Alicyclobacillus acidocaldariusThermophilic Esterase EST2's Activity in Milk and Cheese Models

Abstract

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30°C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

“…Esterase, thioesterase, and peptidase activities were determined according to previously described protocols, using pNP-C12, Smethyl thiobutanoate, and Ac-Leu-p-nitroanilide as substrates, respectively (Mandrich et al, 2006;Yang et al, 2009). The standard enzymatic assay was performed in 50 mmol/L phosphate buffer (pH 8.0) at 60°C.…”

Identification of a Novel Alkaliphilic Esterase Active at Low Temperatures by Screening a Metagenomic Library from Antarctic Desert Soil

“…strain B1-11 isolated from Alaskan soil (7), which showed greatest activity at (and was unstable above) 45°C. The k cat /K m ratio of CHA3 was 30-fold lower than that of a mesophilic esterase from Lactococcus lactis (18), although CHA3 was assayed at suboptimal temperature and pH values because of assay limitations. It has been proposed that cold-adapted enzymes might trade off substrate affinity for catalytic velocity, seen as markedly high K m FIG.…”

Enzyme Promiscuity in the Hormone-sensitive Lipase Family of Proteins

“…Furthermore, for EST2 a thioesterase activity was also discovered (EC 3.1.2.X) that can be clearly classified as catalytic promiscuity [77]. This activity (at 25°C) was observed in a study ( Table 2) aimed at determining the ability of EST2 to work in milk and cheese standardized models and testing if this enzyme had thioesterase and ester-synthesizing activities, like those found in Lactococcus lactis EstA [78].…”