细胞酶促转化过程中胸苷磷酸化酶(TPase)活性测定方法的研究

Research on the Determination of Thymidine Phosphorylase in Enzymatic Conversion Processes

β-胸苷在乙酰短杆菌胸苷磷酸化酶的作用下,反应生成胸腺嘧啶。该文利用紫外分光光度法建立了细胞酶促转化过程中胸苷磷酸化酶活性的测定方法。β-胸苷在胸苷磷酸化酶作用下生成胸腺嘧啶,由于β-胸苷与胸腺嘧啶在300 nm处均有吸光度,反应后通过测量反应液300 nm处的吸光度,可测定反应中生成的胸腺嘧啶的量,从胸腺嘧啶的生成量的多少来判断胸苷磷酸化酶的活性。研究结果表明,在5%菌体浓度、10 mmol/Lβ-胸苷、0.1 mol/L的磷酸缓冲液(pH 6.9)中,37℃反应20 min,即可准确测定胸苷磷酸化酶的活性。利用上述方法测定胸苷磷酸化酶活性,活性能达80%,高于前人报道的方法所测得的酶活性。其中,乙酰短杆菌来源的胸苷磷酸化酶的表观米氏常数Km为11.9 mmol/L。

Under the action role of thymidine phosphorylase in Brevibacterium acetyl,thymidine can be transferred into thymine. In this paper,a method which is used to determine the activity of thymidine phosphorylase in a cellular enzymatic conversion process was established by UV spectrophotometry. By measuring the reaction solution at 300 nm,the amount of thymine could be determined because β-thymidine and thymine are absorbed at 300 nm,then the activity of thymidine phosphorylase activity could be determined.The results showed that with the concentration of 5% Brevibacterium acetyl,10 mmol /L β-thymidine,0. 1 mol / L phosphate buffer( pH 6. 9),reacting for 20 min at the temperature of 37℃,thymidine phosphorylase activity could be accurately measured. The activity of thymidine phosphrylase could reach the level of 80%,which was higher than the result measured by the methods reported previously. The Michaelis constant of thymidine phosphorylase from Brevibacterium acetyl was 11. 9 mmol / L.