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肌氨酸氧化酶的酶学性质及失活机理 被引量:6

On the Enzymatic Properties and Deactivation Mechanism of Sarcosine Oxidase
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摘要 将来源于Bacillus sp.的肌氨酸氧化酶(SOX)基因在E.coli成功表达,并探索了肌氨酸氧化酶(sarcosine oxidase)酶学性质和失活机理。Bacillus sp.的肌氨酸氧化酶基因克隆入载体p ET28a,并转化至E.coli BL21(DE3)表达。经IPTG诱导8 h,对菌体破壁液进行SDS-PAGE电泳分析,发现在43 kDa处有明显蛋白质条带。采用亲和介质分离纯化获得较高纯度SOX。SOX酶学性质分析结果表明,其相对分子质量为43.8 kDa,最适反应温度40℃,最适反应pH值为8.0;20 mmol/L的Mn^(2+)对SOX具有明显激活作用;其动力学参数分析表明,肌氨酸作为底物时的K_m值为141.6 mmol/L,V_(max)为0.115 mmol/(L·min)。结合SDS-PAGE凝胶电泳、荧光光谱和圆二色性光谱分析,探明了液态SOX的失活机理。在37℃恒温条件下,随储存时间延长,SOX酶分子内部疏水基团逐渐暴露且二级结构被破坏,导致酶分子变性以及最终酶活力下降。为进一步提高SOX的酶活稳定性提供了理论依据。 The sarcosine oxidase(SOX) gene from Bacillus sp. was expressed in E. coli,and the properties and deactivation mechanism were further analyzed. After transformation of the cloned DNA into host E. coli BL21(DE3),the SOX gene was induced by IPTG for 8 h,a protein band of 43 k Da appeared in SDS-PAGE electrophoresis. From the crude enzyme solution,SOX in high purity was obtained through affinitive separation and purification. The analytic results of enzymatic properties showed that its relative molecular mass was 43.8 k Da, and its optimum reaction temperature and p H value were 40 ℃ and 8.0,respectively. Moreover,20 mmol/L Mn^(2+) exhibited an obvious activation effects on recombinant SOX. The kinetic parameters of Km and Vmax were determined as 141.6 mmol/L and 0.115 mmol/(L·min) when sarcosine used as a substrate. With the SDS-PAGE gel electrophoresis,fluorescence and circular dichroism spectroscopy,the deactivation mechanism of SOX was further studied. At constant temperature 37 ℃,the internal hydrophobic group of SOX molecule was gradually exposed and its secondary structure was destroyed along with the prolonged storage time,leading to the denaturation of SOX protein and the lowering down of its activity. The above results provide a theoretical basis for improving SOX stability in future.
出处 《食品与生物技术学报》 CAS CSCD 北大核心 2015年第12期1239-1247,共9页 Journal of Food Science and Biotechnology
基金 国家自然科学基金项目(21306064) 江苏省创新计划项目(CXZZ12_0752) 江南大学博士研究生科学研究基金项目(JUDCF12013)
关键词 肌氨酸氧化酶 酶学性质 失活机理 sarcosine oxidase enzymatic properties deactivation mechanism
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