摘要
木质素是地球上廉价、丰富的可再生资源,其生物降解是自然界碳素循环关键的一步,但由于它是一种高度复杂的、不定型的芳香环聚合物,很难被降解,因此给全球造成了严重的环境污染。由于传统的物理和化学方法并不能从根本上解决这一问题,生物技术便成为环境界和生物界愈发关注的新生力量。如何利用微生物产生的木质素降解酶系来分解和转化木质素成了新的研究课题,并引发了一轮又一轮的研究热潮。目前人们对木质素酶的研究多集中于白腐菌,并以黄孢原毛平革菌(P.chrysosporium)为模式菌。黄孢原毛平革菌(P.chrysosporium)合成的木质素降解酶系中起主要作用的是胞外过氧化物酶系,包括木质素过氧化物酶(Lignin Peroxidase,LiP)和锰过氧化物酶(ManganesePeroxidase,MnP),其中MnP在工业和农业中具有广泛的应用前景。但由于从自然界筛选出的黄孢原毛平革菌其产MnP较低,无法直接应用于生产实践,故对黄孢原毛平革菌进行改良,使其获得更高的酶活是广泛应用的前提。同时,迄今为止,大多关于黄孢原毛平革菌发酵的研究多为液体发酵,成本较高且对环境条件有一定要求,无法有效应用在大规模生产中。因此,对黄孢原毛平革菌的固体发酵条件进行研究更具有重要的现实意义。
本研究从长有褐色或者乳白色锈斑的树皮上分离出4株可以降解染料的菌株,结合乳酸钠法测酶活,筛选出1株产酶能力较高的菌株W3,其MnP活力为8.59Wg,经形态鉴定为黄孢原毛平革菌。由正交试验确定出该菌原生质体制备和再生的最佳条件:酶浓度为1.5mg/mL,酶解时间为2h,酶解温度为30℃。其中所采用的酶液是将蜗牛酶(S)(5mg/mL)、纤维素酶(C)(4mg/mL)用pH5.0的0.4 mol/L NH_4Cl-0.3%β-巯基乙醇0.2mol/L磷酸缓冲液溶解,按S:C=5:4配比而成。随后用波长为632.8nm,输出功率为12mw,光斑直径为1.5mm的He-Ne激光在照射距离30cm处对制备出的原生质体进行激光诱变。通过对突变株的酯酶同功酶谱、产酶能力及遗传稳定性进行分析,选育出产酶能力较强、遗传稳定性良好的7#菌株作为工业发酵的新出发菌株,其MnP活力为14.28U/g。通过单因素试验确定固体发酵培养基优化方案为:添加浓度为0.015mmol/g的Mn~(2+)和浓度为0.1%的Tween-20;通过正交试验确定菌体的固体发酵条件为:菌种驯化时间5d,接种量0.5%,发酵温度37℃,发酵基质含水率85%。在该条件下对黄孢原毛平革菌进行固体发酵,其MnP活力达到20.71U/g。实验证明,亲本菌株经过激光诱变和固体发酵条件优化,其MnP活力提高了141.1%。
Lignin is a kind of inexpensive and affluent renewable resource on the earth.The biodegradation of lignin is the pivotal step in the circulation of carbon in nature.However, due to its extremely complicated and indefinite structure,such polymer is hard to be degraded. Consequently,serious environment pollution has been resulted in around the world. Traditional ways,both physical and chemical,can not resolve this problem effectively and thoroughly.In such conditions,biotechnology is an optimum choice for scientists.How to decompose and convert lignin into unharmful matter with catabolic enzymes produced by microorganism has become a new topic in scientific field,which strongly attracted scientists' eyes.In recent years,most studies related to lignin's catabolic enzymes have been focused on white-rot fungi and Phanerochaete chrysosporium has been regarded as mode fungus.The catabolic enzymes produced by Phanerochaete chrysosporium include lignin peroxidase(Lip) and manganese peroxidase(MnP) and MnP has a broad prospect in application,both in industry and agriculture.Yet,the MnP produced by natural strains exhibit low activity,which has been restrict its practical use in production.In such a case,finding a new way to improve strains' MnP activity is in urgent demand and it's the base of widely application.What's more, so far,most studies on fermentation of Phanerochaete chrysosporium have been concentrated on fluid fermentation,which is highly cost and has strict requirements of circumstances.Thus, making studies on Phanerochaete chrysosporium's solid fermentation is of practical importance.
Four strains which could effectively degrade dye were separated from bark with brown or ivory white locus ferrugineus.One strain with the strongest MnP activity was selected by measuring enzyme activity with natrium lacticum,which is 8.59U/g.According to morphous characteristics,it was regarded as Phanerochaete chrysosporium.Orthogonal experiment was carried out to determine the optimum conditions of Phanerochaete chrysosporium protoplast production and regeneration.The results showed that the strains should be treated with 1.5mg/mL enzyme,at 30℃,for 2 hours.The enzyme was a mixture of glusulase and cellulase. They were dissolved with 0.4 mol/L amchlor-0.3%β-ME and 0.2mol/L phosphate buffer, according to S:C=5:4.Then,He-Ne laser(λ=632.8nm,output power was 12mw,the diameter of light spot was 1.5nm) irradiated protoplasts for different time in a distance of 30cm.7# strain with 14.28U/g MnP activity and better genetic stability was selected after analysis of esterase isozymogram,MnP activity and genetic stability.The solid fermentation medium should be added with 0.015mmol/g Mn~(2+) and 0.1%Tween-20 and the fermentation conditions were as follows:domestication time of strains was 5d,inoculation rate was 0.5%,temperature was 37℃and water ratio was 85%.Under such circumstances,the MnP activity of Phanerochaete chrysosporium hit 20.71U/g.
The study showed that after laser mutagenesis and optimization of solid fermentation conditions,strains' MnP activity could be raised by 141.1%.
引文
[1]岑沛霖,蔡谨.工业微生物学[M].北京:化学工业出版社,2000
[2]Orth A B,Royse D J,Tien M.Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi.Appl Environ Microbiol,1993,59:4017-4023
[3]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,2000,34(4):349-354
[4]张朝晖,夏黎明,柯世省等.固定化培养黄抱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
[5]Martin Hofrichter.Review:lignin conversion by manganese peroxidase(MnP)[J].Enzyme.Microb.Tech.,2002,30(4):454-466
[6]邬义明.植物纤维化学(第二版)[M].中国轻工业出版社,1995:1-6
[7]陶用珍,管映亭.木质素的化学结构及其应用[J].纤维素科学与技术,2003,11(1):42-55
[8]陈立祥,章怀云.木质素生物降解及其应用研究进展[J].中南林学院学报,2003,23(1):79-85
[9]李慧蓉.白腐真菌在碳素循环中的地位和作用[J].微生物学通报,1996,23(2):105-109
[10]Buswell J A,Odier E.Lignin biodegradation[J].CRC Crit Rev Biotechnol,1987,17(11):46-49
[11]Kirk T.K.,Farrell R.L..Enzymatic "combustion":the microbial degradation of lignin [J].Ann Rev Microbiol,1987,41:465-505
[12]黄峰,陈嘉翔,余家鸾等.制浆造纸工业中的微生物及其酶[J].中国造纸学报,1997.12:109-116
[13]Thurston C.F..The structure and function of fungal laccase[J].Microbiology,1994,140:19-26
[14]李慧蓉.白腐真菌的研究进展[J].环境科学进展,1996,4(6):69-77
[15]Buswell JA,OdierE.Lignin biodegration[J].CRC CritRev Biotechnol,1987,(6):1-60
[16]Tomati U,Galli E,Pasetti L,et al.Bioremediation of olive-mill wastewaters by composting[J].Waste Manag Res,1995,13:509-518
[17]Galli E,Pasetti L,FiorelliF,et al.Olive-millwastewater composting:microbiologicalaspects[J].Waste Management& Research,1997,15:323-330
[18]Tuomela M,Vikman M,Hatakka A,et al.Biodegra-dation of lignin in a compost environment:a review.Bioresource Technology,2000,72:169-183
[19]席北斗,刘鸿亮,白庆中等.堆肥中纤维素和木质素的生物降解研究现状[J].环境污染治理技术与设备,2002,3(3):19-23
[20]Lopez M J,Elorrieta M A,Vargas-Garcia M C,et al.The effect of aeration on the biotransformation of lignocellulosic wastes by white-rot fungi[J].Bioresource Technology,2002,81:123-129
[21]Tuomela M,Oivanenl P,Hatakka A.Degradation of synthetic 14C-lignin by various white-rot fungi in soil[J].Soil Biology & Biochemistry,2002,34:1613-1620
[22]黄丹莲,曾光明等.白腐菌应用于堆肥处理含木质素废物的研究[J].环境污染治理技术与设备,2005,6(2):29-32
[23]李慧蓉.白腐真菌生物学和生物技术[M].北京:化学工业出版社,2005:14
[24]黄峰,方靖.纤维二糖脱氢酶对锰过氧化物酶降解木素的促进作用[J].科学通报,2001,18(46):523-528
[25]Sundararnoorthy 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
[26]Michel JRFC,Dass SB,Grulke EA,et al.Role of MnP and LiP of Phanetochaete chrysosporium[J].Appl,Environ,Mierobiol,1991,57:2368-2375
[27]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
[28]池玉杰,伊洪伟.木材白腐菌分解木质素的酶系统-锰过氧化物酶、漆酶和木质素过氧化物酶催化分解木质素的机制[J].菌物学报,2007,16(1):153-160
[29]Wariishi H.Akileswaran L,Gold M H.Manganese peroxidase from basidiomycete Phanerochaete chrysosporium spectral characterization of the oxidized states and the catalytic cycle.Biochemistry,1988,27:5365-5370
[30]Andersson L.A.,Renganathan V.,Chiu A.A.,et al.Spectral Characterization of Diarylpropane oxygenase,a Novel peroxidase-dependent,lignin-degrading heme enzyme[J]. Biol.Chem.1985,266(10):6080-6087
[31]Gletin J.K.,M.,Gold M.H.Purification and characterization of an extrcellul ar Mn(Ⅱ)- depedent peroxidase from the lignin-degrading Basidiomycete Phanerochaete chrysosporium.Arch.Biochem BioPhys.1985,242:329
[32]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一血红密孔菌K-2352[J].微生物学通报,1994,21(3):152-156
[33]Kuwahara M,et al Purification and characterization of two lignin peroxidase isozymes produced Bjerkandera sp.strain Bos55[J].FEBSLett,1984,168-247
[34]Gold M.H..MnP from P.chrysosporium:Biochemical and Genetic Charaterization[J].Intemational Conference on Bio-technology in Pulp and Paper Industry,447-455
[35]谢慧芳,李忠正.与锰过氧化物酶相关的硫酸盐浆的生物漂白[J].生物漂白,2003,22(6):46-49
[36]PAICEM G,REID I D,BOURBONNAISR,et al.Manganese peroxidase produced by Trametes versicolor during pulp bleaching,demethylates and delignifies kraft pulp[J].Appl Environ Microbio,J,1993,59(1):260-265
[37]MOREIRAM T,FEIJOO G,MESTER T,et al.Biobleaching of oxygen delignified pulp by several white rot fungal strains[J].Journal of Biotechnology,1997,53(2/3):237-251
[38]MOREIRAM T,FEIJOO G;SIERRA R,et al.Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi[J].Bioresource Technology,1999,70(3):255-260
[39]TIMOFEEVSKI S L,NIE G,READING N S,et al.Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase[J].Arch Biochem Biophys,2000,373(1):147-153
[40]SASAKI T,KAJINO T,LI B,et al New pulp biobleaching system involving manganese peroxidase immobilized in a silica support with controlled pore sizes[J].Appl Environ Microbiol,2001,67(5):2208-2212
[41]Kondo R,Harazona K,Sakai K.Bleaching of hardwood kraft pulp with manganese peroxide secreted from Phanerochaete sordida YK-642[J].Appl Environ.Mierobiol,1994,60(12):4359
[42]Evaggeli Billa,Bernard Kurek,Evkoxia Koutsoula,et al.Effeet of Manganese Peroxidase on the Cell Wall Components of Wheat Straw and Sorghum Organosolv Pulps[J].Cellulose Chem.Technol,2000,34:131
[43]Hirai H,Kondo R,Sakai K.Screening of lignin-degrading fungi and ligninolytic enzyme activites during biological bleaching of kraft pulp[J].Mokuzai Gakkaishi,1994,40:980
[44]DEGUCHI T,KITAOKA Y,KAKEZAWA M,et al Purification and characterization of a nylon-degrading enzyme[J].Appl Environ Microbio,1998,64(4):1366-1371
[45]MANJI S,ISHIHARA A.Screening of tetrarchlorodibenzo-p-dioxin-degrading fungi capable of producing extracellular peroxidases under various conditions[J].Applied Microbiology and Biotechnology,2004,63(4):438-445
[46]KOICHI H,YOSHIO W,TAKEMA F,et al.Trapping of 2,7-dichlorodibenzo-p-dioxin in aqueous solution by enzymatic reaction of fungal manganese peroxidase in the presence of polyunsaturated fatty acids[J].Current Microbiology,2003,47(3):250-254
[47]YOSHIDA S,CHATANI A,HONDA Y,et al.Reaction of manganese-dependent peroxidase from Bjerkandera adusta in aqueous organic media[J.].Journal of Molecular Catalysis B:Enzymatic,2000,9(4/5/6):173-182
[48]JUNJIM,UCHIMURA Y,ICHINOSE H,et al.Activation of manganese peroxidase in an organic medium using a mediator[J].Hiroyuki Biochemical Engineering Journal,2004,(19):43-46
[49]张景来,常冠钦,陆爱军.白腐工程菌处理TNT炸药废水的研究[J].中国矿业大学学报,2001,30(2):161-164
[50]谢慧芳,方林,赵林果.白腐菌P.sordida YK-462及其锰过氧化物酶对2-氯酚的转化[J].环境污染与防治,2008,30(5):51-54
[51]王业耀,袁彦肖,田仁生.白腐真菌降解焦化废水的试验研究[J].工业水处理,2004,24(2):26-28
[52]刘波,张晓昱,王宏勋.产酸白腐菌处理碱性造纸黑液的机制探析[J].环境科学与技术,2005,28(2):76-78
[53]吴涓,肖亚中,王怡平.挂膜生长的白腐真菌处理草浆造纸黑液废水[J].应用与环境生物学报,2004,10(3):370-374
[54]Pointing S.B.,Bucher V.V.C.,Vrijmoed L.L.P.Dye decolorization by sub-tropical basidiomycetous fungi and the effect of metals on dye degradation[J].World Journal of Microbiology,2000,16(2):199-205
[55]Masanori Fujita,Akira Era,Michihilo lke,et al.Decolorization of Heat-Treatment Liquor of Waste Sludge by a Bioreactor Using Polyurethane Foam-Immobilized White Rot Fungus Equipped with an Ultramembrane Filtration Unit[J].Journal of Bioscience and Bioengineering,2000,90(4):387-394
[56]曾斌,宁大亮,王慧.白腐真菌降解五氯酚的初步研究[J].环境化学,2008,27(2):181-185
[57]Vishal Shah,Frantisek Nerud.Lignin degrading system of white-rot fungi and its exploitation for dye decolorization[J].Canadian Journal of Microbioligy,2002,48:857-570
[58]MARTINSM A M,LIMA N,SILVESTRE A J D,et al.Comparative studies of fungal degradation of single ormixed bioaccessible reactive azo dyes[J].Chemosphere,2003,52(6):967-973
[59]CENEK N,SVOBODOVA K,ERBANOVA P,et al.Ligninolytic fungi in bioremediation:extracellular enzyme production and degradation rate[J].Soil Biology&Biochemistry,2004,36(10):1545-1551
[60]BIAOM,MAYFIELD M B,GOLD M H.Homologous expression of Phanerochaete chrysosporium manganese peroxidase,using bialaphos resistance as a dominant selectable marker[J].Current Genetics,2003(43):407-414
[61]章燕芳,李华钟,华兆哲等.孢原毛平革菌合成的木质素过氧化物酶、锰过氧化物酶及其菌球在染料脱色过程中的作用[J].过程工程学报,2003,2(3):246-251
[62]程晓宾,荚荣,李平生等.锰过氧化物酶的耦合固定化及其性质研究[J].生物工程学报,2007,23(1):90-95
[63]Cohen M S,GabrieIe P D.Degradation of Coal by Fungi Polyporus Versicolor and Poria Monticolor[J].Appl Environ Microbiol,1982,44:23-27
[64]WILLMANN G,FAKOUSSA RM.Extracellular oxidative enzyme of coal-attacking fungi[J].Fuel Process Technol,1997,52(1/2/3):27-41
[65]Hofrichter M,Scheibner K,Schneega I.Mineralization of Synthesis Humic Substances by Manganese Peroxidase from the White Rot Fungus Nematolont Frowardii[J].Appl Microbiol Biotechnol,1998,49:584-588
[66]闫论.腐植酸及其广泛应用[J].新疆农业科学,2004,41(专刊):130-132
[67]陈谊,杭怡琼.白腐真菌降解稻草转化饲料的研究[J].上海交通大学学报,2001,19(2):151-153
[68]白彩霞.玉米秸秆饲料加工开发现状[J].黑龙江畜牧兽医职业学院学报,2006,1:31-32
[69]刘玲,叶博,刘长江.白腐真菌及其木质素酶的研究进展[J].生物技术,2006,27(8):25-28
[70]胡建宏,丁海荣,王立强.利用白腐真菌开发农作物秸秆饲料的研究[J].饲料工业,2005,26(11):57-59
[71]单洪涛,吴跃明,刘建.新秸秆饲料化技术的研究进展[J].中国饲料,2007(4):34-36
[72]李慧蓉.白腐真菌在碳素循环中的地位和作用.微生物学通报,1996,23(2):105-109
[73]刘晓牧,李福昌,王中华等.白腐真菌与秸秆饲料的有效利用[J].饲料研究,2000,1:42-43
[74]赵华,齐刚,代彦等.白腐真菌对稻草秸秆生物降解的研究[J].饲料工业,2003,24(11):37-40
[75]赵红霞,杨建军,詹勇.白腐真菌在秸秆作物资源开发中的研究[J].饲料工业,2002,23(11):40-42
[76]IWAHARA K,HONDA Y,WATANABE T,et al.Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta aqueous organic solvents[J].Appl Microbiol Biotechnol,2000,54(1):104-111
[77]陈丽萍,宗兆锋,李春玲等.用原生质体融合技术提高植物内生放线菌*的抗菌活性[J].西北农林科技大学学报(自然科学版),2008,36(10):158-162
[78]杨谦,孙冬梅,张晶.黄绿木霉原生质体诱变育种研究[J].微生物学通报,2005,32(4):62-67
[79]杜海英,于宏伟,韩军等.原生质体诱变选育乳糖酶高产菌株[J].微生物学通报,2006,33(6):48-51
[80]李秀珍,薄泉,杨平平等.利用原生质体诱变筛选植酸酶高产菌株[J].饲料工业,2007,28(8):27-29
[81]胡杰,潘力,罗立新等.米曲霉孢子原生质体复合诱变及高活力蛋白酶菌株选育[J].食品工业科技,2007,28(5):116-119
[82]彭益强,吕凤萍,贺淹才等.激光诱变筛选高产植酸酶黑曲霉菌株的研究[J].激光生物学报,2002,11(6):434-437
[83]陈志高,沈建新,胡叶碧.紫外线和氯化锂对核糖苷链霉菌原生质体诱变作用的探讨[J].中国医药工业杂志,2001,32(4):150-152
[84]朱林东,金志华.普那霉素产生菌的原生质体诱变育种[J].中国抗生素杂志,2006,31(10):591-594
[85]李萍,宛晓春,陶文沂等.复合诱变黑曲霉选育β-葡萄糖甘酶高产菌株[J].菌物系统,2000,19(1):117-121
[86]郭鲁宏,杨顺楷.黑曲霉单宁酶高活性菌株的诱变选育[J].微生物学通报,2000,27(2):105-108
[87]吴剑波主编.微生物制药[M].北京:化学工业出版社,2002:46-55
[88]章冬春主编.工业微生物诱变育种[M].北京:科学出版社,1984:100-163
[89]向洋.激光生物学[M].长沙:湖南科学技术出版社,1995:31,224
[90]Franz Hoffmann.Laser microbeams for the manipulation of plant cells and subcellular strctures[J].Plant Science,1996,113:1-11
[91]William F.Grant,Elizabeth T.Owens.Lycopersicon assays of chemical/ radiation genotoxicity for the study of environmental mutagens[J].Mutation Research,2002,511:207-237
[92]Csaba Koncz.Plant biotechnology from genome projects to molecular breeding editorial overvies[J].Current Opinion in Biotechnology,2003,14:133-135
[93]Passarella S,Casamassima E,Molinari S,et al.Increase of Proton eleetro-chernical potential and ATP synthesis in rat liver mitoehondria irradiated in vitro by helium-neon laser[J].FEBS Let,1984,175:95-99
[94]Karu TI.Photobiological fundamentals of low-power laser therapy[J].IEEE Journal of Quantum Electronics,1987,QE-23(10):1703-1717
[95]Gordon SA,Surrey K.Red and far-red action on oxidative phosphorylation[J].Radiat Res,1960,1(12):325-339
[96]Van Breugel H,Bar PRD.Power density and exposure time of He-Ne laser irradiation are more important total energy dose in photo-biomodulation of human fibroblasts in vitro[J].Laser in Surgery and Medicine,1992,12:528-537
[97]Quero JMO,Villamandos RG,Millan MM.Sister chromatid exchange induction in sheep peripheral blood mononuclear cells by helio-neon laser radiation[J].Mutation Research,1997,377:69-75
[98]Polo L,Presti F,Schindl A.Schindl L.Role of ground and excited singlet state oxygen in the red light-induced stimulate on of escherichia coli cell growth[J].Biochemical and Biophysical Rsearch Comunications,1999,257:753-758
[99]Kohli R,Bose B,Gupta PK.Induction of phr gene expression in E.coli strain KY706/Ppl-1 by He-Ne laser irradiation[J].J Photochem Photobiol B:Biol,2001,60:136-142
[100]张俊国,张三元,张学臣等.水稻激光育种的研究Ⅰ.种子及减数分裂期幼穗激光处理诱发变异分析[J].吉林农业科学,2005,30(2):17-20
[101]陈震古.激光诱导杨树单性生殖的研究[J].激光生物学报,1998,7(1):11-13
[102]陈震古,刘小刚,许成林等.激光诱导榆树单倍体植株及无融合生殖的研究[J].量子电子学,1996,13(4):391-396
[103]任作鍈,文明发.桑树激光育种方法的研究[J].激光生物学报,1993,2(3):328-331
[104]陈恒雷,徐辉,吕长武等.激光诱变育种的研究概况[J].激光生物学报,2006,15(4):436-440
[105]谭石慈,朱延彬,马俊富等.激光辐照诱变鱼类育种[J].中国激光,1993,20(9):718-720
[106]何家林,谢瑞生,章之蓉.激光辐照鱼类胚胎导致畸变[J].激光生物学报,1993,2(1):199-203
[107]邵耀椿,杨晓锋,丁海东等.激光辐照DNA导入番茄细胞色素氧化酶的研究[J].应用激光,2003,23(4):223-225
[108]刘永军,南照东,孙海涛等.氦氖激光辐照对黄瓜萌发过程影响的研究[J].应用激光,1995,15(2):83-85
[109]张建东,陈怡平,王勋陵.CO_2激光处理对大豆种子萌发及生理的影响[J].西北植物学报,2004,24(2):221-225
[110]Gerd Weber,Karl Otto Greulich.Manipulation of Cells,Organelles,and Genomes by Laser Microbeam and Optical Trap[J].International Review of Cytology,1992,133:1-41
[111]朱宏莉,郭爱莲,宋纪蓉等.氦-氖激光诱变细菌细胞及原生质体选育果胶酶高产菌株[J].光子学报,2005,34(11):1693-1696
[112]Wenyu Lu,Jianping Wen,et al.Effect of He-Ne laser irradiation on hydrogen production by Enterobacter aerogenes[J].International Journal of Hydrogen Energy,2008,33:34-42
[113]王雪松,王莹,于晓迪等.茁芽短梗霉原生质体激光诱变及高产菌株筛选[J].生物技术,2005,15(1):29-31
[114]孙毅.激光照射与氮离子注入对绿色木霉纤维素酶的生物效应研究[D].新疆:新疆大学,2006
[115]韩建荣,董仙芳.激光诱变青霉PT95原生质体选育类胡萝卜素高产菌株[J].微生物学通报,2002,29(2):31-34
[116]韩建荣,郝建国,邢茂生.黑木耳激光诱变及营养缺陷型突变体筛选.山西大学学报(自然科学版),2002,25(1):71-73
[117]罗英等.激光复合诱变黑曲霉原生质体选育热稳定菊粉酶高产菌[J].四川师范大学学报,1998,21(5):581-586
[118]戴德慧,郭爱莲,蒋家新.He-Ne激光对红曲霉M_(9X)的原生质体诱变育种[J].食品科学,2005,26(11):75-77
[119]张建军,甘秀英,侯娟等.He-Ne激光复合诱变选育柠檬酸高产菌[J].石河子大学学报(自然科学版),2007,25(2):225-228
[120]陈五岭,罗海峰,张小里.He-Ne激光诱变的香菇变异株遗传稳定性分析[J].光子学报,2000,29(4):380-384
[121]李耀维,冯文新,张素梅.He-Ne激光对金针菇SOD高产株的诱变效应[J].激光生物学报,2002,11(4):283-286
[122]胡能书等.同工酶技术及其应用[M].长沙:湖南科学技术出版社,1985
[123]Alma,S&Sandal..PC.,1969:CorPSei,9(2):157-159
[124]HallR,ZentmyerGA.,1967:phystopathology,57:813
[125]江涓.八个香菇品种脂酶同工酶的研究[J].中国食用菌,1999,18(3):15-17
[126]杨宗渠,王柏楠.食用菌诱变育种研究进展[J].中国食用菌,1998,17(2):6-9
[127]傅四清,魏蓉城.同工酶技术在真菌分类中的应用[J].中国食用菌,1998.17(1): 8-9
[128]李中振.灵芝超氧化物歧化酶同工酶的研究[J].中国食用菌,1997,4:30-33
[129]邓功成,刘朝贵.鸡腿蘑菌丝体的同工酶研究[J].中国食用菌,2003,22(6):42-44
[130]赵桂东.江苏大麦纹枯病酷酶同工酶的测定[J].江苏农业科学,1993,74:163-170
[131]管萍,景建洲等.同工酶技术在金霉素链霉素鉴别中的应用[J].西北大学学报(自然科学版),1996,26(6):533-536
[132]张瑾,华秀英,王海英.13个香菇品种间酯酶同功酶的研究[J].沈阳师范学院学报(自然科学版),2000,18(4):35-39
[133]霍丽云,陈宝金,刘青.利用聚丙烯酰胺凝胶平板电泳对杂交细胞酯酶同功酶的分析研究[J].山东教育学院学报,2001,1:72-73
[134]曹丽华,叶华智.禾谷镰刀菌的酯酶和酸性磷酸酯酶同功酶电泳分析[J].河南农业大学学报,2001,35(3):262-265
[135]胡加付,缪凯,柴守权等.用酯酶同功酶分析白僵菌的林间扩散能力[J].中国生物防治,2005,21(4):238-241
[136]陈文强,邓百万,丁锐等.猴头不同菌株酯酶同功酶的研究,安徽农业科学,2006,34(5):916-917
[137]傅安涛,宋爱荣,丁伟等.15个灰树花菌株的酯酶同功酶分析.中国食用菌,2007,26(1):40-42
[138]胡能书等.激光辐照水稻的生理生化基础研究[J].应用激光,1981,1(3):22-24
[139]胡能书等.经He-Ne激光照射的水稻芽中同工酶的电泳分析[J].应用激光,1981,8(3),39-42
[140]胡能书,向阳,吴秀山.激光辐照水稻的生理生化基础研究-(Ⅱ)He-Ne激光辐照对水稻不同发育期同工酶的影响[J].应用激光联刊,1982,2(5):37-40
[141]廖映粉等.经He-Ne激光照射的甜橙酷酶同工酶的电泳分析[J].应用激光联刊,1985,5(2),11-15
[142]向洋.激光生物学[M],长沙:湖南科技出版社,1995,2(4):373
[143]陈五岭等.He-Ne激光在香菇速生高产菌株选育中的应用,Ⅱ酯酶同工酶分析及中试栽培[J].光子学报,1997,26(11):977
[144]张卉,佟俊生,刘长江.~(60)Co-γ射线辐照菌丝选育多糖高产菌株[J].中国食用菌,2003,22(5):13-14
[145]Rodriguez C S,Rivela I,Munoz M R,et al.Ligninolytic enzyme production and the ability of decolourisation of Poly R2478 in packed-bed bioreactors by Phanerochaete Chrysosporium[J].Bioprocess Engineering,2000,23(3):287-293
[146]李华钟,章燕芳,华兆哲等.黄孢原毛平革菌选择性合成木质素过氧化物酶和锰过氧化物酶[J].过程工程学报,2002,2(2):137-141
[147]Jimenez-Tobon G,Kuratkowski W,Rozbicka B,et al.In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete Chrysosporium[J].Microbiology,2003,149(11):3121-3127
[148]P K Gill,D S Arora.Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi[J].J Ind Microbiol Biotechnol,2003,30:28-33
[149]Memory Tekere,Remigio Zvauya,John S Read.Ligninolytic enzyme production in selected sub-tropical white rot fungi under different culture conditions[J].Journal of Basic Microbiology,2001,41(2):115-129
[150]Yoshida S,Yonehara S,Minami S,et al.Production and characterization of ligninolytic enzymes of Bjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter[J].Mycoscience,2002(37):417-425
[151]Rao S K,Tien M.Oxidation of guaiacol by lignin peroxidase[J].Journal of Biology Chemical,1995,270(22):254-258
[152]林鹿,高杨,周学飞等.白腐菌对制浆黑液中硫酸盐木素的降解[J],环境科学,1997,18(1):23-26
[153]王宜磊.碳源和氮源对彩绒革盖菌Coriolus versicolor木质纤维素酶和木质素酶分泌的影响[J].微生物学杂志,2000,20(1):19-31
[154]王佳玲,余惠生,黄秀瑜等.碳源和Mn(Ⅱ)对白腐菌Panus conchatus产木素降解酶的影响[J].纤维素科学与技术,1998,6(3):27-31
[155]王佳玲,付时雨,余惠生等.氮源浓度和芳香化合物对白腐菌Panus conchatus 产木素降解酶的影响[J].纤维素科学与技术,1997,5(4):25-32
[156]Pickard M.A.,Vandertol H.,Romand R,et al.High production of ligninolytic enzymes from white rot fungi in cereal bran liquid medium[J].Canadian Journal of Microbiology,1999(45):627-631
[157]Dosoretz C G;Chen A H C,Grethlein H E.Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium[J].Appl.Microbial,Biotechnol.,1990(34):131-137
[158]Bonnarme P,Jeffries T W.Mn(Ⅱ)regulation of lignin peroxidases and manganese dependent peroxidases from lignindegrading white rot fungi[J].Appl.Environment Microbiology,1990,56(1):210-217
[159]Bar-lev S S,Kirk T K.Effects of molecular Oxygen on lignin degradation by P.chrysosportum[J].Biochemical and Biophysical Research Communications,1981(99):373-378
[160]Gold M H,Cheng T M.Induction of colonial growth and replica plating of the white rot basidiomycete Phanerochaete chrysosporium[J].Appl Environ Microbiol,1978,35(6):1223-1225
[161]管萍.同功酶技术在微生物和植物杂交种鉴定中的应用[M],1994,15-16
[162]Thomas W Jeffries et al.Nutritional Regulation of Lignin Degradation by Phanerochaete chrysosporium[J].Applied and Environmental Microbiology,1981,42(2):290-296
[163]邢来君等.普通真菌学[M].北京:高等教育出版社,1999
[164]杭怡琼,薛惠琴,郁怀丹.白腐真菌对稻草秸秆的降解及其有关酶活性的变化[J].菌物系统,2001,403-407
[165]李多伟等.生物技术实验与仪器操作指南[M].西安:西北大学出版社,1996