Cloning and Characterization of GDSL Esterases from Bacillus paralicheniformis T7

TL;DR

This study identifies and characterizes two GDSL esterases from a soil bacterium, showing their potential for use in sustainable technologies that degrade fats.

Contribution

The paper reports the cloning and characterization of two novel GDSL esterases from Bacillus paralicheniformis T7.

Findings

The recombinant esterases rEST-24 and rEST-28 showed maximum activity at 40 °C and pH 7.0.

rEST-28 was resistant to detergents like Tween-20, Tween-80, and Triton X-100.

Submerged fermentation on feather medium produced an esterase extract with 17,618 ± 610 U/g activity.

Abstract

Various contemporary biotechnological and industrial procedures rely on enzymes that effectively degrade lipids and associated substances. However, enzymes that maintain activity across diverse conditions remain scarce. Microorganisms are a key source of these enzymes; however, numerous bacterial enzymes remain insufficiently studied. In this study, we focused on the identification and characterization of novel enzymes derived from bacteria isolated from natural environments. We specifically examined a soil bacterium, Bacillus paralicheniformis T7, which synthesizes enzymes known as esterases. Two esterases produced by this bacterium were identified, isolated, and comprehensively examined. The study findings indicated that B. paralicheniformis T7 represents a promising biological source of enzymes for sustainable technologies that depend on environmentally benign degradation of fat-based materials.

Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca2+, Zn2+, Cu2+, and Fe2+ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes.

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