黄蓝状菌几丁质酶E2纯化、性质及抗菌活性研究.docVIP

黄蓝状菌几丁质酶E2纯化、性质及抗菌活性研究 山东林业科技2011年第4期总195期SHANDONGFORESTRYSCIENCEANDTECHNOI()GY2011.No.4 文章编号:1002—2724(2Ol1)04一O01304 黄蓝状菌几丁质酶E纯化,性质及抗菌活性研究 陈述,王志伟!,解文科,唐帅,李慧 (1.临沂市林业局,临沂276000;2.临沐县林业局) 摘要:黄蓝状茵在SMCS液体培养基中置于恒温摇床(200r/min,27.5℃)上培养13天,诱导产生大量的几丁质酶.培养 滤液经硫酸铵分级沉淀,DEAE--SepharoseFastFlow阴离子交换层析,Pheny|一SepharoseFastFlow疏水层析,SephaerylS 一 100分子筛层析得到了凝胶电泳(SDS—PAGE)谱带单一的几丁质酶E.,其分子量为32KD其最适反应温度为50~C,最适 pH值是5.Mn对其有明显的激活作用,Cu,FeCu对其有强烈的抑制作用.抗菌活性显示.其对供试病原菌具有明显 的抑菌作用. 关键词:黄蓝状菌;几丁质酶;性质 中图分类号:S7l8.43文献标识码:A Purification,propertiesandantifungalactivityofonechitinaseproduecedbyTalaromycesflavus Chenshueta1. (ForestryBureauofIinyiCounty,ShandongProvinee.276000,China) Abstract:OneextracellularchitinaseE!waspurifiedtOSDS—PAGEhomogeneousbyammoniumsulfatefraction,DEAE SepharoseFastFlowChromatography.Phenyl—SepharoseFastFlowChromatographyandSephacrylS～10()charomatogra phywhenTalaromycesflavuswasgrowninSMCSbrothat27.5℃onarotaryshakerat200r/minforl3days.Themolecular weightofthechitinasewasabout32kD.Thepropertyandantifungalactivityofchitinaseweretestedinthisstudy,theresults wereasthefollowings:TheoptimalpHandtemperatureoftheenzymeforhydrolysisofchitinare5andat50~Crespectively. Differentmetalionsshoweddifferenteffectsonthechitinaseactivities.MnevidentlyincreasedactivitYofthechitinases, Na一,CaandKincreasedpartiallyactivitiyofthechitinase,whereasCu,FeCucausedinhibitionheavilv.Thechitinas— esappearedtobeantifungalactivityagainsttestedfungiandmoreeffectiveagainstfungiofFusariumoxyspormnSchlecht,Bot— ryosphaeriaberengerianaf.sp.PiricolaAlternarialogipesandVerticilliumdahliae. KeyWords:Talaromycesf~~1VUS;chitinase;property 黄监状菌是一种重要菌核寄生菌,无性态为 Penicilliu.2voer*nlculatum.它可以抑制大丽轮枝 孢菌丝的生长,并增加其微菌核的死亡率(Fravel, 1987).黄蓝状菌(Talaromyces,,lavus)同盾壳霉 (Coniothyriuminitans),木霉(,厂r]~oder-Dla sp.),粘帚霉(Glioclacliumsp.)一样是具有一定应 用潜力的菌核寄生菌.因此,分离纯化黄蓝状菌几 丁质酶有利于这种寄生机制的阐明,亦有利于拓展 森林保护学研究思路,提高病虫害防治水平. 1材料与方法 1.1材料和试剂 1.1.1菌株:黄蓝状菌 种病原真菌:尖镰孢 (Talaromycesflavus)及4 菌(Fusariumo:cyspop Schlecht.),贝伦格葡萄座腔菌梨牛专化型(Botry— osphaeriaber