind of on porous polyurethane are tested , the third is the most effective in the fermentation experiment.The mycelium immobilized stablely in the foam of the following and fermentation liquor was clear, which is beneficial on the post-treatment, Polyurethane foam is a suitable carrier for its cheap price and effectiveness.
We optimize three parameters which are the most effective in the lignin peroxidase production through orthogonal method : the amount of the carrier , the amount of ammonium tartrate and the the rotation. The optimal value of the three parameters are 4g/L, 2g/L and 190r/min respectively. Under which the production was increased and the activities of the lignin peroxidase can reach 200U/L Additionally , inoculation amount test show that it influence little in the fermentation.
Repeated batch fermentation of the immobilized Phanerochaete chrysosporiun indicated that we can get the lignin peroxidase if we adding initial fermentation medium or adding medium which is lower in glucose or ammonium tartrate, We can reused the immobilized mycelium and bypass the incubation period after immobilization. Furthermore , reducing glucose or ammonium tartrate is an economic way in reducing production cost.
We did some crude separation and purification of the lignin peroxidase.In the research we found that the concentration of 45% ammonium
sulfate is the best when precipitation of the lignin peroxidase, the acquirement rate of which is 86% , We optimize three parameters that are the most effective in the activity of immobilized lignin peroxidase production through orthogonal method : The optimal value of the three parameters are 3% sodium alginate, 0.4% glutaraldehyde and 0.2mol/L Calcium Chloridere respectively, under which the spectrums of the optional pH and optional temperature were expand and had a better stability.
When the four kind of dyes were degraded directively by the enzyme solution. They all had different extents of the degradation . In the test of using immobilized lignin peroxidase degrade dyes , We chose disperse Rubine as the object and investigated the potential of our immobilization system. In the comparison of the decolorization results of the three concentrations of the disperse Rubine we tested , we could conclude that the decolorization rate of the higher concentration are lower than those of the lower concentration..The result indicated that the immobilized lignin peroxidase could be reused at least of 7 times. In the test of the decolorization we found the colorization rate is declined gradully which due to the reducement of the concentration of H2O2.
引文
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71. Nathan Rothschild ,Yitzhak Hadar ,and Carlos Dosoretz, Ligninlytic system Formation by PHanerochaete chrysosprium in air ,Appplied and environmental Microbiology ,May 1995,pp. 1833-1838
72. Philip J.Kersten and T. Kent Kirk, Involvment of new Enzyme ,Glyoxal oxidase in extracellular H_2O_2 production by Phanerochaete chrysosprium ,Applied of Bacteriology, May 1987,p,2195-2201
73. Jeffrey K.Gleen, Michael H.Gold .Purification and characterization of an extracellar Mn(Ⅱ)-dependent peroxidase from the lignin-dagrading basidiomycete, Phanerochaete chrysosporium, Biochem biophys Arch. 1995,242(2):239-241
74. A. Paszczynski, A.B. Huynh and R. Crawford, Comparison of liguinase and peroxidase M2 from whiterot fungus Phanerochaete chrysosporium. Arch. Biochem. BiopHys. 26 1986, p. 477.
75. Matthias S. Fawer, Jrg Stierli, StepHen Cliffe and Attain Fiechter, The characterisation of immobilised lignin peroxidase by flow injection analysis , Biochimica et BiopHysica Acta (BBA)-Protein Structure and Molecular Enzymology Volume 1076, Issue 1,8 January 1991, Pages 15-22
76. Asther, M; Meunier, J C, Immobilization as a tool for the stabilization of lignin peroxidase produced by Phanerochaete chrysosporium INA-12 , Applied Biochemistry And Biotechnology Volume 38, Issue 12 January-February 1993, Pages 57-67
77. Peralta-zamora p.,Gomes de Moraes S.;Esposita E.;Antunes R,; Reyes J.;Duran N, Decolorization of pulp mill effluents with immobilized lignin and manganese peroxides from Phanerochaete chrysosporium ,Environmental Technology Volumn 19,Issue 5,1998,p. 521-528
78. Marcia Dezotti, Lucia H. Innocentini-Mei and Nelson Durán, Silica immobilized enzyme catalyzed removal of chloroliguins from eucalyptus kraft effluent, Journal of Biotechnology, Volume 43, Issue 3, 15 December 1995, Pages 161-167
79. Timothy L. Presnell, Harold E. Swaisgood, Thomas W. Joyce and Hou-min Chang,, Investigation into the kinetic properties of immobilized lignin peroxidases, Journal of Biotechnology, Volume 35, Issue 1, 15 June 1994, Pages 77-85
80. Grabski, A C; Rasmussen, J K; Coleman, P L; Burgess, R R, Immobilization of manganese peroxidase from Lentinula edodes on alkylaminated EmpHaze AB 1 polymer for generation of Mn~(3+) as an oxidizing agent; Applied Biochemistry And Biotechnology , Volume 60, Issue 1, July 1996, Pages 1-17 , ISSN: 0273-2289
81. W. Edwards, W. D. Leukes and J. J. Bezuidenhout, Ultrafiltration of petrochemical industrial wastewater using immobilised manganese peroxidase and laccase: application in the defouling of polysulp Hone membranes, Desalination, Volume 149, Issues 1-3,10 September 2002, Pages 275-278
82. Mielgo, C. Palma, J. M. Guisan, R. Fernandez-Lafuente, M. T. Moreira G. Feijoo and J. M. Lema, Covalent immobilisation of manganese peroxidases (MnP) from Phanerochaete chrysoaporium and Bjerkandera sp. BOS55, Enzyme and Microbial Technology , Volume 32, Issue 7, 12 June 2003, Pages 769-775
83. Grabski A.C.; Grimek H.J.; Burgess R.R, Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of Mn(Ⅲ)-chelate as a chemical oxidant of chloropHenols , Biotechnology and Bioengineering , Volume 60, Issue 2, 20 October 1998, Pages 204-215
84. Maynard ,C.W.,Jr..Dye application ,manufacture of dye intermediates and dyes In, J.A.Kent(ad.). Riegel's handbook of industrial chemistry. Van Nostrand Reinhold ,New York. 1983,p.809-861.
85. Vaidya ,A.A.,and K.V.Datye ..Environmental pollution during chemical processing of synthetic fibers .Colourage 1982 14:3-10
86. Zollinger .H. 1987.Color chemistry-syntheses ,properties and applications of orangnic dyes and pigments, VCH publishers ,New york. ,p.92-102
87. McCann ,J., and B.N.Ames.Detection of carcinogens as mutagens in the salmonella/microsome test .Assay of 300 chemicals :discussion .Proc.Natl .Acad. Sci.USA. 1975 73:950-954
88. Fujita ,S,,and J.persach .Electron transfer between liver microsomal cytochrome b_5 and cytochrome P-450 in the azo reductase reaction .Biochem .BiopHys. Res .Commun 1977.78:328-335
88. Tarpley ,W.G.,J.A.Miller and E.C.Miller Addcuts from the reaction of N-benzyloxy-N-methyl-4-aminoazobenzene with deoxyguanosine or DNA in virto from hepatic DNA of mice treated with N-methyl-or N,N-dimethyl-4-aminoazobenzene, Cancer Res,1980,40:2493-2499
90 Kulla ,H..,F.Klausener ,U.Meyer ,B.Ludeke ,and T.Leisinger, Interference of aromatic sulfo groups in the microbial degradation of the azo dyes orange Ⅰ ,1983
91. Jack T.Spadaro ,Michael H.Gold ,and V.Renganathan, Degradation of azo dyes by the lignin-degrading Fungus Phanerochaete chrysosporium, Applied and Environmental Microbiology, Aug 1992,p.2397-3401
92 Mielgo, M. T. Moreira, G. Feijoo and J. M. Lema, A packed-bed fungal bioreactor for the continuous decolourisation of azo-dyes (Orange Ⅱ), Journal of Biotechnology Volume 89, Issues 2-3,23 August 2001, Pages 99-106
93. Mielgo, M. T. Moreira, G. Feijoo and J. M. Lema, Biodegradation of a polymeric dye in a pulsed bed bioreactor by immobilised Phanerochaete chrysosporium, Water Research Volume 36, Issue 7, April 2002, Pages 1896-1901
94. Sami Sayadi, and Radhouane Ellouz ,Role of lignin peroxidase and Manganese peroxidase from Phanerochaete chrysosporium in the Degradation of olive Mill wastewaters, Applied and Environmental Microbiology ,Mar, 1995,p, 1098-1103
95. Hèla Zouari, Marc Labat and Sami Sayadi, Degradation of 4-chlorophenol by the white rot fungus Phanerochaete chrysosporium in free and immobilized cultures , Bioresouree Technology , Volume 84, Issue 2, September 2002, Pages 145-150
96. Knapp,J.S.,Newby, P.S. and Reece, L.P, Decolorization of dye s by wood-rooting basidiomycete fungi ,Enzyme and Microb. Technol., 1995,17:664～668
97. Pasti-Grigsby ,M,B., Paszcynski, A., Goszczynski,s., crawford ,D.L. Crawford ,R.L., Infulence of aromatic subsititution patters on azo dye degradability by Streptomyces spp.and Phanerochaete chrysosprium. Appl.Environ. Microbiol. 1992,58,3605-3613
98. Andrezj Paszczynski, Marlia B. Pasti-grigsby, Mineralization of sulfonated Azo dyes and sulfanilic acid by Phanerochaete chrysosprium and Streptomyces chromofuscus Applied and enviromental Microbiology , Nov,1992,p.3598～3604
99. Goszczynski.A. Paszczynski ,New pathway for Degradation of Suifonated Azo Dyes by Microbial peroxidases of Phanerochaete chrysosprium and Streptomyces Chromofuscus, Journal of Bacteriology, Mar. 1994,p. 1339～1347
100. Ming Tien and T.kent kirk, Lignin Peroxidase of Phanerochaete chrysosprium
101．郑婉玲，邹云珍，臧向莹 强化海藻酸钠凝胶制备固定化酶 工业微生物 1998年第25卷 第三期
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103. Stablility testing of ligninase and Mn-peroxidase from Phanerochaete chrysosprium Biotechnol Bioeag., 1989, 34:1251～1260
2．曹玲．全金英，李忠政 氧化氨解木质素结构的研究 南京林业大学学报 Vol．22，No 2，Jun 1998
3．管筱武，张甲耀，罗宇煊 木质素降解酶及其调控机理的研究进展 上海环境科学 1998．(11)：17
4．卢雪梅，李越中，高培基 黄孢原毛平革菌木素过氧化物酶类在天然木素降解中作用的研究菌物系统1998：17(2) 179～184
5. Aitken ,D. M., R. venkatadri, and R. L. Irvine , Oxidation of pHenolic pollutants by a lignin dearading enzyme from the white-rot fungus Phanerochaete chrysosporium Water Res, 1989,23:443-450
6. Satyendra K. Grag and Dinesh R. modi, Decolorization of Pulp-paper Effluent by white-rot Fungi Critical Reviews in Biotechnology, 1999,19(2):85-112
7．张朝晖，夏黎明，柯世省，岑沛霖 固定化黄孢原毛合成木素过氧化物酶 高校化学工程学报April 1999 Vol 2．No 13
8．卢雪酶．檀俊利，高培基 黄孢原毛平革菌对美洲黑杨的生物制浆的研究 广州造纸 2000 No．1
9．张朝晖，夏黎明，林建平．岑沛霖 木素过氧化物酶降解偶氮染料卡布龙红的动力学及过程模型化工学报April 2002 Vol 53，No．4
10．陈一飞，施成良，印染废水的成分分析及净化处理技术 四川丝绸 2002年第三期
11．李慧蓉 白腐真菌及其技术的潜在应用 工业微生物 1996．28(2)
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18. Adinarayana Reddy and Trevor M. D'Souza, Physiology and molecular biology of the lignin peroxidases of Phanerochaete chrysosporium, FEMS Microbiology Reviews Volume 13, Issues 2-3, Match 1994, Pages 137-152
19. Balachandra Dass and C. Adinarayana Reddy, Characterization of extracellular peroxidases produced by acetate-buffered cultures of the lignin-degrading basidiomycete Phanerochaete chrysosporium, FEMS Microbiology Letters Volume 69, Issue 3, I June 1990, Pages 221-224
20. Sovan Sarkar, Angel T. Martínez and María Jesús Martínez, Biochemical and molecular characterization of a manganese peroxidase isoenzyme from Pleurotus ostreatus, Bioehimiea et BiopHysica Acta (BBA)-Protein Structure and Molecular Enzymology Volume 1339, Issue I, 25 April 1997, Pages 23-30
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47 李越中，高培基，王祖农，黄孢原毛平革菌合成木素过氧化物酶的营养调控，微生物学报，1994，34(1)：29-36
48. T. Kent Kirk, Suki Croan and Ming TienKaren E. Murtagh and Roberta Lee Farrell, Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain, Enzyme and Microbial Technology, Volume 8, Issue 1, January 1986, Pages 27-32
49. Yasumi Akamatsu, and Mikio Shimada, Suppressive effect of-pHenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium, FEMS Microbiology Letters , Volume 131, Issue 2,1 September 1995, Pages 185-188
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51．柯世省，夏黎明，张朝晖，岑沛霖 氧浓度对固定化黄孢平革菌合成木素过氧化物酶的影响林产化学与工业 2000年9月，第20卷，第3期(Vol 20，No 3，Sept 2000)
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53. Nathan Rothschild ,Yitzhak Hadar ,and Carlod Dosoretz ,Ligninalytic System Formation by Phanerochaete chrysosporium in Air ,Aplied and Environmental ,May 1995,p, 1833-1838
54. Liebeskind, H. Hǒcker, C. Wandrey and A. G Jiger, Strategies for improved lignin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer FEMS Microbiology Letters , Volume 71,Issue 3, 15 September 1990, Pages 325-330
55. Marcel Asther, Cecile capdevila.and Georges Corrieu, Control of lignin Peroxidase Production Phanerochaete chrysosporium INA-12 by temperature shifting, Applied and Environmental Microbiology. Dec, 1988,p.3194-3196
56 Susan Linko ,Continuous production of lignin peroxidase by immobilized Phanerochaete chrysosporium in a pilot scale bioreactor, Journal of Biotechnology ,Volume 8, Issue 2, June 1988, Pages 163-170
57. Pascal Bonnarme, Michèle Asther and Marcel Asther, Influence of primary and secondary proteases produced by free or immobilized cells of the white-rot fungus Phanerochaete chrysosporium on lignin peroxidase activity, Journal of Biotechnology, Volume 30, Issue 3, September 1993, Pages 271-282
58. Kirk T.K.,Croan S.,Tien M.,Murtagh K.E.and Farrel R.L Production pf multile ligninase by Phanerochaete chrysosporium:Effect of selected growth conditions and use of a mutant strain. Enzyme Microb.Technol 1986,8,27-32
59. Willershausen H,.Jager A.and Graf H ,Ligninase production of Phanerochaete chrysosporium bioreactors, Biotechnil .6.. J. 1987 239-243
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61. Janskekar H.and Fiechter A. Cultivation of Phanerochaete chrysosporium and production of ligninase in submerged stirred tank reactors, Biotechnol 8, ,J 1988 97-112
62. Bonnarme P.and Jeffries T.WSelective production of extracellular peroxidase from Phanerochaete chrysosporium in an air-lift bioreactor, Ferm ,Bioenging 70, J, 1990 158-163
63. Linko s .and Hujanen M enzyme production by an immobilized fungus bioreactor, Ann.N.Y.Acad, Sci.613, 1990 764-770
64. Polvinen K.,Lehtonen P.,Leisola M.and Visuri KPilot-scale production and properties of lignin peroxidase ,ACS Symp ,ser ,460, 1991 223-235
65. Xu Fujian, Chen Hongzhang and Li Zuohu, Solid-state production of lignin peroxidase (LiP) and manganese peroxidase (MnP) by Phanerochaete chrysosporium using steam-exploded straw as substrate, Bioresource Technology, Volume 80, Issue 2, November 2001, Pages 149-151
66. Susana Rodriguez Couto, Marjaana Rtt, Alberto Dominguez and Angeles Sanromán, Strategies for improving ligninolytic enzyme activities in semi-solid-state bioreactors, Process Biochemistry, Volume 36, Issue 10, April 2001, Pages 995-999
67. Clayton D.Barclay, kaymond L.legge .and Grahame Ffarqujar, Modelling the Growth kinetics of PHanerochaete chrysosporium in submerged static culture Applied and Environmental microbiology, June 1993,p 1887-1892
68. John A. Buswell , YiJin Cai , Shu-ting Chang , Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes , FEMS Microbiology Letters Volume 128 , Issue 1,15 April 1995, Pages 81-87
69．李华钟，章燕芳，华兆哲，陈坚 黄孢原毛平革菌合成锰过氧化物酶工艺的研究 食品与与生物技术 Vol 21，No2， Jun 2002
70. Philip J.Kersten ,Glyoxal oxidase of Phanerochaete chrysosprium :It's charcterization and activation by lignin peroxidase ,Vol.87,pp.2936-2940,April 1990
71. Nathan Rothschild ,Yitzhak Hadar ,and Carlos Dosoretz, Ligninlytic system Formation by PHanerochaete chrysosprium in air ,Appplied and environmental Microbiology ,May 1995,pp. 1833-1838
72. Philip J.Kersten and T. Kent Kirk, Involvment of new Enzyme ,Glyoxal oxidase in extracellular H_2O_2 production by Phanerochaete chrysosprium ,Applied of Bacteriology, May 1987,p,2195-2201
73. Jeffrey K.Gleen, Michael H.Gold .Purification and characterization of an extracellar Mn(Ⅱ)-dependent peroxidase from the lignin-dagrading basidiomycete, Phanerochaete chrysosporium, Biochem biophys Arch. 1995,242(2):239-241
74. A. Paszczynski, A.B. Huynh and R. Crawford, Comparison of liguinase and peroxidase M2 from whiterot fungus Phanerochaete chrysosporium. Arch. Biochem. BiopHys. 26 1986, p. 477.
75. Matthias S. Fawer, Jrg Stierli, StepHen Cliffe and Attain Fiechter, The characterisation of immobilised lignin peroxidase by flow injection analysis , Biochimica et BiopHysica Acta (BBA)-Protein Structure and Molecular Enzymology Volume 1076, Issue 1,8 January 1991, Pages 15-22
76. Asther, M; Meunier, J C, Immobilization as a tool for the stabilization of lignin peroxidase produced by Phanerochaete chrysosporium INA-12 , Applied Biochemistry And Biotechnology Volume 38, Issue 12 January-February 1993, Pages 57-67
77. Peralta-zamora p.,Gomes de Moraes S.;Esposita E.;Antunes R,; Reyes J.;Duran N, Decolorization of pulp mill effluents with immobilized lignin and manganese peroxides from Phanerochaete chrysosporium ,Environmental Technology Volumn 19,Issue 5,1998,p. 521-528
78. Marcia Dezotti, Lucia H. Innocentini-Mei and Nelson Durán, Silica immobilized enzyme catalyzed removal of chloroliguins from eucalyptus kraft effluent, Journal of Biotechnology, Volume 43, Issue 3, 15 December 1995, Pages 161-167
79. Timothy L. Presnell, Harold E. Swaisgood, Thomas W. Joyce and Hou-min Chang,, Investigation into the kinetic properties of immobilized lignin peroxidases, Journal of Biotechnology, Volume 35, Issue 1, 15 June 1994, Pages 77-85
80. Grabski, A C; Rasmussen, J K; Coleman, P L; Burgess, R R, Immobilization of manganese peroxidase from Lentinula edodes on alkylaminated EmpHaze AB 1 polymer for generation of Mn~(3+) as an oxidizing agent; Applied Biochemistry And Biotechnology , Volume 60, Issue 1, July 1996, Pages 1-17 , ISSN: 0273-2289
81. W. Edwards, W. D. Leukes and J. J. Bezuidenhout, Ultrafiltration of petrochemical industrial wastewater using immobilised manganese peroxidase and laccase: application in the defouling of polysulp Hone membranes, Desalination, Volume 149, Issues 1-3,10 September 2002, Pages 275-278
82. Mielgo, C. Palma, J. M. Guisan, R. Fernandez-Lafuente, M. T. Moreira G. Feijoo and J. M. Lema, Covalent immobilisation of manganese peroxidases (MnP) from Phanerochaete chrysoaporium and Bjerkandera sp. BOS55, Enzyme and Microbial Technology , Volume 32, Issue 7, 12 June 2003, Pages 769-775
83. Grabski A.C.; Grimek H.J.; Burgess R.R, Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of Mn(Ⅲ)-chelate as a chemical oxidant of chloropHenols , Biotechnology and Bioengineering , Volume 60, Issue 2, 20 October 1998, Pages 204-215
84. Maynard ,C.W.,Jr..Dye application ,manufacture of dye intermediates and dyes In, J.A.Kent(ad.). Riegel's handbook of industrial chemistry. Van Nostrand Reinhold ,New York. 1983,p.809-861.
85. Vaidya ,A.A.,and K.V.Datye ..Environmental pollution during chemical processing of synthetic fibers .Colourage 1982 14:3-10
86. Zollinger .H. 1987.Color chemistry-syntheses ,properties and applications of orangnic dyes and pigments, VCH publishers ,New york. ,p.92-102
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