Abstract
Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure to elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring following in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retains high levels of catalytic activity after exposure to temperatures as high as 95°C for several hours. Furthermore, it exhibits very good stability against exposure to high concentrations of a variety of organic solvents. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modeling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold that seems to include a “subdomain insertion”, which is similar to the one present in its closest homolog of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a range of organic solvents and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications.
Highlights
Lipolytic enzymes (EC 3.1.1.x) catalyze the hydrolysis of ester bonds in lipids, and depending on their substrate preference, they are divided in two main classes, carboxylesterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) (Brockerhoff, 2012)
The most characteristic example of an industrially relevant esterolytic enzyme is that of the naproxen carboxylesterase from Bacillus subtilis, which is utilized for the biocatalytic synthesis of the non-steroidal drug naproxen (Quax and Broekhuizen, 1994)
After exposure to 50% acetonitrile, EstDZ3 was found capable of retaining about 60% of its maximal activity (Figure 3B). These results demonstrate that EstDZ3 is an esterolytic enzyme with remarkable kinetic thermostability and very good stability against prolonged exposure to high concentrations of organic solvents
Summary
Lipolytic enzymes (EC 3.1.1.x) catalyze the hydrolysis of ester bonds in lipids, and depending on their substrate preference, they are divided in two main classes, carboxylesterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) (Brockerhoff, 2012). In non-aqueous media, many of these enzymes are capable of performing the inverse reaction and catalyze the synthesis of ester bonds (Bornscheuer and Kazlauskas, 2006). These characteristics, complemented by their ability to modify a very broad range of substrates with high chemo-, regio-, and enantio-selectivity, render lipolytic enzymes a very attractive class of catalysts for conducting biotransformations (Bornscheuer, 2002). The most characteristic example of an industrially relevant esterolytic enzyme is that of the naproxen carboxylesterase from Bacillus subtilis, which is utilized for the biocatalytic synthesis of the non-steroidal drug naproxen (Quax and Broekhuizen, 1994)
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