斑点叉尾鮰源嗜麦芽寡养单胞菌胞外蛋白酶的特性研究

THE CHARACTER OF EXTRACELLULAR PROTEASE OF STENOTROPHOMONAS MALTOPHILIA ISOLATED FROM ICTALURUS PUNCTATUS

  • 摘要: 为了研究嗜麦芽寡养单胞菌胞外蛋白酶的酶学特性,本实验检测了不同的修饰剂、温度、pH、EDTA及Ba2+、Co2+、Cd2+、Ca2+、Mn2+、Hg2+、Cu2+及Mg2+等金属离子对酶活的影响,结果表明Ch-T、NBS和2-ME能显著抑制酶活性,而N-AI、PMSF和PCMB对酶活的影响不大,说明蛋氨酸残基、色氨酸残基和二硫键是酶活性的必需基团,而酪氨酸残基、丝氨酸残基和巯基与酶活性无直接关系。酶的最适温度为20℃,在pH为9.0时酶活最高,EDTA能显著影响酶活性,Ca2+、Hg2+、Cu2+能显著降低酶活性,而Co2+能使酶活增强,证实该酶为一种金属蛋白酶。同时检测了该酶的致病性,结果表明,该酶对小鼠及斑点叉尾鮰具有明显的致死作用,其LD50分别为4.33μg/g体重和3.49μg/g体重。

     

    Abstract: Many researches reported that Stenotrophomonas maltophilia infected human in last twenty to thirty years,but we found it could make Ictalurus punctatus dead for the first time.Extracellular protease was one of the important pathogenic factors of Stenotrophomonas maltophilia.In order to investigate the character of extracellular protease and the optimal environment,the effects of protein modification reagents,tmperature,pH and metal iron on the S.maltophilia extracellular protease activity had been studied.The protein modification of extracellular protease of S.maltophilia was studied with PMSF,PCMB,N-AI,Ch-T,NBS and 2-ME as the modification reagents.The results indicated that the protease was not affected by N-AI,PMSF and PCMB,but Ch-T,NBS and 2-ME could reduce the activity significantly.It implied that Ser,Tyr and sulfhydryl residues were not activity dependent groups,while Met,Trp and disulfide linkage groups played an important role on the activity of extracellular protease.Meanwhile,we studied the effect of different environments on the protease activity.It showed that the optimal temperature was 20℃ and the optimal pH was 9.0.PMSF could not affect the activity but EDTA,Ca2+,Hg2+and Cu2+ could reduce the activity of protease greatly,however,Co2+ could enhance the activity.It demonstrated that the protease was a metal-protease.Meanwhile,the purified protease was injected to rats and Ictalurus punctatus intraperitoneally,which came out a huge death.The LD50 of extracellular protease to rats and Ictalurus punctatus were 4.33μg/g和3.49μg/g respectively.

     

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