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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。
Determination of serum creatinine concentration is an important indicator of clinical diagnosis of renal function. The quality control of a key enzyme, sarcosinate oxidase (ECX) (EC 1.5.3.1), is an important issue that we must first address in the process of determination by coupled enzymatic methods. In this paper, lactose-induced recombinant sarcosine oxidase (r-SOX) gene was highly expressed in E. coli, 300L fermenter fermentation culture, the final density of bacteria reached OD600 value of 22, wet cell weight 30g / L, wet Cell containing activity 20U / g. Recombinant sarcosine oxidase expression accounted for about 25% of bacterial soluble protein. The cell lysate was purified by selective thermal denaturation at 55 ℃ and Ni-Sepharose FF chromatography. The purity of the enzyme was over 97% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Specific activity of 25U / mg, purification fold was 14.4, the activity yield was 92.4%. The purified enzyme was determined by SDS-PAGE and molecular sieve chromatography, the molecular weight of 53kD and 55kD, suggesting that the enzyme structure of a single peptide chain. Purified enzyme solution and freeze-dried powder were yellow, trichloroacetic acid and heat denaturation, and found that it covalently bonded with riboflavin. In addition, the stability of the enzyme and one of the enzymes that interfere with catalase have also been further investigated. The results obtained above have laid the foundation for the development and utilization of this enzyme.