微生物蛋白质谷氨酰胺酶的初步分离纯化

生物技术通报 ›› 2014, Vol. 0 ›› Issue (2): 161-165.

微生物蛋白质谷氨酰胺酶的初步分离纯化

关静, 孙仁强, 宋佳雯, 黄迪, 成楠, 岑院汉凤, 张扬, 陈曦乐, ,赵怡姗, 朱晨旦, 郑烨, 康立, 高红亮

华东师范大学生命科学学院，上海 200241

收稿日期:2013-08-01 出版日期:2014-02-27 发布日期:2014-02-27
作者简介:关静，女，研究方向：食品微生物；E-mail：k378897850@163.com
基金资助:
自然科学基金项目（81072422），国家大学生创新创业训练计划项目（1310269039），上海市大学生创新基金项目（KY2012-60S）

Extraction and Purification of Microbial Protein—Glutaminase

Guan Jing, Sun Renqiang, Song Jiawen, Huang Di, Cheng Nan, Ceng yuan Hanfeng, Zhang Yang, Chen Xiyue, Zhao Yishan, Zhu Chendan, Zheng Ye, Kang Li, Gao Hongliang

（School of Life Science，East China Normal University，Shanghai 200241）

Received:2013-08-01 Published:2014-02-27 Online:2014-02-27

摘要/Abstract

摘要： 蛋白质谷氨酰胺酶可以特异性地水解蛋白质和多肽的谷氨酰胺残基，研究了产吲哚金黄杆菌产生的微生物蛋白质谷氨酰胺酶的代谢曲线和初步的分离纯化。发酵过程中pH升高，氨浓度增加，到14 h时酶活达到最高为0.359 U/mL。发酵液离心去上清液经3 kD滤膜超滤4倍时，纯化倍数和得率都最高。超滤液加入4倍体积无水乙醇沉淀蛋白质，酶的得率最高为72.7%。沉淀用缓冲液复溶后，经过SP-Sepharose Fast Flow离子交换层析分离，得到单一峰。经过多步纯化后酶得率为31.72%，纯化倍数为124倍，经SDS-PAGE电泳鉴定为单一条带，分子量约为20 kD。

关键词: 微生物蛋白质谷氨酰胺酶, 超滤, 乙醇沉淀, 离子交换层析, SDS-PAGE

Abstract: Protein glutaminase（PG）is an enzyme that catalyzes the specific hydrolysis of glutaminyl residues in proteins and peptide. The metabolic pattern of Chryseobacterium indologenes in PG synthesis and primary purification of PG were studied. The pH value and ammonia concentration increased during the fermentation. The maximal PG activity reached 0.359 U/mL for 14 hours culture. The efficiency of purification and yield of PG were maximal when culture borth was centrifuged and the supernatant was concentrated for four times by ultrafiltration with 3 kD cut-off membrane. Ultrafiltrate was precipitated by 4 times volumes ethanol and the maximal yield of PG was 72.7%. Precipitated PG by ethanol was dissolved in PBS buffer and further purified by SP-Sepharose Fast Flow ion-exchange chromatography. A single peak was got in elution profile. The final yield of PG was 31.72% and the PG was purified 124 times after multiple purification steps. The purified sample contained a single band in SDS-PAGE and corresponding molecular weight was about 20 kD.

关静, 孙仁强, 宋佳雯, 黄迪, 成楠, 岑院汉凤, 张扬, 陈曦乐, ,赵怡姗, 朱晨旦, 郑烨, 康立, 高红亮. 微生物蛋白质谷氨酰胺酶的初步分离纯化[J]. 生物技术通报, 2014, 0(2): 161-165.

Guan Jing, Sun Renqiang, Song Jiawen, Huang Di, Cheng Nan, Ceng yuan Hanfeng, Zhang Yang, Chen Xiyue, Zhao Yishan, Zhu Chendan, Zheng Ye, Kang Li, Gao Hongliang. Extraction and Purification of Microbial Protein—Glutaminase[J]. Biotechnology Bulletin, 2014, 0(2): 161-165.

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