Amperometric biosensor for diamine using diamine oxidase purified from porcine kidney

被引:37
作者
Bouvrette, P [1 ]
Male, KB [1 ]
Luong, JHT [1 ]
Gibbs, BF [1 ]
机构
[1] UNIV WATERLOO, DEPT CHEM ENGN, BIOTECHNOL RES INST, NATL RES COUNCIL CANADA, MONTREAL, PQ H4P 2R2, CANADA
关键词
porcine kidney diamine oxidase; enzyme immobilization; diamines; histamine; putrescine; cadaverine; enzyme purification;
D O I
10.1016/S0141-0229(96)00064-6
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Diamine oxidase was purified over 2300-fold from porcine kidney to a specific activity of 1 U mg(-1). The final preparation exhibited a single 103 kDa protein band and contained relatively large amounts of Asx and Glx acidic residues. When stored at -80 degrees C, soluble enzyme retained its original catalytic activity for at least five months. The optimal pH and temperature of the enzyme immobilized by intramolecular cross-linking via gill taraldehyde activation and deposited onto a preactivated nylon membrane were 7.4 and 60 degrees C with cadaverine as substrate. The apparent K-m(n) values (Michaelis-Menten constants) of immobilized diamine oxidase with histamine, putrescine, and cadaverine as substrates were estimated to be 0.27. 3.2, and 0.64 mM, respectively. Artificial mediators such as ferrocene derivatives, 2,6-dichlorophenolindophenol, potassium ferricyanide and 4-aminodiphenylamine were nor observed to facilitate electron transfer from the reduced enzyme to the electrode. The biosensor using the immobilized diamine oxidase and a platinum working electrode (poised at +700 mM vs Ag/AgCl for determination of hydrogen peroxide released from the enzymatic oxidation) was linear up to 6 mM histamine, cadaverine, or putrescine with a lower detection limit of 25 mu M. Each analysis could be performed in 3 min including washing and time for the current to return to baseline. The enzyme membranes were stable at 5 degrees C for at least two months and could be used for more than 60 repeated analyses without significant loss of sensitivity. (C) 1997 by Elsevier Science Inc.
引用
收藏
页码:32 / 38
页数:7
相关论文
共 28 条
[1]  
ALLAIN CC, 1974, CLIN CHEM, V20, P470
[2]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[3]  
BUFFONI F, 1966, PHARMACOL REV, V18, P1163
[4]  
Dawson R.M. C., 1986, DATA BIOCH RES
[5]  
FOUTS JR, 1957, J BIOL CHEM, V225, P1025
[6]  
GLAZER AN, 1975, CHEM MODIFICATION PR, P13
[7]   DUAL FUNCTIONALITIES OF 4-AMINODIPHENYLAMINE IN ENZYMATIC ASSAY AND MEDIATED BIOSENSOR CONSTRUCTION [J].
GROOM, CA ;
LUONG, JHT ;
THATIPALMALA, R .
ANALYTICAL BIOCHEMISTRY, 1995, 231 (02) :393-399
[8]  
Hill H.A., 1990, BIOSENSORS PRACTICAL, P19
[9]  
HUGLI TE, 1972, J BIOL CHEM, V247, P2828
[10]  
Jacober L. F., 1982, In 'Chemistry & biochemistry of marine food products' G [see FSTA (1983) 15 G7R466]., P347