Discrimination Between Lipases And Esterases

The fluorogenic lipids can also be used for the discrimination of lipases and esterases (Figure 5). The assay is lipase-specific, when the long-chain alkyldiacylglycerols are utilized as substrates. The short-chain glycerolipids may be substrates for lipases and esterases. In contrast, the fluorogenic fatty acid estej"£jy$ mainly esterase substrates. The latter compounds represent pyrenemethyl esters of trinitrophenyaminofatty acids and are single-chain lipids. They work according to the same spectroscopic principle as compared to the fluorogenic glycerolipids. They contain the same chromophors and produce a fluorescence signal upon hydrolytic cleavage.

Figure 6 shows the hydrolytic activities of four different variants of Bacillus subtilis EST 4 cell lysates. Fluorogenic alkyldiacylglycerols and fatty acid esters were used as lipase and esterase substrates as outlined above.Three different lipolytic activitities have been detected. Cell lysates from variant A hydrolyzed the long- and short-chain glycerolipids as well as the carboxylic acid ester. Variant C was active towards the short-chain glycerolipid and the carboxylic acid ester, whereas variants B and D degraded the carboxylic acid ester only. The activities toward long- and short chain glycerolipids must be ascribed to different enzymes because the former one was only found in A but not in C. Hydrolysis of the short-chain glycerolipid is most probably due to the same enzyme in A and C since the respective stereopreferences (for the sn-1 -acyl enantiomer) are almost identical. A separate enzyme is responsible for carboxylic acid ester hydrolysis, because it produces the sole activity in B and D.

Lipases Lipases and Esterases

Esterases

Figure 5. Chemical structures of substrates for lipases and esterases. Long-chain alkyldiacylglycerol (for lipases); short-chain alkyldiacylglycerol (for lipases and esterases), trinitrophenylaminohenanoic acid pyrenemethyl ester (for esterases).

Esterases

Figure 5. Chemical structures of substrates for lipases and esterases. Long-chain alkyldiacylglycerol (for lipases); short-chain alkyldiacylglycerol (for lipases and esterases), trinitrophenylaminohenanoic acid pyrenemethyl ester (for esterases).

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