Lipases colorimetric methods

Conceptually, assays for lipase activity using the colorimetric method (copper-soap procedure Basic Protocol 2) are similar to titrimetry in that liberated fatty acids are being measured however, the colorimetric method is more specific for fatty acids (Lowry and Tinsley, 1976). Quenched subsamples of emulsified acylglycerol/lipase reaction mixtures are combined with the biphasic mixture of cupric acetate/pyridine and benzene. Cupric salts of the fatty acids are formed (molar stoichiometry of fatty acid to Cu2+ of 4 2) and these soaps, which are blue in color, are partitioned into benzene to allow for quantification by measuring absorbance of the clear benzene phase at 715 nm. 

Kwon, D.Y. and Rhee, J.S. 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 63 89-92. 

Blake, M.R., Koka, R., Weimer, B.C. 1996. A semiautomated reflectance colorimetric method for the determination of lipase activity in milk. J. Dairy Sci. 79, 1164-1171. 

Since the proposed method was optimized for human serum spiked with porcine pancreas lipase, it was applied to real samples to determine its estimated practicability. The seven samples from healthy volunteers were measured by the proposed method and the colorimetric method. The average and median values obtained with the proposed method were 7.76 and 7.67 Uhdi> respectively. The colorimetric method is based on the determination of liberated methylresorufm from... 

The antioxidants in serum matrices such as ascorbic acid, uric acid and bilirubin have been known to interrupt CL emission. This inhibition of CL emission was completely eliminated by introducing the SPE procedure. HDI-laurate was successfully converted to the active enhancer, HDI, by lipase in serum, and HDI enhanced the luminol-H202-HRP CL system. The results of the proposed method should good correlation with those of the colorimetric method although the results... 

OF and Lipase P were also shown to aggregate with block copolymers made of poly[(A-acetylimino)ethylene] and poly[(A-pentanoylimino)ethylene], with about 30% increased activity for the ester hydrolysis and 200% increased activity for the ester synthesis in aqueous solutions compared to the native enzymes. The activity of egg white lysozyme located in the core of PEG—poly(o ,j8-aspartic acid) micelles was investigated by colorimetric methods and revealed an increase for p-nitrophenyl-penta-A-acetyl-/ -chitopentaoside as substrate. By the determination of kinetic constants of enzymatic reaction, it was demonstrated that the increase in the enzymatic activity was... 

Kwon, D., and J. Rhee. 1986. A Simple and Rapid Colorimetric Method for Determination of Free Fatty Acids for Lipase Assay. Journal of the American Oil Chemists Society 63 (l) 89-92. 

Lipase activity was measured by a colorimetric method ( adapted from Nixon and Chan. 1979 ). The substrate (sunflower oil) was purified.The reaction was carried out at 30°C in a shaker water bath. 

A number of methods are available for following lipase activity. Although numerous modifications and variations have been introduced, the basic methods are (1) titration of the liberated fatty acids, (2) changes in surface tension, (3) colorimetric determination of the fatty acids, (4) use of gas-liquid chromatography, and (5) use of radioactive substrates. Kuzdzal-Savoie (1980) has reviewed the subject. 

Basic Protocol 1 Titrimetric Determination of Lipase Activity Basic Protocol 2 Colorimetric Assay of Lipase Activity Using the Copper Soap Method... 

COLORIMETRIC ASSAY OF LIPASE ACTIVITY USING THE COPPER SOAP METHOD... 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

Tributyrin 2. Colorimetric (a) (3-Naphthol methods NaCl Extraction with ether-petroleum ether, titration to visual endpoint Milk lipase Bacterial lipase Castberg et al. (1975) Fitz-Gerald and Deeth (1983)... 

Because the molecular basis of enantioselectivity is poorly understood, directed evolution seems to be an excellent choice for engineering enantioselective biocatalysts. Several impressive examples have been documented. In a classical study, Reetz and coworkers used error-prone PCR coupled with a 96-well plate based colorimetric screening method to increase the enantioselectivity of a Pseudomonas aeruginosa lipase toward 2-methyldecanoate. After several rounds of directed evolution, the enantioselectivity of the lipase increased from E = 1.04 (2% enantiomeric excess) to E = 25 (90-93% enantiomeric excess, ee) (E is the enantioselectivity factor). Using a similar approach. 

Wang D.Wang J, Wang B,Yu H. A new and efficient colorimetric high-throughput screening method for triacylglycerol lipase directed evolution. Mol Catal B Enzym 2012 82 18-23. 

Garcia-Roman, M., Teixeira, J.A., and Belo, I. (2011) Optimization of a colorimetric assay for yeast lipase activity in complex systems. Anal. Methods, 3, 1008 1013. 

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