壳聚糖固定化S-腺苷甲硫氨酸合成酶

Study on Characterizations of Immobilized S-adenosylmethionine Synthetase on Chitosan Microspheres

DOI：10.3969/j.issn.1673-1689.2013.09.008

中文关键词: S-腺苷甲硫氨酸合成酶 壳聚糖 固定化酶 酶学性质

英文关键词: S-adenosylmethionine synthetase chitosan immobilized enzyme enzymatic properties

基金项目:国家自然科学基金项目(20802057);陕西省科技攻关项目(2011K08-12);西安市科技支撑计划项目(CXY1134Wl25);西北工业大学基础研究基金项目(JC201161);西北工业大学“翱翔之星”计划资助

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中文摘要:

在大肠杆菌中重组表达了S-腺苷甲硫氨酸合成酶,然后以壳聚糖为载体,戊二醛为交联剂,对酶进行固定化研究。结果表明,最佳固定化条件为:壳聚糖质量浓度为2.5 g/dL、戊二醛体积分数为0.8%、固定化酶量为6 mg/mL、固定化时间为12 h,该条件下所得固定化酶活性回收率为76%,固定化酶热稳定性较好,在55℃下保温5 h仍保留53%的活力,而游离酶在50℃下保温5 h则完全失活。固定化酶在碱性溶液环境的稳定性较高,在pH 7.5~9.0的缓冲液中4℃保温10 h酶活性仍保留80%以上。将固定化酶用于S-腺苷甲硫氨酸的合成,连续反应5批次后,酶活性仍保留64%。在底物三磷酸腺苷(ATP)浓度为30 mmol/L的条件下,固定化酶催化底物ATP的转化率超过95%。

英文摘要:

S-adenosylmethionine(SAM) synthetase was expressed in E. coli,and then the purified SAM synthetase was immobilized on chitosan microspheres with glutaraldehyde crosslinking.Properties of the immobilized enzyme were identified. The results showed the optimal conditions for the immobilization of the enzyme were as follows:the mass fraction of chitosan was 2.5%;the volume fraction of glutaraldehyde was 0.8%;the amount of enzyme was 6 mg/mL gel;the immobilization time was 12 hours. Under these conditions,the activity yield of the immobilized enzyme was 76%. The immobilized enzyme showed a good stability compared with the free enzyme.After incubation at 55 ℃ for 5 h the immobilized enzyme maintained 53% of the original activity while the free enzyme lost all activity at 50 ℃ for 5 h. The immobilized enzyme also showed a good stability in alkaline solution. It still kept more than 80% of the original activity when incubated in the buffer of pH 7.5~9.0 at 4 ℃ for 10 h. The immobilized enzyme was employed to synthesize the SAM and it remained 64% activity after five times repeated operations. The substrate conversion rate was more than 95% in the presence of 30 mmol/L adenosine triphosphate(ATP)。

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