双齿围沙蚕蛋白酶的纯化及其性质
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汪靖超, 赵 峰, 李荣贵, 王 斌
纯化;蛋白酶;纯化双齿围沙蚕;性质汪靖超,赵峰,李荣贵,王斌.双齿围沙蚕蛋白酶的纯化及其性质. 世界华人消化杂志2007;15(8)800-806,汪靖超,赵峰,李荣贵,王斌,汪靖超,通讯作者:,电话:,收稿日期:,接受日期:,Purif
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汪靖超, 赵峰, 李荣贵, 青岛大学医学院生物系 山东省青岛市 266071
王斌,青岛大学医学院微生物学教研室 山东省青岛市 266021
汪靖超, 硕士, 讲师, 主要从事分子生物学研究.国家重点基础研究前期专项基金资助项目, No. 2004CCA02400
通讯作者: 汪靖超, 266071, 山东省青岛市, 青岛大学医学院生物系. wangjc@qdu.edu.cn
电话: 0532-85953710
收稿日期: 2006-11-10 接受日期: 2007-01-10
Purification and properties of the protease from Perinereis aibuhitensis Grube
Jing-Chao Wang, Feng Zhao, Rong-Gui Li, Bin wang
Jing-Chao Wang, Feng Zhao, Rong-Gui Li, Department of Biology, Qingdao University Medical College, Qingdao 266071, Shandong Province, China
Bin Wang, Department of Microbiology, Qingdao University Medical College, Qingdao 266021, Shandong Province, China
Correspondence to: Jing-Chao Wang, Department of Biology, Qingdao University Medical College, Qingdao 266071, Shandong Province, China. wangjc@qdu.edu.cn
Received: 2006-11-10 Accepted: 2007-01-10
AbstractAIM:To purify a new protease from the clamworm Perinereis aibuhitensis Grube, and study its properties.
METHODS: After homogenization, Superdex-75 chromatography, MonoQTM chromatography, and SP-Sepharose 4B chromatography were used ordinally to purify the protease from the clamworm Perinereis aibuhitensis Grube. A new gelatin zymography method was used to study the proteolytic activity of the protease: protease separated by non-reductive SDS-PAGE was electrophoretically transferred to a gel containing gelatin, and the proteolytic activity of the protease was determined by the clear zone of the electrophoretic protein band. The effect of pH value, temperature, protease inhibitor, organic solvents and metal ions on the proteolytic activity of the protease was studied.
RESULTS: After purification, one kind of protease was obtained, which showed two protein bands with molecular weights of 34 900 and 33 900 separately by reductive SDS-PAGE, while three bands with molecular weights of 65 600, 57 900, and 43 500 separately by non-reductive SDS-PAGE. Gelatin zymography showed that the proteolytic activity mainly appeared around the band of Mr 66 000. The protease was stable and displayed high proteolytic activity within the range of pH 8 to 10 and at 20 - 35℃ It was strongly inhibited by phenylmethyl sulfonyl fluoride (PMSF), while stable in the presence of organic solvents.
CONCLUSION: The protease is composed of two subunits, whose molecular weights are 33 900 and 34 900, respectively. Gelatin zymography is sensitive and effective in studying the proteolytic activity of proteases.
Key Words: Purification; protease; Perinereis aibuhitensis Grube; properties
Wang JC, Zhao F, Li RG, Wang B. Purification and properties of the protease from Perinereis aibuhitensis Grube. Shijie Huaren Xiaohua Zazhi 2007;15(8):800-806
摘要目的:纯化双齿围沙蚕(Perinereis aibuhitensis Grube)蛋白酶, 并对其性质进行研究 ......
汪靖超, 赵峰, 李荣贵, 青岛大学医学院生物系 山东省青岛市 266071
王斌,青岛大学医学院微生物学教研室 山东省青岛市 266021
汪靖超, 硕士, 讲师, 主要从事分子生物学研究.国家重点基础研究前期专项基金资助项目, No. 2004CCA02400
通讯作者: 汪靖超, 266071, 山东省青岛市, 青岛大学医学院生物系. wangjc@qdu.edu.cn
电话: 0532-85953710
收稿日期: 2006-11-10 接受日期: 2007-01-10
Purification and properties of the protease from Perinereis aibuhitensis Grube
Jing-Chao Wang, Feng Zhao, Rong-Gui Li, Bin wang
Jing-Chao Wang, Feng Zhao, Rong-Gui Li, Department of Biology, Qingdao University Medical College, Qingdao 266071, Shandong Province, China
Bin Wang, Department of Microbiology, Qingdao University Medical College, Qingdao 266021, Shandong Province, China
Correspondence to: Jing-Chao Wang, Department of Biology, Qingdao University Medical College, Qingdao 266071, Shandong Province, China. wangjc@qdu.edu.cn
Received: 2006-11-10 Accepted: 2007-01-10
AbstractAIM:To purify a new protease from the clamworm Perinereis aibuhitensis Grube, and study its properties.
METHODS: After homogenization, Superdex-75 chromatography, MonoQTM chromatography, and SP-Sepharose 4B chromatography were used ordinally to purify the protease from the clamworm Perinereis aibuhitensis Grube. A new gelatin zymography method was used to study the proteolytic activity of the protease: protease separated by non-reductive SDS-PAGE was electrophoretically transferred to a gel containing gelatin, and the proteolytic activity of the protease was determined by the clear zone of the electrophoretic protein band. The effect of pH value, temperature, protease inhibitor, organic solvents and metal ions on the proteolytic activity of the protease was studied.
RESULTS: After purification, one kind of protease was obtained, which showed two protein bands with molecular weights of 34 900 and 33 900 separately by reductive SDS-PAGE, while three bands with molecular weights of 65 600, 57 900, and 43 500 separately by non-reductive SDS-PAGE. Gelatin zymography showed that the proteolytic activity mainly appeared around the band of Mr 66 000. The protease was stable and displayed high proteolytic activity within the range of pH 8 to 10 and at 20 - 35℃ It was strongly inhibited by phenylmethyl sulfonyl fluoride (PMSF), while stable in the presence of organic solvents.
CONCLUSION: The protease is composed of two subunits, whose molecular weights are 33 900 and 34 900, respectively. Gelatin zymography is sensitive and effective in studying the proteolytic activity of proteases.
Key Words: Purification; protease; Perinereis aibuhitensis Grube; properties
Wang JC, Zhao F, Li RG, Wang B. Purification and properties of the protease from Perinereis aibuhitensis Grube. Shijie Huaren Xiaohua Zazhi 2007;15(8):800-806
摘要目的:纯化双齿围沙蚕(Perinereis aibuhitensis Grube)蛋白酶, 并对其性质进行研究 ......
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