Recombinant Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: Enzyme Expression and Design of a Reliable Experimental Procedure for the Stereoselective Hydration of Oleic Acid

Abstract

Different microbial strains are able to transform oleic acid (OA) into 10-hydroxystearic acid (10-HSA) by means of the catalytic activity of the enzymes oleate hydratase (EC 4.2.1.53). Lactobacillus rhamnosus ATCC 53103 performs this biotransformation with very high stereoselectivity, affording enantiopure (R)-10-HSA. In this work, we cloned, in Escherichia coli, the oleate hydratase present in the above-mentioned probiotic strain. Our study demonstrated that the obtained recombinant hydratase retains the catalytic properties of the Lactobacillus strain but that its activity was greatly affected by the expression procedure. According to our findings, we devised a reliable procedure for the hydration of oleic acid using a recombinant E. coli whole-cell catalyst. We established that the optimal reaction conditions were pH 6.6 at 28 °C in phosphate buffer, using glycerol and ethanol as co-solvents. According to our experimental protocol, the biocatalyst does not show significant substrate inhibition as the hydration reaction can be performed at high oleic acid concentration (up to 50 g/L).

Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: A FADH2-Dependent Enzyme with Remarkable Industrial Potential

“…To this end, we first ran a number of experiments using different formulations of the above-mentioned recombinant OLH (Table 1). As previously reported [26], a PBS buffer containing glycerol and ethanol effectively stabilize the enzyme. In these experimental conditions, the protein is highly stable and can be easily handled by the operator without observing any precipitation or denaturation, even after different freeze/thaw cycles.…”

“…Afterwards, a number of other microorganisms proved to be able to perform this transformation [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] but the enzymes responsible for the hydration step (oleate hydratases) have been characterized only recently [24]. Oleate hydratases (OLH, EC 4.2.1.53) convert oleic acid (OA) into (R)-10-hydroxystearic acid (10-HSA) [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39], a high value commercial molecule due to its potential application as surfactant, lubricant, additive in cosmetics industries, and as a starting material in polymer and flavor chemistry. In addition, this enzyme can catalyze the hydration of different UFAs [36,[40][41][42][43][44][45][46][47] with the same regio-and stereoselectivity, affording exclusively the corresponding 10-hydroxy derivatives.…”

“…Being involved in a research project aimed at the valorization of the vegetable oils waste, we studied the biocatalytic hydration of the latter fatty acids using both microbial transformations [21][22][23] and isolated hydratase [26]. In this context, we demonstrated the versatility of the probiotic bacteria Lactobacillus rhamnosus ATCC 53103 [22], which is able to hydrate oleic, linoleic, and linolenic acid affording (R)-10-hydroxystearic acid, (S)-(12Z)-10-hydroxy-octadecenoic acid, and (S)-(12Z,15Z)-10-hydroxy-octadecadienoic acid, respectively, in very high enantiomeric purity (ee > 95%), without the formation of other regioisomers or polyhydroxylated fatty acids.…”

“…The first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD) provided the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor [48]. Oleate hydratases (OLH, EC 4.2.1.53) convert oleic acid (OA) into (R)-10-hydroxystearic acid (10-HSA) [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39], a high value commercial molecule due to its potential application as surfactant, lubricant, additive in cosmetics industries, and as a starting material in polymer and flavor chemistry. In addition, this enzyme can catalyze the hydration of different UFAs [36,[40][41][42][43][44][45][46][47] with the same regio-and stereoselectivity, affording exclusively the corresponding 10-hydroxy derivatives.…”

“…Since the biocatalytic activity of this Lactobacillus strain is due to the expression of an oleate hydratase previously identified from the bacteria genome [41], we cloned and overexpressed the latter enzyme using Escherichia coli BL21(DE3) as a heterologous host [26]. Our study demonstrated that the obtained recombinant C-terminal His-tagged hydratase retains the catalytic properties of the Lactobacillus strain and we devised a reliable whole-cell-based procedure for the hydration of oleic acid [26]. Unfortunately, we also observed that the purified OLH is completely inactive and the catalytic activity can be restored only marginally through the addition of flavin adenine dinucleotide (FAD) to the reaction mixture.…”