摘要
目的从节杆菌Arthrobacter sp．XL2胞内中初步提取黄嘌呤氧化酶（XOD）。方法探讨了不同破壁方法和硫酸铵盐析提取粗酶的条件。采用响应面分析法优化了超声波法抽提XOD的条件。结果菌体用超声波破壁，在频率25KHz，细胞浓度80mg／mL下每次辐射2s，间隔2s，总辐射时间18min，输出功率400W，破碎产酶量达到（4．69±0．11）U／mL。用40％～60％饱和度硫酸铵阶段盐析提取粗酶效果最好。将盐析粗酶用Sephadex G-25脱盐后，比酶活达到6．95U／mg，纯度提高了3．43倍，然后进行XOD粗酶部分酶学性质研究。XOD的稳定pH和温度范围分别为：pH7．0～9．0和低于50℃。XOD的最适反应温度和pH分别为：40℃及pH8．0。Zn^2＋，Cd^2＋，Cu^＋，Pb^2＋，Hg^2＋强烈抑制酶的活性。结论此粗酶提取工艺可以有效地将XOD从细胞内释放出来，提高了粗酶抽提得率。为后续的进一步纯化研究奠定了基础。
Purpose To purify xanthine oxidase in vivo from cells of Arthrobacter sp. XL2 preliminarily. Methods Xanthine oxidase produced from Arthrobacter sp. XL2 was purified by three steps including breaking cell wall, ammonium sulfate precipitation, and gel filtration chromatography（GFC）. Response surface methodology（RSM） was applied to optimize the process paramrters which were the important variables effecting the xanthine oxidase production. Results h had better results when the following methold was used： supersonic wave frequency 25 KHz, ultrasonic power 400 W, cell density 80 mg/mL, radiate 2 second each time, total radiate 18 minutes, 2 second interval to break cell wall, and 40% - 60% ammonium sulfate precipitation to remove contaminating proteins and extracting crude enzyme products, which were desalted by GFC. The specific activity of initial pure enzyme was 6.95 U/mg. The xanthine oxidase was stable in the pH range between 7.0-9.0 and below 50℃ . h was markedly inactivated by incubation with 2 mmol/L Zn^2＋ , Cd^2＋ , Cu^2＋ , Pb^2＋ ,Ag^＋ , Hg^2＋ . Conclusion The process can be used to purify preliminarily the crude xanthine oxidase from Arthrobacter sp. XL2 efficiently, then further deeply studying purification and characteristics of this xanthine oxidase.
出处
《中国生化药物杂志》
CAS
CSCD
2007年第5期322-326,共5页
Chinese Journal of Biochemical Pharmaceutics
基金
工业微生物教育部重点实验室基金(KLIB-KF200502)
福建省自然科学基金(C0410006)资助项目
关键词
黄嘌呤氧化酶
超声波
硫酸铵盐析
稳定性
xanthine oxidase
supersonic wave
ammonium sulfate precipitation
stability
