catalysts Article Recombinant Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: Enzyme Expression and Design of a Reliable Experimental Procedure for the Stereoselective Hydration of Oleic Acid Antonio Castagna 1, Davide De Simeis 1,*, Erica E. Ferrandi 2 , Stefano Marzorati 1 , 2 1, 1, Daniela Monti , Stefano Serra * and Mattia Valentino y 1 Istituto di Scienze e Tecnologie Chimiche, C.N.R., Via Mancinelli 7, 20131 Milano, Italy; [email protected] (A.C.); [email protected] (S.M.); [email protected] (M.V.) 2 Istituto di Scienze e Tecnologie Chimiche, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy; [email protected] (E.E.F.); [email protected] (D.M.) * Correspondence: [email protected] (D.D.S.); [email protected] (S.S.); Tel.: +39-02-2399-3076 (S.S.) The authors are listed in alphabetical order. y Received: 27 August 2020; Accepted: 24 September 2020; Published: 1 October 2020 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). Keywords: oleate hydratase; heterologous expression; probiotics; Lactobacillus rhamnosus; biotransformation; biocatalysis 1. Introduction The enzymatic addition of water to the double bond of free fatty acids was first described, in 1962, by Wallen et al. [1] in the course of a study on the ability of the Pseudomonas sp. strain 3266 to catalyze the conversion of oleic acid 1 (OA) into 10-hydroxystearic acid 4 (10-HSA) (Figure1). Catalysts 2020, 10, 1122; doi:10.3390/catal10101122 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 1122 2 of 16 Catalysts 2020, 10, x FOR PEER REVIEW 2 of 16 FigureFigure 1. The 1. The most most common common C C1818 unsaturatedunsaturated fatty acids: fatty oleic acids: (1), linoleic oleic ( (12),) and linoleic linolenic (2 )(3 and) acid linolenicand (3) acid andtheir their transformation transformation into (R into)-10-hydroxystearic (R)-10-hydroxystearic acid (4), (S acid)-(12Z ()-10-hydroxy-octadecenoic4), (S)-(12Z)-10-hydroxy-octadecenoic acid (5) acid (5)andand (S ()-(12S)-(12Z,15ZZ,15)-10-hydroxy-octadecadienoicZ)-10-hydroxy-octadecadienoic acid (6), acid respectively. (6), respectively. Reagents and Reagents conditions: and conditions:(a) (a) anaerobicanaerobic fermentation fermentation withwith LactobacillusLactobacillus rhamnosus rhamnosus ATCCATCC 53103. 53103. Afterward,Afterward, several several microorganisms microorganisms were were identifiedidentified as as possessing possessing this this particular particular capability capability [2– [2–7], 7], including different probiotics species [8]. This hydration reaction is catalyzed by the enzymes includingoleate di ffhydrataseerent probiotics (EC 4.2.1.53), species whose [ 8first]. This isolation hydration and characterization reaction is was catalyzed performed by by the Hager enzymes [9], oleate hydratasealmost (EC half 4.2.1.53), a century whoseafter the firstpublication isolation of Wall anden’s characterization research. Since the was enzyme performed identification, by Hager a [9], almostlarge half number a century of new after oleate the hydrat publicationases (OLHs) of Wallen’shave been research.discovered [10–32]. Since theThese enzyme recent studies, identification, a largebesides number describing of new the oleate new hydratasesenzymes’ structures, (OLHs) report haveed been on their discovered catalytic activity [10–32 either]. These in terms recent of studies, besidessubstrate describing specificity the new or the enzymes’ stereoselectivity structures, of the reportedhydration onreaction. their catalytic activity either in terms of The physiological function of this particular enzymatic class is still unclear. In fact, only a few substrate specificity or the stereoselectivity of the hydration reaction. studies have speculated on the protective role of these enzymes against potentially toxic unsaturated Thefatty physiological acids [10,20,33,34]. function of this particular enzymatic class is still unclear. In fact, only a few studies haveThe speculated most common on theunsaturated protective fatty role acids of (U theseFAs), enzymes shown in againstFigure 1, potentiallyare oleic (1), toxiclinoleic unsaturated (2) fatty acidsand linolenic [10,20,33 (3,)34 acid,]. and they are the main components (as triglycerides) of the vegetable oils used Theformost human common consumption. unsaturated fatty acids (UFAs), shown in Figure1, are oleic ( 1), linoleic (2) and linolenic (3From) acid, an and applicative they are point the of main view, componentshydroxy fatty acids (as triglycerides) (HFAs) are important of the compounds vegetable oilsused used for for many industrial applications, such as starting materials for biodegradable polymers, lubricants, human consumption. emulsifiers, drugs, cosmetic ingredients, and flavors [2,13,30,35–41]. From anFor applicativeexample, 12-hydroxystearic point of view, acid hydroxy (12-HSA), fatty as well acids as 10-HSA, (HFAs) is aremostly important used as a compoundsthickening used for manyagent industrial for the production applications, of lubricants such as and starting skincare materials preparations for biodegradable [35–39]; and ricinoleic polymers, acid lubricants,is emulsifiers,commonly drugs, used cosmetic as starting ingredients, material forand the producti flavorson [2 ,of13 surfactants,,30,35–41]. resins and polymers (nylon- For11) example, [40,41]. 12-hydroxystearic acid (12-HSA), as well as 10-HSA, is mostly used as a thickening agent for theMoreover, production HFAs of and lubricants some of andtheir skincarederivatives preparations also possess relevant [35–39]; biologic and ricinoleical activities. acid In isfact, commonly hydroxy fatty acids are plant self-defense substances [42] and are important in the control of allergy, used asinflammation starting material and immunity for the [43,44]. production Recently, of particular surfactants, esters resins of hydroxy and polymersfatty acids possessing (nylon-11) anti- [40,41]. Moreover,diabetic and HFAs anti-inflammatory and some of activities their derivatives were also identified also possess in bioactive relevant lipids biological (FAHFAs) activities. [45]. In fact, hydroxy fattyFrom acids a synthetic are plant point self-defense of view, a number substances of chemic [42al reactions] and are can important be used to inhydrate the control the easily of allergy, inflammationavailable andUFAs immunity 1–3. Unfortunately, [43,44]. these Recently, transformations particular la estersck stereo- of hydroxyand regioselectivity, fatty acids which possessing anti-diabeticafford complex and anti-inflammatory mixtures of isomers. activities In the simplest were alsocase, identifiedthe chemical in hydration bioactive of lipids oleic acid (FAHFAs) leads [45]. to a mixture of two racemic isomers, namely, 9-HSA and 10-HSA. Of course, the chemical hydration From a synthetic point of view, a number of chemical reactions can be used to hydrate the easily of polyunsaturated fatty acids leads to a much more complex mixtures of HFAs, including the availablepossibility UFAs 1 of–3 the. Unfortunately, formation of diols these and transformations triols. lack stereo- and regioselectivity, which afford complex mixturesOtherwise, of fatty isomers. acid hydratases In the simplestare often highly case, selective the chemical and are hydrationable to introduce of oleic the hydroxyl acid leads to a mixturegroup of two in a racemicspecific position isomers, of the namely, UFAs carbon 9-HSA chain. and Few 10-HSA. studies Of have course, been carried the chemical out about hydration the of polyunsaturatedfeasibility of fatty using acids these leadsenzymes to afrom much an applicative more complex point mixturesof view [21]. of HFAs, including the possibility of the formationWe have of previously diols and demonstrated triols. the versatility of the probiotic bacteria Lactobacillus rhamnosus ATCC 53103 [6], which is able to hydrate oleic, linoleic and linolenic acid with high stereoselectivity Otherwise, fatty acid hydratases are often highly selective and are able to introduce the hydroxyl group in a specific position of the UFAs carbon chain. Few studies have been carried out about the feasibility of using these enzymes from an applicative point of view [21]. We have previously demonstrated the versatility of the probiotic bacteria Lactobacillus rhamnosus ATCC 53103 [6], which is able to hydrate oleic, linoleic and linolenic acid with high stereoselectivity and in good yields. More specifically, the latter strain catalyzes the hydration