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题名: On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis
作者: Jiang, HH;  Zou, HF;  Wang, HL;  Ni, JY;  Zhang, Q;  Zhang, YK
关键词: immobilized trypsin reactor ;  glycidyl methacrylate-modified membrane ;  reaction kinetics ;  frontal analysis ;  enzymes
刊名: JOURNAL OF CHROMATOGRAPHY A
发表日期: 2000-12-15
卷: 903, 期:1-2, 页:77-84
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine ;  Physical Sciences
类目[WOS]: Biochemical Research Methods ;  Chemistry, Analytical
研究领域[WOS]: Biochemistry & Molecular Biology ;  Chemistry
英文摘要: A microreactor by immobilized trypsin on the activated glycidyl methacrylate-modified cellulose membrane packed column was constructed, Immobilized trypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the immobilized enzyme has been developed based on the frontal analysis of enzymatic reaction products, which was performed by the on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-alpha -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis-Menten kinetics constant (K-m) and V-max values measured by the frontal analysis method were, respectively, 0.12 mM and 0.079 mM min(-1) mg enzyme(-1). The former is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher than that of the static method. Inhibition of the immobilized trypsin by addition of benzamidine into substrate solution has been studied by the frontal analysis method. The apparent Michaelis-Menten constant of BAPNA (K-m), the inhibition constant of benzamidine (K-m) and V-max were determined. It was indicated that the interaction of BAPNA and benzamidine with trypsin is competitive, the K-m value was affected but the V-max was unaffected by the benzamidine concentration. (C) 2000 Elsevier Science B.V. All rights reserved.
关键词[WOS]: AFFINITY-CHROMATOGRAPHY ;  GLUCOSE-OXIDASE ;  PURIFICATION ;  MEMBRANES ;  RETENTION ;  TRYPSIN ;  SPACER
语种: 英语
WOS记录号: WOS:000165977800008
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/138773
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromat R&A Ctr, Dalian 116011, Peoples R China

Recommended Citation:
Jiang, HH,Zou, HF,Wang, HL,et al. On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis[J]. JOURNAL OF CHROMATOGRAPHY A,2000,903(1-2):77-84.
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