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重组脯氨酰内肽酶提取方法改进及活性研究

Impact of Extraction Methods on the Catalytic Activities of Recombinant Sphingomonas Capsulata Prolyl Endopeptidase
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摘要 脯氨酰内肽酶具有水解多肽中脯氨酰残基的能力,具有治疗乳糜泻的潜力,构建高表达量的重组脯氨酰内肽酶、建立保持其高活性的提取方法对于脯氨酰内肽酶的药物开发至关重要。该研究经密码子优化、基因合成,构建了重组鞘氨醇单胞菌脯氨酰内肽酶(SCPEP)的大肠杆菌BL21(DE3)工程菌。探索建立改进型渗透休克法提取SCPEP,经Ni-NTA纯化、超滤浓缩得到产物。采用分光光度法基于底物Suc-Ala-Pro-pNA评价SCPEP的活性,同时与高压均质法提取的产物进行对比研究。结果显示,在pH 6. 0和pH 7. 0的反应体系中,采用改进型渗透休克法提取的SCPEP水解Suc-Ala-Pro-pNA的kcat/KM分别为(56. 58±12. 00)(mmol/L)-1·s-1和(55. 82±15. 33)(mmol/L)-1·s-1,而高压均质法提取的SCPEP水解Suc-Ala-Pro-pNA的kcat/KM分别为(24. 78±10. 63)(mmol/L)-1·s-1和(48. 69±13. 79)(mmol/L)-1·s-1,改进型渗透休克法提取的SCPEP在pH6. 0和pH 7. 0下的活性比高压均质法提取的SCPEP高2. 30和1. 15倍,故改进型渗透休克法有利于提取的SCPEP保持更高的活性。 Prolyl endopeptidase(PEP)has an ability to hydrolyze prolyl residues in polypeptides;therefore,it has the potential to treat celiac disease.Optimizing the recombinant expression and purification methods of PEP is very important to the drug development of prolyl endopeptidase.The purpose of this study is to construct a high-expression recombinant SCPEP by molecular biology techniques and to explore a new extraction method to maintain the high activity of SCPEP.And the effect of the new extraction method-modified osmotic shock method and traditional high-pressure homogenization extraction method on the activity of recombinant SCPEP is compared and analyzed.In this study,recombinant Escherichia coli BL21(DE3)engineered strain were constructed by codon optimization and gene synthesis.In the exploration of the establishment of a new extraction method,this study reduced the centrifugal speed of the traditional osmotic shock method and established a modified osmotic shock method.Then SCPEP was extracted by modified osmotic shock method and high pressure homogenization.At last SCPEP was purified by Ni-NTA and concentrated by ultrafiltration.The activity of SCPEP extracted by modified osmotic shock method was evaluated by spectrophotometry based on the substrate Suc-Ala-Pro-pNA,and compared with the products extracted by high pressure homogenization.The results showed that at pH 6.0 and pH 7.0,the kcat/KMof SCPEP extracted by modified osmotic shock method was(56.58±12.00)(mmol/L)-1·s-1 and(55.82±15.33)(mmol/L)-1·s-1,respectively;while the kcat/KMof SCPEP extracted by high pressure homogenization method was(24.78±10.63)(mmol/L)-1·s-1 and(48.69±13.79)(mmol/L)-1·s-1,respectively.SCPEP extracted by modified osmotic shock method has 2.30 times and1.15 times higher catalytic activity at pH 6.0 and pH 7.0 than SCPEP extracted by high pressure homogenization.In summary,this study successfully constructed a high expression recombinant wild-type SCPEP,and compared the two methods of extracting recombinant SCPEP,and optimize
作者 毛建平 肖斌 宋晓彤 余蓉 郑永祥 MAO Jian-ping;XIAO Bin;SONG Xiao-tong;YU Rong;ZHENG Yong-xiang(West China School of Pharmacy,Sichuan University,Key Laboratory of Drug Targeting and Drug Delivery Systems,Ministry of Education,West China School of Pharmacy,Sichuan University,Chengdu 600041,China)
出处 《药物生物技术》 CAS 2019年第2期100-104,共5页 Pharmaceutical Biotechnology
基金 国家自然科学基金面上项目(No.81773623).
关键词 鞘氨醇单胞菌属 脯氨酰内肽酶 提取方法 渗透休克法 高压均质法 催化活性 Sphingomonas capsulate(SC) Prolyl endopeptidase(PEP) Extraction method Modified osmotic shock method High pressure homogenization Catalytic activity
作者简介 毛建平(1994-),男(汉族),四川成都人,硕士研究生,生物制药学,Tel:13880963726,E-mail:maijap@126.com;通讯作者:郑永祥(1986-),男,福建建阳人,讲师,主要研究方向:蛋白质工程药物与酶类药物的理性设计和评价,Tel:17358514961,E-mail:yongxiangzheng@scu.edu.cn。
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