An active-site titration method for lipases

被引:53
作者
Rotticci, D
Norin, T
Hult, K [1 ]
Martinelle, M
机构
[1] Royal Inst Technol, Dept Biotechnol, SE-10044 Stockholm, Sweden
[2] Royal Inst Technol, Dept Chem Organ Chem, SE-10044 Stockholm, Sweden
来源
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS | 2000年 / 1483卷 / 01期
关键词
concentration; immobilized lipase; aqueous medium; organic medium; interfacial activation; detergent; inhibitor;
D O I
10.1016/S1388-1981(99)00168-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate, This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:132 / 140
页数:9
相关论文
共 21 条
[1]  
Barros RJ, 1998, BIOTECHNOL BIOENG, V59, P364
[2]  
Bjorkling F, 1994, Bioorg Med Chem, V2, P697, DOI 10.1016/0968-0896(94)85020-8
[3]   N-BUTYL-N-METHYL-4-NITROPHENYL CARBAMATE AS A SPECIFIC ACTIVE-SITE TITRATOR OF BILE-SALT-DEPENDENT LIPASES [J].
FOURNERON, JD ;
ABOUAKIL, N ;
CHAILLAN, C ;
LOMBARDO, D .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1991, 196 (02) :295-303
[4]   Covalent inhibition of digestive lipases: An in vitro study [J].
Gargouri, Y ;
Ransac, S ;
Verger, R .
BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM, 1997, 1344 (01) :6-37
[5]   A FLUOROGENIC ACTIVE-SITE TITRANT FOR SERINE PROTEASES [J].
GREEN, DPL .
ANALYTICAL BIOCHEMISTRY, 1985, 147 (02) :487-490
[6]   ANALOGS OF REACTION INTERMEDIATES IDENTIFY A UNIQUE SUBSTRATE-BINDING SITE IN CANDIDA-RUGOSA LIPASE [J].
GROCHULSKI, P ;
BOUTHILLIER, F ;
KAZLAUSKAS, RJ ;
SERREQI, AN ;
SCHRAG, JD ;
ZIOMEK, E ;
CYGLER, M .
BIOCHEMISTRY, 1994, 33 (12) :3494-3500
[7]   Active-site titration of serine proteases using a fluoride ion selective electrode and sulfonyl fluoride inhibitors [J].
Hsia, CY ;
Ganshaw, G ;
Paech, C ;
Murray, CJ .
ANALYTICAL BIOCHEMISTRY, 1996, 242 (02) :221-227
[8]   RULES FOR OPTIMIZATION OF BIOCATALYSIS IN ORGANIC-SOLVENTS [J].
LAANE, C ;
BOEREN, S ;
VOS, K ;
VEEGER, C .
BIOTECHNOLOGY AND BIOENGINEERING, 1987, 30 (01) :81-87
[9]  
Longhi S, 1997, PROTEIN SCI, V6, P275
[10]   Chiral preference of cutinase in the reaction with phosphonate inhibitors [J].
Mannesse, MLM ;
deHaas, GH ;
vanderHijden, HTWM ;
Egmond, MR ;
Verheij, HM .
BIOCHEMICAL SOCIETY TRANSACTIONS, 1997, 25 (01) :165-170