摘要
测定血清中肌酐的浓度是临床上诊断肾功能的一项重要指标。在利用偶联酶法测定的过程中,一种关键酶——肌氨酸氧化酶(SOX)(EC 1.5.3.1)的质量控制是我们首先要解决的一个重要问题。本文利用乳糖诱导重组肌氨酸氧化酶(r-SOX)基因在E.coli中高效表达,经300L发酵罐发酵培养,菌体终密度达到OD600值22,菌体湿重达30g/L,湿菌体含活性20U/g。重组肌氨酸氧化酶表达量占菌体可溶性蛋白的25%左右。菌体破碎液经55℃选择性热变性处理和Ni-Sepharose FF层析二步纯化,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析酶的纯度达97%以上,比活力达25U/mg,纯化倍数为14.4,活性收率为92.4%。纯化的酶经SDS-PAGE和分子筛层析法测定,其分子量分别为53kD和55kD,提示该酶结构为单一肽链。纯化的酶液和冻干粉均呈黄色,经三氯乙酸和热变性处理,发现其与核黄素以共价键的方式结合。此外,对该酶的稳定性和其中的干扰酶过氧化氢酶也做了进一步的考察。以上所取得的研究结果为此酶的开发和利用奠定了基础。
An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum.In the analytical process applied with coupled-enzyme,the quality control of sarcosine oxidase(SOX)as a key enzyme is the first problem to be solved.In order to establish an efficient and laboratory-scale production of SOX,the recombinant sarcosine oxidase(r-SOX)gene was a high-level expression in E.coli induced with lactose on a large-scale fermentation in 300 Lfermenter.The results suggested that the biomass concentration reached OD600 of 22and the expression of recombinant sarcosine oxidase in E.coli accounted for about 25%of total soluble protein in culture after fermentation.The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography.The sarcosine oxidase with 97% purity,25U/mg specific activity and 92.4% activity recovery was obtained.The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography.This sarcosine oxidase was found to be a conjugated protein,yellow enzyme,which combined with FAD as prosthetic group by covalent linkage.The contaminant of catalase was not detected in the sample pool of this enzyme.In addition,a further test to the thermal stability of sarcosine oxidase was done.According to the above results,the development and utilization of this enzyme has been set up on a reliable foundation.
出处
《生物医学工程学杂志》
CAS
CSCD
北大核心
2014年第5期1090-1096,共7页
Journal of Biomedical Engineering
基金
四川省大学生创新创业训练计划资助项目(201313705020)
关键词
重组肌氨酸氧化酶
诱导表达
高密度发酵
recombinant sarcosine oxidase
inducible expression
high density fermentation