Research Article: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past

Date Published: July 25, 2017

Publisher: Public Library of Science

Author(s): Judit Ribera, Mónica Estupiñán, Alba Fuentes, Amanda Fillat, Josefina Martínez, Pilar Diaz, Monika Oberer.

http://doi.org/10.1371/journal.pone.0181029

Abstract

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

Partial Text

Lipolytic enzymes (EC 3.1.1.-), widely distributed in nature, are a diverse group of hydrolases catalysing the cleavage or formation of ester bonds [1,2]. Grouped under the general term of lipases, lipolytic enzymes include “true” lipases (EC 3.1.1.3, triacylglycerol hydrolases) and esterases (EC 3.1.1.1, carboxyl ester hydrolases), which differ in both, their kinetics and chain-length substrate preferences [1,2]. Esterases are active on short-chain length esters partially soluble in water, and lipases have optimal activity towards long-chain triacylglycerides, not soluble in aqueous environments [2]. Therefore, esterases display a typical Michaelis-Menten behaviour, whereas many lipases show interfacial activation when lipids reach an equilibrium between the monomeric, micellar and emulsified states [2]. Although the physiological role of many microbial lipases remains still unclear, in bacteria most esterases are intracellular enzymes involved in lipid metabolism and turnover [3,4]. On the contrary, most bacterial true lipases are secreted enzymes and perform their role extracellularly, probably by procuring nutrients to the cell or by executing synthesis reactions aimed at releasing modified lipid compounds that could act as extracellular cell communication signals, or have a role in detoxification of biocides [3–6].

We provide here evidence that the newly isolated strain Bacillus sp. JR3 from the Laurisilva Forest of El Hierro Island displays a complex thermophilic and thermostable extracellular lipolytic system, with great potential for industrial applications requiring biocatalysts adapted to harsh temperature conditions. PCR prospection of JR3 genome using degenerated consensus primers for thermophilic lipases allowed cloning and characterization of the new esterase LipJ. The cloned enzyme displays a mesophilic profile, showing preference for short chain-length substrates, with a kinetics profile typical of an esterase. Although being a member of the bacterial lipase family I.5 and bearing several traits of thermophilic lipases, identified in the amino acid sequence and the 3D structure model, LipJ is indeed a mesophilic enzyme. The mixture of thermophilic plus mesophilic features shown by LipJ allows to hypothesise that this enzyme could have evolved from a thermophilic lipase after adaptation to the present mild environment.

 

Source:

http://doi.org/10.1371/journal.pone.0181029

 

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