Abstract
The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (k cat/K m = 737±77 s−1 mM−1). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.
Highlights
Carboxylesterases (EC 3.1.1.1) and lipases are important industrial enzymes with numerous applications in biotechnology
Enzymes from psychrophilic microorganisms have attracted much attention because of their high catalytic activity at low temperatures and their inherently broad substrate specificity relative to their thermophilic counterparts [28]. These properties allow the use of cold-active lipases and carboxylesterases in numerous applications of the detergent and dairy industries, in addition to the organic synthesis of fragile pharmaceutical compounds [28]
A great deal of effort has been devoted to the discovery and characterization of new lipolytic enzymes from various psychrophilic microorganisms [29,30,31,32,33,34,35,36]
Summary
Carboxylesterases (EC 3.1.1.1) and lipases (triacylglycerol hydrolases; EC 3.1.1.3) are important industrial enzymes with numerous applications in biotechnology (reviewed in [1]). Esterases hydrolyze water-soluble or emulsified esters with relatively short fatty acid chains (,than 10 carbons) while ‘‘true’’ lipases are generally more active toward emulsified, long-chain fatty acids [2,3] These enzymes, which catalyze reactions in a regio- and/or enantioselective fashion [4], can catalyze the release of free fatty acids from triglycerides and under certain conditions can perform esterification and transesterification reactions. These biocatalysts are generally stable in organic solvents, allowing their use in numerous industrial applications [5]. Bacterial genome mining of versatile microorganisms offers an attractive opportunity to uncover new lipolytic biocatalysts displaying interesting biochemical properties
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