Bacillus subtilis Lipase A—Lipase or Esterase?

Abstract

The question of how to distinguish between lipases and esterases is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic solvents as a criterion is probed on a minimal α/β hydrolase fold enzyme, the Bacillus subtilis lipase A (BSLA), and compared to Candida antarctica lipase B (CALB), a proven lipase. Both hydrolases show activity in dry solvents and this proves BSLA to be a lipase. Overall, this demonstrates the value of this additional parameter to distinguish between lipases and esterases. Lipases tend to be active in dry organic solvents, while esterases are not active under these circumstances.Catalysts 2020, 10, 308 2 of 17 lipase A (BSLA) [9]. BSLA is a small (181 amino acids, 19 kDa) serine hydrolase ( Figure 1). It is neither interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not water-miscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low a w [1,3].Catalysts 2019, 9, x FOR PEER REVIEW 2 of 17 interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not watermiscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low aw [1,3].

Recent Trends in Biomaterials for Immobilization of Lipases for Application in Non-Conventional Media

“…Later, Ali et al [19] suggested classifying carboxylic ester hydrolases differently, dividing them into lipolytic esterases (EC 3.1 L) and non-lipolytic esterases (EC 3.1 NL). More recently still, Bracco et al [20] suggested classifying lipases and esterases based on their ability to act in organic solvents that are immiscible in water (e.g., toluene), with low water activity (a w ). In this review, we use the definition whereby lipases hydrolyze triacylglycerols or acyl esters with acyl chains of 10 carbons or more.…”

Revisiting the scope and applications of food enzymes from extremophiles

“…Esterase break ester bonds of shorter chain fatty acids, while lipase display a much broader substrate range, including long‐chain fatty acids (Delgado‐García et al., 2018). Lipases are capable of hydrolyzing insoluble or aggregated substrates, while esterases are highly effective toward soluble substrates (Bracco et al., 2020; Reyes‐Duarte, Coscolín, Martínez‐Martínez, Ferrer, & García‐Arellano, 2018). Because both enzymes have similar substrate specificity, they can be expressed from the same microorganism.…”

“…Because both enzymes have similar substrate specificity, they can be expressed from the same microorganism. Majority of lipases and esterases share the same α/β hydrolase fold and are sometimes difficult to differentiate by primary sequence comparisons, structural features, and kinetic parameters (Bracco et al., 2020; Vaquero, de Eugenio, Martínez, & Barriuso, 2015). For instance, Candida antarctica lipase B (Cal B) acts as a lipase when adsorbed to an acylglyceride interface and as an esterase when exposed to an aqueous environment (Benson & Pleiss, 2017).…”

Biocatalytic Approach for Direct Esterification of Ibuprofen with Sorbitol in Biphasic Media

“…However, biphasic systems that contain large amounts of water allow adequate hydration of the biocatalyst. Recently, Bracco et al have shown that a w is a suitable parameter to distinguish between lipases and esterases [ 58 ]. Although some lipases (such as those derived from CALB and R. arrhizus ) maintain good activity at low water content, other lipases require higher water activity levels to show good catalytic activity—e.g., lipase from P. cepacian [ 59 ].…”