EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

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.

Discovering novel hydrolases from hot environments

“…The combination of different environmental samples, different substrates and different incubation conditions resulted in a huge number of enrichment cultures. Detailed recipes of culture media utilized for enrich-ment have been described (Zarafeta et al, 2016b;Gavrilov et al, 2016;Kovaleva et al, 2015;Podosokorskaya et al, 2014). Although more than one thousand enrichments were set up, less than 10% of these survived three transfers.…”

“…For cloning into the pUC18 vector, DNA was subjected to partial digestion with Bsp143I and Hin1II and fragments of >2 kb were gel extracted and ligated with BamHI/SphI digested pUC18. The ligated DNA was transformed into the highly competent E. cloni ® 10G SUPREME cells (Lucigen) (Zarafeta et al, 2016b).…”

“…For typical expression library sizes (10 4 -10 5 clones), a commonly used and robust approach is to examine bacterial colonies on agar plates that contain a screening substrate (Zarafeta et al, 2016b;Popovic et al, 2017). The substrate is often a colourless compound that produces a colour reaction, either because a chromophore or fluorophore is released upon enzymatic hydrolysis, or because the enzyme product can be indirectly detected by means of a colour reaction.…”

“…As an example, esterases and lipases can be readily screened for on agar plates that contain emulsified triglycerides, tributyrin for esterase and triolein or olive oil for lipase activity. A clear halo can be observed in the turbid tributyrin medium around the esterase producing colonies, exemplified by the esterase EstDZ1 from a new Dictyoglomus isolate (Zarafeta et al, 2016b). Lipase activity screening can be aided by the addition of rhodamine B, a fluorescent dye that is presumed to form a complex with free fatty acids yielding bright orange fluorescence (Kouker and Jaeger, 1987).…”

“…Other new thermostable esterase enzymes have been identified by bioinformatics analysis and these have provided knowledge of the increased diversity within this enzyme class. A very thermostable esterase has been identified in a microbial community isolated from a Chinese hot spring (Zarafeta et al, 2016b). Genomic screening identified it to originate from a Dictyoglomus bacterium.…”