properties, structure, and applications of microbial sterol esterases

MINI-REVIEW

Properties, structure, and applications of microbial sterolesterases

Maria Eugenia Vaquero1 & Jorge Barriuso1 & María Jesús Martínez1 & Alicia Prieto1

Received: 11 November 2015 /Revised: 14 December 2015 /Accepted: 17 December 2015 /Published online: 7 January 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract According to their substrate preferences, carboxylicester hydrolases are organized in smaller clusters. Amongthem, sterol esterases (EC 3.1.1.13), also known as cholesterolesterases, act on fatty acid esters of cholesterol and other ste-rols in aqueous media, and are also able to catalyze synthesisby esterification or transesterification in the presence of organ-ic solvents. Mammalian cholesterol esterases are intracellularenzymes that have been extensively studied since they areessential in lipid metabolism and cholesterol absorption, andthe natural role of some microbial sterol esterases is supposedto be similar. However, besides these intracellular enzymes, anumber of microbes produce extracellular sterol esterases,which show broad stability, selectivity, or wide substrate spec-ificity, making them interesting for the industry. In spite ofthis, there is little information about microbial sterol esterases,and only a small amount of them have been characterized.Some of the most commercially exploited cholesterol ester-ases are produced by Pseudomonas species and by Candidarugosa, although in the last case they are usually describedand named as Bhigh substrate versatility lipases.^ From astructural point of view, most of them belong to the α/β-hy-drolase superfamily and have a conserved Bcatalytic triad^formed by His, an acidic amino acid and a Ser residue that islocated in a highly conserved GXSXG sequence. In this re-view, the information available on microbial sterol esteraseshas been gathered, taking into account their origin, production

and purification, heterologous expression, structure, stability,or substrate specificity, which are the main properties thatmake them attractive for different applications. Moreover, acomprehensive phylogenetic analysis on available sequencesof cholesterol esterases has been done, including putative se-quences deduced from public genomes.

Keywords Sterol esterase . Biocatalysts . Hydrophobicenzymes . Bacteria . Fungi

Introduction

Carboxylic ester hydrolases (EC 3.1.1) are a large class ofenzymes catalyzing the hydrolysis or synthesis of ester bonds.Their ecological and physiological relevance can be deducedfrom the fact that they have been described in all life domains,prokaryotic and eukaryotic (Levisson et al. 2009), as intra- orextracellular proteins. But besides this, many of them are ex-ceptionally robust catalysts able of acting under conditionsdrastically different from those of their natural environment,for example in the presence of organic solvents (Villeneuveet al. 2005). This is the reason why this group includes thebiocatalysts with the highest number of industrial applicationssuch as lipolytic enzymes, used in an array of sectors as oilsand fats, detergents, bakery, cheese, textile, leather and paper,etc. (Jaeger and Reetz 1998; Hasan et al. 2006). From a struc-tural point of view, most of them belong to the α/β-hydrolasesuperfamily and have a conserved Bcatalytic triad^ formed byHis, an acidic amino acid and a Ser residue that is located in ahighly conserved GXSXG sequence. During hydrolysis, thecatalytic Ser will start the nucleophilic attack of the substratehelped by the other two residues from the triad, which are inclose spatial vicinity. These are presumed to facilitate thehydrolysis of esters by a mechanism similar to that of

* María Jesús Martí[email protected]

* Alicia [email protected]

1 Centro de Investigaciones Biológicas, Consejo Superior deInvestigaciones Científicas, Madrid, Spain

Appl Microbiol Biotechnol (2016) 100:2047–2061DOI 10.1007/s00253-015-7258-x

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chymotrypsin-like serine proteases (Appel 1986). Anothercharacteristic feature is the presence of an amino acidic regionwhose sequence is not as conserved as that of the catalytictriad, the oxyanion hole, which serves to stabilize a transitionstate generated during catalysis. In addition, these enzymesgenerally do not require cofactors.

According to their substrate preferences, carboxylic esterhydrolases are organized in smaller clusters. Among them,sterol esterases (EC 3.1.1.13), also known as cholesterol es-terases, act on fatty acid esters of cholesterol and other sterolsin aqueous media, and are also able to catalyze their synthesisby esterification or transesterification in the presence of organ-ic solvents (Weber et al. 2001). As explained above, they arewidespread in nature and have been identified frommammals’tissues such as the pancreas, intestinal mucosa, liver, placenta,aorta, and brain (Brockerhoff and Jensen 1974; Mas andLombardo 1994), to filamentous fungi, yeast, and bacteria(Kaiser et al. 1994; Sugihara et al. 2002).

Mammalian cholesterol esterases have been extensivelystudied and their role is mainly related to lipid metabolismand cholesterol absorption (Rudd and Brockman 1984;Mukherjee 2003). They usually contain over 500 amino acidresidues, with a molecular mass >60 kDa, and the catalytic Serresidue is included in the GESAG sequence. Microbial sterolesterases are supposed to play a similar function as their mam-mals’ counterparts, and their natural role could be related tolipid metabolism and use of lipids as carbon sources. Forexample, one of the few reports on these enzymes demon-strates that three membrane-anchored lipases with sterol ester-ase activity from Saccharomyces cerevisiae are essential tomaintain the sterols homeostasis in vivo (Köffel et al. 2005).Similarly, after a large screening for sterol esterase and lipaseactivities in Aspergillus spp., the GRAS strains Aspergillusoryzae NRRL 6270 and Aspergillus sojae NRRL 6271showed to produce intracellular enzymes with homology tothose of S. cerevisiae, with a very similar signal-anchor motiffor type III membrane proteins (Töke et al. 2007).

Besides the intracellular sterol esterases, some microbessecrete these enzymes to the environment. This is the case ofseveral plant pathogens or saprophytes, in which the functionof these extracellular esterases is mostly related to degradationof target compounds from plant envelopes (Juniper and Jeffree1983). In general, the secreted enzymes show broad stability,selectivity, or wide substrate specificity, making them interest-ing for the industry (Jaeger and Reetz 1998). In spite of this,there is little information about microbial sterol esterases, andonly a small amount of them have been characterized. This isthe reason of the paucity of protein sequences and structuralinformation available for deducing general features beyondthose described for other carboxyl ester hydrolases. A listof extracellular microbial sterol esterases is summarizedin Table 1. Some of them have been isolated and char-acterized, although without structural details, and the known

structures correspond to sterol esterases from the prokaryotesBurkholderia glumae and Chromobacterium viscosum, theyeast Candida rugosa (three isoforms) (Grochulski et al.1993, 1994; Ghosh et al. 1995; Mancheño et al. 2003), andthe filamentous fungus Ophiostoma piceae (Gutiérrez-Fernández et al. 2014). Two common structural features ofthese proteins are also shared by lipases. Firstly, the existenceof a lid covering the active site whose displacement is promot-ed in the presence of a substrate or interface, and secondly,their tendency to form aggregates. This owes to their highlyhydrophobic character, which in turn is necessary for estab-lishing interactions with very hydrophobic substrates. Thisproperty can make difficult the purification and characteriza-tion of these proteins, and even induce to mistakes on theirmolecular mass assignation if measured under non-denaturingconditions, since the calculated value may correspond to aprotein aggregate.

In terms of substrate specificity, many sterol esterases areable to catalyze the hydrolysis or synthesis of a rather broadrange of other substrates containing ester linkages, such asacylglycerols, aryl esters (Gray et al. 1992; Svendsen et al.1995; Calero-Rueda et al. 2002b; Kontkanen et al. 2006c;Du et al. 2010), and in some cases alcohol esters, cinnamyl

Table 1 Sources of microbial sterol esterases

Organism Reference

Bacteria

Acinetobacter Du et al. (2010)

B. cepacia Takeda et al. (2006)

C. trachomatis Peters et al. (2012)

C. viscosum Kontkanen et al. (2004)

P. aeruginosa Sugihara et al. (2002)

P. fluorescens Uwajima and Terada (1976)

P. mendocina Svendsen et al. (1995)

P. pseudoalcaligenes Svendsen et al. (1995)

S. aureus Harvie (1977)

Streptomyces sp. Xiang et al. (2006)

S. avermitilis Xiang et al. (2007)

S. griseus Xiang et al. (2007)

S. lavendulae Kamei et al. (1979)

Fungi

C. rugosa Rúa et al. (1993)

F. oxysporum Okawa and Yamaguchi (1977)

M. albomyces Kontkanen et al. (2006c)

N. haematococca Vaquero et al. (2015b)

O. piceae Calero-Rueda et al. (2002b)

P. glomerata Pollero et al. (2001)

Trichoderma sp. Maeda et al. (2008)

T. reesei Vaquero et al. (2015b)

2048 Appl Microbiol Biotechnol (2016) 100:2047–2061

esters, xhantophyl esters (Zorn et al. 2005; Maeda et al. 2008),or synthetic polymers (Barba Cedillo et al. 2013b). In thiscontext, the difficult distinction between lipases and sterolesterases is a matter of controversy and, although it is some-thing merely formal, is sometimes confusing even for the sci-entists involved in this field. As will be demonstrated acrossthis paper, most enzymes with sterol esterase activity are ver-satile catalysts able to act on different substrates includinginsoluble, long-chain fatty acid acylglicerols, which are thestandard substrates described for lipases. Here, we will dealwith enzymes with described sterol esterase activity, regard-less of their categorization as sterol esterase or lipase in theoriginal publication or by the provider company, in the case ofcommercial catalysts. Many enzymes initially described aslipases for their ability to hydrolyze/synthesize triglyceridescan also act on sterol esters, as shown in further works inwhich these compounds were assayed as substrates.Probably, these two catalysts categories comprise a continuumof enzymes whose substrate specificity range betweenthose of true lipases, with activity only against insolubleacylglycerols, and true sterol esterases, specific for sterol es-ters. This broad substrate specificity makes them attractivefrom an industrial and biotechnological perspective.

In this review, the information available on microbial sterolesterases has been gathered, taking into account their origin,production and purification, heterologous expression, struc-ture, stability, or substrate specificity, which are the mainproperties that make them attractive for different applications.Moreover, a comprehensive phylogenetic analysis on avail-able sequences of cholesterol esterases has been done, includ-ing putative sequences deduced from public genomes (Fig. 1).

Biotechnological relevance of microbial sterolesterases

As mentioned above, microbial sterol esterases are very inter-esting catalysts for several biotechnological applications. Theuse of cholesterol esterase as diagnostic reagent for measuringcholesterol in human blood serum (Allain et al. 1974)pioneered the industrial use of this kind of enzymes and, morerecently, non-clinical applications have also been postulated.For example, their use in the paper pulp industry was pro-posed for eliminating or decreasing lipidic accumulations(denominated pitch) in pulps and process waters (Calero-Rueda et al. 2002a; Kontkanen et al. 2006c). Wood extrac-tives, composed of lipophilic wood resin components (triglyc-erides, sterol esters, resinic acids, free fatty acids, and sterols),are known to produce problems during paper pulp productionand reduce paper quality (Gutiérrez et al. 2001). Commerciallipases, such as Resinase (Novozymes, Denmark), have beenused to efficiently hydrolyze the triglycerides of pulp, increas-ing operation stability and paper strength (Hata et al. 1996;

Gutiérrez et al. 2009). However, this enzyme preparation lackssterol esterase activity and does not act on wood sterol esters(Mustranta et al. 2001), yielding a paper of poor quality andphysical characteristics from woods rich in these compounds(Gutiérrez et al. 2001; Kokkonen et al. 2002, 2004).Nevertheless, the use of crudes containing the sterolesterase/lipase secreted by O. piceae showed suitable resultsat laboratory scale for degradation of triglycerides and sterolesters present in hardwood and softwood pulps, and was pat-ented to reduce pitch problems during papermaking process(Calero-Rueda et al. 2002b). Similarly, mixtures of residualsubstances from waxes and adhesives (mainly formed by es-ters), known as stickies, are deposited in recycled paper pulps,constituting a serious concern for these industries. The crudeenzyme preparation from O. piceae demonstrated its hydro-lytic effect on polyvinyl acetate, one of the compounds char-acteristic of these deposits, and its application for treatment ofrecycled pulps and process waters was also proposed to palli-ate stickies problems (Barba Cedillo et al. 2013b).

Microbial sterol esterases can be used as clean catalysts forthe synthesis of different kinds of sterol esters of industrialrelevance, in cosmetics (Panitch 1997), or as additives in func-tional foods. This is the case of the fatty acid esters of phytos-terols or phytostanols, currently commercialized asnutraceuticals because of their ability to reduce blood choles-terol levels (Plat and Mensink 2005), being more soluble,stable, and effective than the corresponding non-esterified ste-rols and then generally preferred in the food industry (Weberet al. 2002; Villeneuve et al. 2005; Cantrill and Kawamura2008). Several patents and research papers report the enzymat-ic synthesis of phytosterol/stanol esters (Weber et al. 2002;Negishi et al. 2003; Norinobu et al. 2003; Basheer and Plat2004; Villeneuve et al. 2005; Seo et al. 2006; Töke et al. 2007;Maeda et al. 2008; Søe and Jørgensen 2010; Barba Cedilloet al. 2013a; Vaquero et al. 2015b) although at the present timethe industrial production of these compounds is still per-formed by chemical procedures due to the high cost of thebiocatalysts. Besides, lipases and esterases are used in laundryproducts or detergents (Sugihara et al. 2002; Masaki et al.2003) and were studied for surface modification of polyestersused in the textile industry (Yoon et al. 2002; Gubitz andPaulo 2003; Vertommen et al. 2005). Polyester (polyethyleneterephthalate, PET) is a synthetic polymer that confers favor-able characteristics to textile products. However, it has severalundesirable properties: high tendency to pill, high glossiness,it is difficult to dye, and resistant to removal of oil and greasestains (Abo et al. 1999; Yoon et al. 2002). Sterol esteraseshave been studied for the surface modification of polyestersto improve these characteristics (Kontkanen et al. 2006b).

Some of the most commercially exploited cholesterol es-terases are produced by Pseudomonas species and byC. rugosa, although in the last case they are usually describedand named as Bhigh substrate versatility lipases^ (Mancheño

Appl Microbiol Biotechnol (2016) 100:2047–2061 2049

et al. 2003). Several patents protect the production of certaincholesterol esterases, demonstrating their interest. All thesedata have been summarized in Tables 1 and 2. Usually, heter-ologous expression of these enzymes for obtaining high

amounts of the biocatalyst at low cost is mandatory, and forspecific applications such as in the food industry, the recom-binant proteins must be produced in hosts BGenerallyRecognized as Safe^ (GRAS).

Fig. 1 Phylogenetic analysis of different families of sterol esterases. Thetree was built with MEGA6 software, using MUSCLE for alignment andMaximum-Likelihood for clustering. The name of each species ispreceded by the sequence accession number. All sequences have a

signal peptide prediction (SignalP 4.0), except the lysosomal, plant andArchaea enzymes. The enzymes that have been expressed andcharacterized are underlined


Bacterial sterol esterases

The first extracellular cholesterol esterase purified and charac-terized was obtained from the bacterium Pseudomonasfluorescens (Uwajima and Terada 1975). Later, the distributionof cholesterol esterase activity in about 500 microbial strainsbelonging to the kingdoms Bacteria and Fungi was examined(Uwajima and Terada 1976). The study revealed that cholester-ol esterase was produced by some Pseudomonas, Bacillus, andStreptomyces strains. One of the P. fluorescens isolates resultedto be the most efficient, and enzyme production in this strainwas later patented because of its high productivity and extra-cellular excretion (Table 2). Shortly thereafter, Harvie (1977)reported the finding of a protein with cholesterol esterase activ-ity from a Staphylococcus aureus strain. Although the informa-tion available for the later is limited, it was described as aprotein with a molecular mass of 25.5 kDa with tendency toaggregate under native conditions. The enzyme was labile, los-ing the activity easily, and hence its potential biotechnological

application is limited. In subsequent years and up to now, a fewextracellular bacterial sterol esterases have been reported, andamong them the number of fully characterized proteins issmall. Nevertheless, in the following sections we will go overthe information recorded in the literature.

Cholesterol esterases from the genus Pseudomonas

Asmentioned above, Uwajima and Terada (1975) reported forthe first time on an enzyme with hydrolytic activity on cho-lesterol esters, secreted by P. fluorescens KY395. It was char-acterized in native conditions as a 129-kDa protein with amarked preference for long chain fatty acid cholesterol esters,very stable at 55 °C, active in a wide range of pH (from 5 to12), and displaying also lipolytic activity. Later on, these au-thors described two cholesterol esterase isoenzymes (I and II)from the strain ATCC 21156 of the same microorganism(Uwajima and Terada 1976), showing different isoelectricpoint and Km value but the same molecular mass, pH stability,

Table 2 Microbial enzymespatented or commercialized assterol esterases

Patents and microorganism Applicant Purpose

US4011138A P. fluorescens Kyowa Hakko Kirin 1975 Production

US4052263 Nocardia cholesterolicum Eastman Kodak Company 1977 ProductionMasurekar and Goodhue 1977

US4343903A List of microorganisms Boehringer Mannheim Gmbh 1982 ProductionBeaucamp et al. 1982a

US 4360596 A Pseudomonas sp. Boehringer Mannheim Gmbh 1982 ProductionBeaucamp et al. 1982b

WO1993010224A1 P. cepacia Novo Nordisk 1993 ProductionBarfoed 1993

For specific purposes

WO 1994023052 A1 Pseudomonas fragi Novo Nordisk 1994 HydrolysisBarfoed 1994

EP0968268 A1Pseudomonas sp. The Procter & Gamble company Laundry products

Vijayarani et al. 1998 Pitch reductionWO2000053843A1 List of microorganisms Buchert et al. 2000

WO 2002075045 B1 O. piceae Calero-Rueda et al. 2002b Pitch reduction

WO 2003066792 A1 Menicon Co. 2003 Lens careMasaki et al. 2003

PCT/ ES 2395582 B1 Barba Cedillo et al. 2013a Sterol esters synthesis

Commercial cholesterol esterases Supplier

C. rugosa Roche

Sigma-Aldrich

Pseudomonas sp. Asahi Kasei

MPBio

Sigma-Aldrich

TOYOBO

P. fluorescens Sigma-Aldrich (formerly Amano)

Boehringer Mannheim

Schizophyllum commune TOYOBO


and preference for long-chain fatty acid esters of cholesterol asthe enzyme from strain KY395. They were thermally stable upto 60 °C and were activated by the surfactant Triton X-100.This detergent reduced their sedimentation coefficient, proba-bly as a result of better solubilization. Both isoenzymes weremore active on unsaturated fatty acid esters and the best sub-strate among the assayed was cholesteryl linoleate. They alsoproved to be absolutely specific against cholesterol esters,since they did not show lipase, lipoprotein lipase, phospholi-pase, or aliesterase activities.

After these initial findings, cholesterol esterase activity hasbeen detected in commercial samples of the lipases fromPseudomonas mendocina (Lumafast, Genecor) (Gray et al.1992), Pseudomonas cepacia (Novo Nordisk), andPseudomonas pseudoalcalígenes (Svendsen et al. 1995).Similarly, P. mendocina 3121 secretes a 30-kDa enzyme withcholesterol esterase activity (Marcinkeviciene et al. 1994) thatis probably the same protein described later by Surinenaiteet al. (2002) as a lipase, with the same molecular mass andsimilar properties, although whose activity against sterolesters was not assayed.

Finally, Sugihara et al. (2002) described a cholesterol es-terase from a strain of P. aeruginosa, with molecular massaround 53 kDa and an isoelectric point of 3.2. It tends to formaggregates in the culture filtrate. Its thermal and pH stabilitywas in the same range as those from P. fluorescens and, sim-ilarly to it, was inhibited by phenylmetanesulfonyl fluoride(PMSF), an inhibitor of serine hydrolases, and preferredlong-chain fatty acid sterol esters as substrates. Nevertheless,the enzyme from P. aeruginosa also hydrolyzed triglyceridesof different length, cleaving the sn-2 ester bond faster than thesn-1,3, and was activated by bile salts instead of by TritonX-100 as in P. fluorescens.

Sterol esterases from the genus Burkholderia

As can be observed in Fig. 1, the sequences of the sterolesterases from this genus are phylogenetically related toPseudomonas enzymes. This is explained from the close sim-ilarity of both genera: Burkholderia was created in 2001(Coenye et al. 2001) and all species currently included thereinwere formerly classified as Pseudomonas. These changes af-fecting the taxonomical classification of the producer speciesmay be misleading when enzymes from different genera arecompared, since some of them appear in the literature withtheir old names. This is the case of the lipases with sterolesterase activity secreted by Pseudomonas (synonymBurkholderia) glumae and P. (synonym Burkholderia)cepacia. In this review, we will refer to them by their currentnames.

Noble et al. (1993) reported the first three-dimensionalstructure of a bacterial lipase. The protein, from B. glumae,was crystallized in its closed conformation revealing a

particular subset of the α/β-hydrolase fold, with a disulfidebond, the catalytic triad (Ser87, Asp263, and His285),and a Ca2+ binding-site in the largest of its three do-mains. Interestingly, few years later, a protein from the bac-teriumChromobacterium viscosum, with 100% identical ami-no acid sequence, was also crystallized (Lang et al. 1996),showing a similar conformation and some differences, amongwhich the most relevant is the presence of a oxyanion hole thatseems to be absent in the B. glumae enzyme. The Ca2+ bindingsite is well described in this work, demonstrating that the ion,essential for enzyme activity, is six-coordinated, contactingwith four oxygen atoms from four residues and two watermolecules. This Ca2+ site was too far from the active site,but could play an important stabilizing role. The presence ofthis ion is not a common feature of the structure of cholesterolesterases/lipases. It has only been described in thePseudomonas family (Noble et al. 1993; Lang et al. 1996;Köffel et al. 2005) and in the human and pancreatic lipase(Hermoso et al. 1996), although the presence of these metalions in Staphylococcus hyicus lipase has been suggested(Simons et al. 1999). It should be also mentioned that anenzymatic cocktail commercialized as C. viscosum lipase, ini-tially produced by Asahi and now distributed by MerckMillipore, shows sterol esterase activity (Kontkanenet al. 2004). As observed in Fig. 1, the lipase alreadydescribed in this species, which is likely responsible forthis activity in the commercial crude, clusters among theγ-proteobacteria group of cholesterol esterases.

A similar enzyme produced by B. cepacia (Svendsen et al.1995) is commercialized by NovoNordisk (Barfoed 1994). Itsstructure in the open conformation was simultaneously pub-lished by Schrag and Cygler (1993) that analyzed the proteinprovided by Genzyme without further purification, and Kimet al. (1997) that purified it from the crude sold by Amano. Intheir respective reports, they confirmed the structural and se-quence similarity (84 % identity) of this enzyme with thosefrom B. glumae and C. viscosum. The molecular basis of itsenantioselectivity for some substrates was later analyzed,based on its crystal structure (Mezzetti et al. 2005). Then,several records deposited in the Protein Data Bank illustratethe structure of this protein (PDB: 3LIP, 1OIL, 2LIP, 1YS1,1YS2). In addition, the studies by Pleiss et al. (1998) de-scribed a funnel-like shape for the substrate binding site ofthe B. cepacia enzyme.

The first protein from Burkholderia described as a choles-terol esterase was purified and partially characterized fromB. cepacia ST200 (Takeda et al. 2006). The lipase activity ofthis enzyme was not tested, but it also had aryl esterase activ-ity and showed great stability to temperature (4–65 °C), pH(5.5–12), and organic solvents. Its molecular mass was around37 kDa (SDS-PAGE), but aggregated in aqueous solution.The analysis of its amino acidic sequence showed high iden-tity (87 %) with the well-studied lipase produced by other


strains of B. cepacia and, on the basis of this homology, wehave elaborated a 3D model for the cholesterol esterase ofB. cepacia ST200 (Fig. 2a, b), whose structure has not yetbeen reported. A Qmean of 0.94, very close to the highestvalue, confirms the fidelity of our model. According to it,the amino acids involved in the catalytic machinery of thisenzyme, Ser87, Asp264, and His286, and the residues of theoxyanion hole, Gln88 and Leu17, would be located exactly inthe same positions as in the lipase template (Fig. 2a). In addi-tion, the model is compatible with the presence of a six-coordinated Ca2+.

Cholesterol esterase from Acinetobacter

Du et al. (2010) screened the production of cholesterol esteraseactivities by bacteria from environmental samples. The strainCHE4-1 from Acinetobacter sp., isolated from Pantherapardus feces, showed the highest activity growing in the pres-ence of cholesteryl oleate as the only carbon source. The puri-fied enzyme, of 6.5 kDa molecular mass, is monomeric. Itsactivity was enhanced in the presence of Ca2+, Zn2+, and boricacid, and it was sensitive to Ag and Hg salts, SDS, and DTT.Nevertheless, PMSF, which inhibits serine-hydrolases, did not

Fig. 2 Stereo view of the general structures of bacterial and fungalcholesterol esterases. a, b Homology model for the molecular structureofB. cepacia ST-200 cholesterol esterase. P. cepacia lipase (PDB: 1YS1),which showed 84 % sequence identity with the CHE of B. cepacia ST-200, was used as template. The lid is colored in light blue. aRepresentation of the α/β hydrolase fold and the most relevantresidues: the catalytic triad in red, the oxyanion hole in yellow, theresidues of the calcium-binding site in cyan, the calcium ion as a pink

sphere. b Surface of the protein with the substrate-binding site occupiedby the inhibitor hexylphosphonic acid (R)-2-methyl-3-phenylpropylester. c, d Crystal structure of C. rugosa Lip3 (C. cylindraceacholesterol esterase, PDB:1CLE). The lid is colored in pink. cRepresentation of the α/β hydrolase fold and the most relevant residuesin the same color code that in a. d Surface of the protein with the substrate(cholesteryl linoleate) inside the catalytic site, where the substrate ispartially visible


affect its activity. These characteristics are markedly differentfrom those of other microbial sterol esterases suggesting thatthe catalytic machinery of this cholesterol esterase could bedifferent. The enzyme was stable in a pH range from 5.5 to8, showing the optimal activity at pH 7, had an optimum tem-perature of 42 °C, and rapidly lost activity at 60 °C. Although itprefers long chain unsaturated fatty acid esters, it has highactivity to both long- and short-chain cholesterol esters, andan outstanding activity on cholesteryl acetate.

Chlamydia trachomatis

Chlamydia is a genus of intracellular pathogens of eukaryotichost cells. They are auxotrophic for a variety of essential me-tabolites and obtain cholesterol and fatty acids from their host.Not many lipid metabolism enzymes have been identified inthis genus. Strain CT149 of Chlamydia trachomatis secretes aprotein that exhibits esterase activity in vitro and, whenexpressed in HeLa cells, hydrolyzes cholesteryl linoleate(Peters et al. 2012). The enzyme was annotated as a conservedputative hydrolase (CT149) but, after in silico analysis of itsamino acidic sequence, we have identified two lipase/esteraseGXSXG motifs, and a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence. The ami-no acidic sequence of this protein differs from that of otherbacterial sterol esterases, grouping independently in the phy-logenetic tree (Fig. 1).

Sterol esterases from Streptomyces

The first cholesterol esterase activity from the actinomyceteStreptomyces lavendulae H-646-SY2 was reported by Kameiet al. (1977). The same authors designed a new purificationmethod based on affinity to palmitoyl cellulose, describing theisolation a 12-kDa protein with lipolytic activity (Kamei et al.1979). This enzyme was sequenced and characterized, show-ing to have a molecular mass of 22 kDa under denaturingconditions, similar to that deduced from its nucleotide se-quence (Nishimura and Sugiyama 1994). More recently, cho-lesterol esterase activity was detected in several species of thisgenus, leading to the isolation, characterization, and sequenc-ing of three novel enzymes from Streptomyces sp. X9,Streptomyces avermitilis JCM5070, and Streptomyces griseusIFO13350 (Xiang et al. 2006, 2007). The molecular masses ofthe purified proteins were 23.6, 19.7, and 25.7 kDa, respec-tively, although under native conditions the measured valueswere 163, 120, and 153 kDa, corroborating their oligomeriza-tion tendency. The addition of Triton X-100 increased theiractivity, probably because this detergent promotes protein sol-ubilization as has also been reported by Vaquero et al. (2015a,b). The three enzymes displayed a wide range of substratespecificity, hydrolyzing cholesterol esters of different chainlength, with preference for cholesteryl linoleate, p-nitrophenyl

esters, triglycerides, and phospholipids. It is noteworthy thatreducing agents strongly reduced their activity, which can berelated to the presence of two conserved Cys residues thatcould form a disulphide bridge, stabilizing the tertiary struc-ture. Nevertheless, they were not affected by PMSF as inAcinetobacter sp. CHE4-1 (Xiang et al. 2006; Du et al.2010). The absence of the GXSXG conserved sequence inthe sterol esterases of actinomycetes suggests a unique cata-lytic mechanism and indicates that they are distantly related tothe known lipases/esterases. Moreover, the lack of a con-served His in the catalytic region points to the presence of acatalytic dyad instead of the usual triad and, according to thesedata, these enzymes should be classified in a different groupamong bacterial sterol esterases (Xiang et al. 2007). Thisagrees with the clustering of this group of enzymes presentedin Fig. 1 that shows the resemblance of the esterases fromStreptomyces and their low sequence homology with otherbacterial and eukaryotic cholesterol esterases.

Fungal sterol esterases

Different fungal species are producers of cholesterol esterases,mostly belonging to the C. rugosa-like lipase family, andsome of them have been characterized and proposed for bio-technological applications. This protein family shows com-mon structural features and frequently presents activity on awide variety of substrates including triglycerides and aryl orsterol esters (Barriuso et al. 2013). The structures already re-solved within enzymes of this family are limited to the iso-forms 1, 2, and 3 of C. rugosa lipase (PDBs: 1CRL, 1CLE,1GZ7, 1LLF, 1LPP, 1LPO, 1LPM, 1LPN, 1TRH, 1LPS) andthe O. piceae sterol esterase (PDBs: 4BE4, 4BE9 and 4UPD).Structurally, its members possess the α/β hydrolase fold, ingeneral with 11-stranded mixed β-sheets and 16 α-helices.The catalytic machinery includes the residues of the catalytictriad (Ser, His, and Glu) and the oxyanion hole. The nucleo-philic serine is located in a very sharp turn (Fig. 2c), the nu-cleophilic elbow, composed of the conserved residues GESAGas cholesterol esterases from mammalians, while the sterolesterases from the yeast S. cerevisiae and the bacteriumP. aeruginosa contain GFSQG and GHS(H/Q)G sequences,respectively (Pleiss et al. 2000; Köffel et al. 2005). These en-zymes are also characterized for having a substrate-binding sitelocated in a long (25–30 Å) internal tunnel (Fig. 2d) formed byaromatic and aliphatic residues, configuring a highly hydro-phobic area along this region (Ghosh et al. 1995; Pleiss et al.1998; Mancheño et al. 2003; Gutiérrez-Fernández et al. 2014).The access to the active site is covered by an amphipathic α-helix that serves as a lid, fixed by a disulfide bond. The phy-logenetic tree in Fig. 1 shows that the sequences of C. rugosa-like proteins group together and with the known fungal se-quences, forming an independent family.


Small amounts of sterol esterase activity have alsobeen reported in crude enzymes from Rhizopus oryzae(Kontkanen et al. 2004) and in Lipase B from Geotrichumcandidum (Charton and Macrae 1992).

Sterol esterases from C. rugosa

C. rugosa is a non-sporogenic, imperfect hemiascomycete thatsecretes a variety of closely related enzymes. The genes forfive isoenzymes (lip1 to lip5) were identified and sequencedand exhibit high identity (77–88 %), encoding 534 aminoacid-proteins with molecular masses around 60 kDa that differin their predicted isoelectric point and degree of glycosylation(Lotti et al. 1994). The isoenzymes Lip1-Lip4 have been pu-rified, characterized (Kaiser et al. 1994; Rúa et al. 1998;Pernas et al. 2000; Tang et al. 2001), and expressed in variousheterologous systems, showing that Pichia pastoris was thebest host (Lee et al. 2002, 2007; Chang et al. 2006a, b; Ferreret al. 2009). As already mentioned, the crystal structures ofseveral C. rugosa isoforms have been reported: Lip1, both inits open (Grochulski et al. 1993) and closed conformations(Grochulski et al. 1994), the closed form of Lip2 (Mancheñoet al. 2003), and Lip3 complexed with cholesteryl linoleate ina dimeric arrangement (Ghosh et al. 1995). The presence ofdimers in solution has only been reported for Lip3 (Pernaset al. 2000, 2009), although higher aggregates have been de-tected in Lip2 (Ferrer et al. 2009). The comparative analysisof these structures revealed that the main differences con-cerned their hydrophobic profiles in the flap andsubstrate-binding pocket regions, which could contributeto their different specificity towards triglycerides andcholesterol esters. The isoenzymes Lip1–Lip4 display activityagainst cholesterol esters at different extent, but Lip2 presentsthe highest cholesterol esterase activity (Tenkanen et al. 2002;Mancheño et al. 2003; Shaw et al. 2009). Revising the resultsfrom different authors, the catalytic activity against sterol es-ters of the C. rugosa isoenzymes can be ordered asLip2>Lip4≈Lip3>Lip1. According to this, a high hydropho-bic content in these two regions favors the catalysis of sterolesters (Mancheño et al. 2003). In addition, all of them hydro-lyze triacylglycerides and p-nitrophenyl esters (López et al.2004; Lee et al. 2007). This promiscuity has also been report-ed for the sterol esterases produced by O. piceae (Calero-Rueda et al. 2002b) and Melanocarpus albomyces(Kontkanen et al. 2006c), and for lipases with similar charac-teristics from Fusarium solani (anamorph of Nectriahaematococca) and Trichoderma reesei (Vaquero et al.2015b), whose characteristics will be explained in subsequentsections.

Commercial cocktails of C. rugosa contain different pro-portions of the isoforms secreted by this yeast. The three iso-enzymes usually detected are Lip1, Lip2, and Lip3, althoughLip1 and Lip3 are the main activities (López et al. 2004).

Nevertheless, altered isoenzyme profiles have been obtainedby growing C. rugosa on different substrates or usingderegulated mutants derived from the producer wild-typestrain (Ferrer et al. 2001).

Sterol esterase of M. albomyces

The strain VTT D-96490 of this thermophilic fungus, isolatedfrom composting soil samples, secretes a very hydrophobicsterol esterase (Kontkanen et al. 2006c) with a molecular massof 64 kDa estimated by SDS-PAGE. However, gel filtrationchromatography analysis showed a single peak around238 kDa, suggesting that it is tetrameric in solution. Its iso-electric point was 4.5 and the glycosylation degree is about5 %. As other sterol esterases of the C. rugosa-like family, thisenzyme has broad substrate specificity and it is also activeagainst olive oil and aryl esters. With lipase substrates, theenzyme worked better at neutral pH, while for sterol estersthe activity was optimal at values around 5–5.5, which arelower than those reported for other fungal sterol esterases,usually in the range 6–8 (Okawa and Yamaguchi 1977;Calero-Rueda et al. 2002b). Trying to improve the enzymeyields of the natural producer, it was cloned and expressedin two eukaryotic heterologous hosts (Kontkanen et al.2006a). Its expression in P. pastoris GS115 (AOX promoter)generated extremely low extracellular sterol esterase activity,even lower than the achieved for the native enzyme, probablybecause it was not completely secreted. T. reeseii was used asalternative host (cellobiohydrolase promoter) producing sim-ilar yields than M. albomyces, but the activity was bound tothe mycelium or exists as aggregates. The high content ofhydrophobic amino acid residues (41.1 %) of this esterasemight propitiate its aggregation, and Triton X-100 was neces-sary to recover the enzyme. The production of the recombi-nant form in T. reeseii was further optimized in a laboratorybioreactor, and the properties of the pure protein analyzed(Kontkanen et al. 2006b). The enzyme was similar to thenative one but seemed to be dimeric, taking into accountits molecular mass after gel filtration chromatography. Itwas less stable under different conditions, showingslightly lower activities on some of the assayed sub-strates. The amino acid sequence deduced from the ste1 genehad high identity with C. rugosa-like lipase isoforms(Kontkanen et al. 2006a) and clusters with them in the phylo-genetic tree depicted in Fig. 1.

The effect of the recombinant enzyme from T. reseei on theproperties of paper sheets and PET fabric was assayed, findingthat the strength and hydrophilicity of the paper increased dueto the hydrolysis of triglycerides and sterol esters. The polarityand dyeing capacity of PET also increased significantly byhydrolyzing the ester bonds in the polyester backbone(Kontkanen et al. 2006b).


Sterol esterase of O. piceae

The ascomycete O. piceae is a wood saprophyte that causesthe Bsap staining^ and produces important losses in the forest-ry industry. The sterol esterase secreted by strain CECT 20416of this dimorphic fungus was purified in a single step byhydrophobic interaction chromatography. It was described asa glycoprotein with molecular mass around 56.5 kDa, 8 % N-linked carbohydrates (calculated after enzymatic deglycosyl-ation), and pI of 3.3, which formed multi-aggregates in nativeconditions (Calero-Rueda et al. 2002b). Its biochemical char-acterization proved that this enzyme hydrolyzed sterol and p-nitrophenol esters as well as triglycerides of different fattyacid chain-length, and then it was classified as a broad sub-strate specificity sterol esterase. The protein maintained 50 %activity after 24 h at acidic pH levels, but its stability was notgood over pH 8 or 60 °C (Calero-Rueda et al. 2002b). Thesequencing and molecular characterization of this enzyme andits comparison with related proteins indicated around 40 %identity with C. rugosa lipase isoforms (Calero-Rueda et al.2009). Its catalytic efficiency showed to be much better thanthose from several commercial lipases and sterol esterases,either fungal or bacterial, on all the substrates assayed(Table 3). In view of its good catalytic properties, the sterolesterase was expressed in different heterologous hosts. Thehighest yields were achieved in P. pastoris that secreted a75-kDa protein with 28 % of N-linked carbohydrates andhigher stability at basic pH than the native form (BarbaCedillo et al. 2012). The recombinant enzyme showed im-proved catalytic efficiency when compared to the native pro-tein. The reason for this was attributed to the strategy used forprotein expression, since the recombinant form incorporated6–8 extra amino acids in its N-terminus, which affected itsaggregation behavior. As a result, a more soluble proteinwas secreted, which was corroborated from the finding thatonly monomers and dimers were detected by analytical ultra-centrifugation (Barba Cedillo et al. 2012). Recently, the

O. piceae sterol esterase was produced in low yields in theGRAS yeast S. cerevisiae. This recombinant enzyme had anintermediate aggregation state between the protein expressedin P. pastoris and the native one, and similar catalytic efficien-cy (Vaquero et al. 2015a).

The resolution of the molecular structure of the deglyco-sylated O. piceae enzyme produced in P. pastoris, in both itsopen and closed conformations, gave very interesting infor-mation (Gutiérrez-Fernández et al. 2014). As described forother sterol esterases and lipases, in the closed form the am-phiphilic lid limits the access to the active site. Displacementof this element from its original position allows the entry ofsubstrates to the active site. They are housed in a large andstraight tunnel-shaped substrate’s pocket rich in hydrophobicamino acids, ending in an area close to the surface, whichcould facilitate the hydrolysis of a great variety of substrates.The open form of OPE is organized as a functional homodi-mer, leaving a large cavity between the two monomers.Analytical ultracentrifugation experiments showed that thetransition from monomer to dimer occurred in the presenceof a substrate or inhibitor (Gutiérrez-Fernández et al. 2014).The active form of the Lip3 from C. rugosa is organized as atight homodimer with small cavities entering into the activesites, whereas the dimer of the recombinant O. piceae sterolesterase shows a packman-like structure with a very largeopening. Considering these features, this enzyme could allowboth the entrance of large substrates and the quick release ofthe reaction products, explaining its higher efficiency on tri-glycerides and cholesterol esters when compared under thesame reaction conditions with commercial sterol esterases(Table 3) (Calero-Rueda et al. 2009) or with other enzymesfrom the C. rugosa-like family (Vaquero et al. 2015b). Theefficiency of crude preparations of this enzyme has been eval-uated in applications of biotechnological interest. Hydrolysisand synthesis reactions were performed at low temperaturesand in the absence of detergents or cofactors, comparing theresults with those obtained for other sterol esterases. In

Table 3 Comparison of thespecific activities (U/mg protein)on aryl esters, cholesterol esters,and triglycerides of commercialenzymes and the non-commercialsterol esterase/lipase of O. piceae

Organism Activity pNPB pNPP TB TO CB CO CE

A. oryzae Lip 0.6 1.3 74.9 18.3 0.1 0.0 0.1

C. antarctica Lip 2.2 0.0 22.3 0.2 0.2 0.2 0.2

R. miehei Lip 0.5 3.3 54.7 1.4 0.3 0.2 0.2

C. rugosa Che 0.0 0.0 126.0 40.4 0.4 10.1 0.4

P. fluorescens Che 0.0 0.0 132.0 31.0 8.1 7.2 9.2

Pseudomonas sp. Che 72.1 7.6 116.2 74.4 1.5 26.1 3.2

O. piceae N.C. 23.0 29.0 133.0 78.3 17.0 53.4 15.3

Adapted from Calero-Rueda et al. (2009). Reactions were carried out using 1 mM substrate in the presence ofGenapol X-100 (1 % for p-nitrophenol esters and 5 % for the rest of substrates). The abbreviations Lip and Checorrespond to enzymes commercialized as lipases and cholesterol esterases, respectively

N.C. non-commercial, pNPB p-nitrophenyl butyrate, pNPP p-nitrophenyl palmitate, TB tributyrin, TO triolein,CB cholesteryl butirate, CO cholesteryl oleate, CE cholesteryl estearate


general, the recombinant O. piceae enzyme produced inP. pastoris gave the best results under the assayed conditions,appearing as a very promising catalyst for these applications(Calero-Rueda et al. 2004; Barba Cedillo et al. 2013a, b;Vaquero et al. 2015b).

Sterol esterases from Fusarium

A search for long chain fatty acid sterol ester hydrolases insoil microorganisms led to the isolation of five Fusariumstrains, which released high activity levels to the culture me-dium (Okawa and Yamaguchi 1977). Specifically, one ofthem, identified as Fusarium oxysporum, gave the best activ-ity yield. Cholesterol esterase was induced with several typesof oils, and although lipase substrates were not assayed, itseems feasible that these enzymes present also lipase activity.Gel filtration chromatography of the pure enzyme gave a peakthat eluted with the void volume, indicating that the proteinaggregated in solution. However, no estimate was made of themolecular mass of the active protein in this study. The esteraseremained active in the pH range 4–10 and at 50 °C, losing theactivity at 60 °C. Detergents as Triton-X100 and Adekatol(nonionic surfactant) markedly activated the enzyme, andalso did sodium deoxycholate and sodium cholate althoughto a lesser extent. In terms of substrate specificity, the enzymehydrolyzed cholesterol linoleate and oleate faster than othersubstrates of smaller fatty acid chain.

Madhosingh and Orr (1981) described in this fungus twointracellular cholesterol esterases and one extracellular. Thelater showed a molecular mass of 75 kDa and the same sub-strate specificity and activation by Triton X-100 as the enzymedescribed above. Moreover, the pH range for optimal hydro-lase activities ranged from 4.3 to 8.6. Since no information isavailable regarding the sequence or structure of these en-zymes, we cannot ascertain if they belong to the C. rugosa-like family or not, despite they fulfill several of the character-istic properties of this family.

The sequence of a hypothetical cholesterol esterase/lipasefrom Nectria haematoccoca (teleomorph of Fusarium solani)was recently identified by in silico genome mining (Barriusoet al. 2013). This protein showed 55 and 61 % sequence iden-tity with the sterol esterases fromO. piceae andM. albomyces,respectively; hence, it was classified into the C. rugosa-likefamily (abH03.01) (Fig. 1). After being cloned, this enzymewas produced in P. pastoris, and its biochemical and catalyticproperties studied (Vaquero et al. 2015b). The purifiedenzyme has a molecular mass of 65 kDa, calculatedby PAGE-SDS, and 6.5 % N-linked carbohydrate content.Analytic ultracentrifugation studies and gel filtration chroma-tography revealed the presence of monomers, dimers, and big-ger multimeric species. The protein was stable in a pH range of4–11, preserving around 50 % activity at pH 11 after 72 h.Temperature stability in the range 30–60 °C was measured

during 24 h, and the T50 value (temperature in which the en-zyme maintained 50 % residual activity) was 46 °C. The sterolesterase from N. haematoccoca hydrolyzed triglycerides andp-nitrophenol and cholesterol esters, showing broad substratespecificity, as other enzymes of the family C. rugosa-like.Nevertheless, this enzyme was not the best when comparedwith other hypothetical enzymes expressed in P. pastoris andwith the sterol esterase from O. piceae (Vaquero et al. 2015b).

Sterol esterases from Trichoderma sp.

The strain AS59 from Trichoderma sp. was also selected froma screening of soil samples, aimed to find microorganisms thatproduced sterol esterases (Maeda et al. 2008). The purifiedextracellular cholesterol esterase had pI of 4.3 and its molec-ular mass measured by MALDI-TOF was 58 kDa, a valuevery similar to that detected by chromatography, indicatingthat aggregated protein forms are not present. After 18 h in-cubation, the enzyme maintained 75 % residual activity in thepH range 4–8, and the T50 value was 40 °C. The enzyme wasable to hydrolyze cholesteryl and aryl esters, and triglycerides,showing preference for the short-chain fatty acid substrates(highest activity for cholesteryl butyrate) and for the positionsn-1,3 of glycerol. Then, its specificity differs from those ofother cholesterol esterases that usually hydrolyze betterlonger-chain esters such as cholesteryl linoleate. In addition,the enzyme was activated by high concentrations of bile salts(40 mM) and was inhibited by PMSF, suggesting its serinehydrolase character. As mentioned before, there is not avail-able information on the sequence or structure of this esterase,and therefore it is impossible to determine whether it belongsto the C. rugosa-like family.

Finally, a hypothetic sterol esterase from T. reesei was alsoidentified as a result of in silico genome mining (Barriusoet al. 2013). Its sequence analysis showed 56 and 59 % iden-tity with O. piceae andM. albomyces sterol esterases, respec-tively. After analyzing the presence of conserved structuralmotifs, it was ascribed to the C. rugosa-like family althoughit lacked the conserved N-glycosylation site characteristic ofmembers of this family. The enzyme was cloned, produced inP. pastoris, purified, and characterized as a glycoprotein witha molecular mass around 63 kDa (SDS-PAGE) and 10 % N-linked carbohydrates (Vaquero et al. 2015b). Sedimentationvelocity experiments confirmed the coexistence of monomersand aggregated forms with higher sedimentation coefficient.The enzyme retained more than 60 % residual activity at pH4–9 after 72 h and had T50 value of 42 °C after 24 h incuba-tion, measured in the interval between 30 and 60 °C. Thesterol esterase from T. reesei hydrolyzes p-nitrophenyl esters,triglycerides, and cholesterol esters. It showed similar efficien-cy than O. piceae esterase on esters of short chain fatty acidsalthough it was less efficient towards long chain fatty acidsesters (Vaquero et al. 2015b).


Sterol esterase from Phoma glomerata

The ascomycete P. glomerata is a ubiquitous conidial fungus(Domsch et al. 1993). Extracellular lipolytic activity was ini-tially detected in its culture supernatants (Pollero et al. 1997),and the enzyme responsible for this activity was purified,showing a molecular mass around 75 kDa (Pollero et al.2001). Nevertheless, this sterol esterase was not further stud-ied and, as in other cases, the lack of information precludesmaking sequence or phylogenetic analysis to ascertain its po-tential relationship with the C. rugosa-like family.

Conclusions

In spite of the importance of microbial sterol esterases as bio-technological tools and at industrial scale, there is little infor-mation about the properties and characteristics of many ofthese biocatalysts. Intracellular cholesterol esterases areindispensible enzymes for the lipolytic metabolism of livingorganisms but, at first glance, this activity is not frequentlyfound extracellularly, as deduced from the results of studiestackling the screening of a wide amount of microbial isolates.Many of these enzymes are extremely versatile, combiningseveral esterase activities, although displaying preferencesfor some types of substrates, and these characteristics, togetherwith their stability, make them attractive for biotechnologicaland industrial applications. Some cholesterol esterases, suchas those produced by Pseudomonas species, Schizophyllumcommune, and C. rugosa are already commercialized.

As a general rule, the characteristics of microbial sterolesterases fit within the features of serine hydrolases. Theyare very hydrophobic proteins and tend to aggregate in watersolution. Apart from these characters, shared by all sterol es-terases, those produced by different bacteria hardly have any-thing else in common. A diversity of individual traits can befound among them, as the peculiar catalytic mechanism sug-gested for Acinetobacter esterase, the catalytic dyad of en-zymes from actinomycetes, the Ca2+ requirement for the pro-teins produced by some Pseudomonas and Burkholderia iso-lates, or the strict cholesterol esterase character of the enzymefrom P. fluorescens ATCC 21156. The reported molecularmasses for the monomeric forms of bacterial sterol esterasesare quite variable, ranging from around 50 to 6.5 kDa, andthey are usually smaller than those of their fungal counter-parts. In contrast, the information on true or hypothetical yeastand fungal sterol esterases suggest that they form a quite ho-mogeneous group, which allows their inclusion into theC. rugosa-like family. These enzymes do not require cofactorsand show wide substrate specificity, although their catalyticefficiency is diverse. Some of them are known to be glyco-proteins with variable amount of N-glycidic chains and mo-lecular mass around 60 kDa. Mixtures of isoenzymes from

C. rugosa are commercially available. However, the sterolesterase from O. piceae is more versatile and shows the bestcatalytic performance when compared with commercial li-pases and sterol esterases in laboratory tests in both hydrolysisand synthesis reactions.

A deeper knowledge of the sequence and structure of thistype of enzymes could permit the development of structure-function studies and rational design approaches, aimed to im-prove or orient enzymatic activity towards a specific goal.Currently, the outcomes from genome sequencing andmetagenomic analysis provide a growing amount of geneand protein sequences. In this context, genome mining of hy-pothetical enzymes and sequence-based functional predictionsfor a specific activity could be a way to follow in the imme-diate future. Nevertheless, to ascertain the verisimilitude ofthese predictions, a true protein must always be expressedand purified to test its real activity.

Acknowledgments This work was funded by the Spanish projectsBIO2012-36372, RTC-2014-1777-3 and S2013/MAE-2907. M.E.Vaquero gratefully acknowledges an FPU fellowship from MINECO. J.Barriuso thanks the financial support from the JAE-DOC CSIC program.

Compliance with ethical standards

Conflict of interest The authors declare that they have not competinginterests.

Ethical statement This article does not contain any studies with ani-mals performed by any of the authors.

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http://dx.doi.org/10.1007/s00253-015-6890-9

properties, structure, and applications of microbial sterol esterases

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