荧光假单胞菌GcM5-1A胞外木质素过氧化物酶的初步纯化及性质研究  

Preliminary purification and characterization of extracellular lignin peroxidase from Pseudomonas fluorescens GcM5-1A

作　　者：孔令营[1] 郭道森[1] 赵博光[2] 李荣贵[1] 

机构地区：[1]青岛大学生物系 [2]南京林业大学森林资源与环境学院

出　　处：《北京林业大学学报》2010年第3期112-116,共5页Journal of Beijing Forestry University

基　　金：国家自然科学基金重点项目(30430580)

摘　　要：本文研究了松材线虫携带的荧光假单胞菌GcM5-1A的培养液经硫酸铵分级沉淀后,各组分中木质素过氧化物酶(LiP)的活性及各组分对黑松悬浮细胞的毒性;还对GcM5-1A菌株胞外LiP进行了初步纯化,并对纯化后的酶学性质进行了研究。结果表明:LiP活性主要出现在50%～70%沉淀组分中;0～20%沉淀组分和50%～70%沉淀组分对黑松悬浮细胞的毒性很强;GcM5-1A菌株培养液经硫酸铵分级沉淀以及DEAE-SepharoseFF两步纯化后,其LiP的比活力从0.81U/mg提高到21.30U/mg;其最适温度为35℃,最适pH为4.0;在15～35℃、pH6.0～9.0范围内,酶活力保持相对稳定;NH2OH·HCl和KCN对酶活力的抑制作用分别达到88%和57%,而NaN3对酶活力的抑制作用只有15%左右;吸收光谱显示该酶在406nm处有一个Soret峰,加入1mmol/LNa2S2O4后,吸光度出现了明显的下降,但Soret峰没有显著移动。In this study,a fermented culture of Pseudomonas fluorescens GcM5-1A carried by the pinewood nematode,Bursaphelenchus xylophilus,was precipitated with ammonium sulfate solutions with different saturation rates and the resulting solution was used to test lignin peroxidase（LiP） activity and its toxicity to a suspension of Pinus thunbergii cells.We also studied the preliminary purification and characterization of extracellular LiP from P.fluorescens GcM5-1A.The results show that LiP activity mainly existed in 50%-70% ammonium sulfate fraction;50%-70% ammonium sulfate fraction also exhibited relatively strong toxicity besides 0-20% ammonium sulfate fraction.The specific LiP activity was raised from 0.81 to 21.30 U /mg after two purification steps：ammonium sulfate fractionation and DEAE-Sepharose FF.The optimum pH and temperature of LiP were about 4.0 and 35℃,respectively.The enzyme was stable at pH of 6.0 to 9.0 and temperatures of 15 to 35℃.The typical peroxidase inhibitors NH2OH·HCl and KCN inhibited 88% and 57% of enzyme activity,respectively,while the inhibitory effect of NaN3 was only about 15%.The absorption spectrum of the native enzyme showed a Soret peak at 406 nm.Introduction by 1 mmol/L Na2S2O4 resulted in a marked decrease in absorbance;however,no shift was evident in the Soret peak.

关 键 词：松材线虫 荧光假单胞菌 木质素过氧化物酶 毒素 

分 类 号：S763[农业科学—森林保护学] Q93[农业科学—林学]