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

MINI-REVIEW

Properties, structure, and applications of microbial sterol

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 preferences, carboxylic and purification, heterologous expression, structure, stability, ester are organized in smaller clusters. Among or substrate specificity, which are the main properties that them, sterol esterases (EC 3.1.1.13), also known as cholesterol make them attractive for different applications. Moreover, a esterases, act on fatty acid esters of cholesterol and other ste- comprehensive phylogenetic analysis on available sequences rols in aqueous media, and are also able to catalyze synthesis of cholesterol esterases has been done, including putative se- by esterification or transesterification in the presence of organ- quences deduced from public genomes. ic solvents. Mammalian cholesterol esterases are intracellular that have been extensively studied since they are Keywords Sterol . Biocatalysts . Hydrophobic essential in lipid metabolism and cholesterol absorption, and enzymes . Bacteria . Fungi the natural role of some microbial sterol esterases is supposed to be similar. However, besides these intracellular enzymes, a number of microbes produce extracellular sterol esterases, Introduction which show broad stability, selectivity, or wide substrate spec- ificity, making them interesting for the industry. In spite of Carboxylic ester hydrolases (EC 3.1.1) are a large class of this, there is little information about microbial sterol esterases, enzymes catalyzing the hydrolysis or synthesis of ester bonds. and only a small amount of them have been characterized. Their ecological and physiological relevance can be deduced Some of the most commercially exploited cholesterol ester- from the fact that they have been described in all life domains, ases are produced by Pseudomonas species and by Candida prokaryotic and eukaryotic (Levisson et al. 2009), as intra- or rugosa, although in the last case they are usually described extracellular proteins. But besides this, many of them are ex- and named as Bhigh substrate versatility .^ From a ceptionally robust catalysts able of acting under conditions structural point of view, most of them belong to the α/β-hy- drastically different from those of their natural environment, drolase superfamily and have a conserved Bcatalytic triad^ for example in the presence of organic solvents (Villeneuve formed by His, an acidic amino acid and a Ser residue that is et al. 2005). This is the reason why this group includes the located in a highly conserved GXSXG sequence. In this re- biocatalysts with the highest number of industrial applications view, the information available on microbial sterol esterases such as lipolytic enzymes, used in an array of sectors as oils has been gathered, taking into account their origin, production and 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- * María Jesús Martínez superfamily and have a conserved Bcatalytic triad^ formed by [email protected] His, an acidic amino acid and a Ser residue that is located in a * Alicia Prieto highly conserved GXSXG sequence. During hydrolysis, the [email protected] catalytic Ser will start the nucleophilic attack of the substrate helped by the other two residues from the triad, which are in 1 Centro de Investigaciones Biológicas, Consejo Superior de close spatial vicinity. These are presumed to facilitate the Investigaciones Científicas, Madrid, Spain hydrolysis of esters by a mechanism similar to that of 2048 Appl Microbiol Biotechnol (2016) 100:2047–2061 chymotrypsin-like serine proteases (Appel 1986). Another Table 1 Sources of microbial sterol esterases characteristic feature is the presence of an amino acidic region Organism Reference whose sequence is not as conserved as that of the , the , which serves to stabilize a transition Bacteria state generated during . In addition, these enzymes Acinetobacter Du et al. (2010) generally do not require cofactors. B. cepacia Takeda et al. (2006) According to their substrate preferences, carboxylic ester C. trachomatis Peters et al. (2012) hydrolases are organized in smaller clusters. Among them, C. viscosum Kontkanen et al. (2004) sterol esterases (EC 3.1.1.13), also known as cholesterol es- P. aeruginosa Sugihara et al. (2002) terases, act on fatty acid esters of cholesterol and other sterols P. fluorescens Uwajima and Terada (1976) in aqueous media, and are also able to catalyze their synthesis P. mendocina Svendsen et al. (1995) by esterification or transesterification in the presence of organ- P. pseudoalcaligenes Svendsen et al. (1995) ic solvents (Weber et al. 2001). As explained above, they are S. aureus Harvie (1977) widespread in nature and have been identified from mammals’ Streptomyces sp. Xiang et al. (2006) tissues such as the pancreas, intestinal mucosa, liver, placenta, S. avermitilis Xiang et al. (2007) aorta, and brain (Brockerhoff and Jensen 1974;Masand S. griseus Xiang et al. (2007) Lombardo 1994), to filamentous fungi, yeast, and bacteria S. lavendulae Kamei et al. (1979) (Kaiser et al. 1994;Sugiharaetal.2002). Fungi Mammalian cholesterol esterases have been extensively C. rugosa Rúa et al. (1993) studied and their role is mainly related to lipid metabolism F. oxysporum Okawa and Yamaguchi (1977) and cholesterol absorption (Rudd and Brockman 1984; M. albomyces Kontkanen et al. (2006c) Mukherjee 2003). They usually contain over 500 amino acid N. haematococca Vaquero et al. (2015b) residues, with a molecular mass >60 kDa, and the catalytic Ser O. piceae Calero-Rueda et al. (2002b) residue is included in the GESAG sequence. Microbial sterol P. glomerata Pollero et al. (2001) esterases are supposed to play a similar function as their mam- Trichoderma sp. Maeda et al. (2008) mals’ counterparts, and their natural role could be related to T. reesei Vaquero et al. (2015b) lipid metabolism and use of lipids as carbon sources. For example, 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 to maintain the sterols homeostasis in vivo (Köffel et al. 2005). structures correspond to sterol esterases from the prokaryotes Similarly, after a large screening for sterol esterase and Burkholderia glumae and Chromobacterium viscosum, the activities in Aspergillus spp., the GRAS strains Aspergillus yeast Candida rugosa (three isoforms) (Grochulski et al. oryzae NRRL 6270 and Aspergillus sojae NRRL 6271 1993, 1994; Ghosh et al. 1995;Mancheñoetal.2003), and showed to produce intracellular enzymes with homology to the filamentous fungus Ophiostoma piceae (Gutiérrez- those of S. cerevisiae, with a very similar signal-anchor motif Fernández et al. 2014 ). Two common structural features of for type III membrane proteins (Töke et al. 2007). these proteins are also shared by lipases. Firstly, the existence Besides the intracellular sterol esterases, some microbes of a lid covering the whose displacement is promot- secrete these enzymes to the environment. This is the case of ed in the presence of a substrate or interface, and secondly, several plant pathogens or saprophytes, in which the function their tendency to form aggregates. This owes to their highly of these extracellular esterases is mostly related to degradation hydrophobic character, which in turn is necessary for estab- of target compounds from plant envelopes (Juniper and Jeffree lishing interactions with very hydrophobic substrates. This 1983). In general, the secreted enzymes show broad stability, property can make difficult the purification and characteriza- selectivity, or wide substrate specificity, making them interest- tion of these proteins, and even induce to mistakes on their ing for the industry (Jaeger and Reetz 1998). In spite of this, molecular mass assignation if measured under non-denaturing there is little information about microbial sterol esterases, and conditions, since the calculated value may correspond to a only a small amount of them have been characterized. This is protein aggregate. the reason of the paucity of protein sequences and structural In terms of substrate specificity, many sterol esterases are information available for deducing general features beyond able to catalyze the hydrolysis or synthesis of a rather broad those described for other carboxyl ester hydrolases. A list range of other substrates containing ester linkages, such as of extracellular microbial sterol esterases is summarized acylglycerols, aryl esters (Gray et al. 1992; Svendsen et al. in Table 1. Some of them have been isolated and char- 1995; Calero-Rueda et al. 2002b; Kontkanen et al. 2006c; acterized, although without structural details, and the known Du et al. 2010), and in some cases alcohol esters, cinnamyl Appl Microbiol Biotechnol (2016) 100:2047–2061 2049 esters, xhantophyl esters (Zorn et al. 2005; Maeda et al. 2008), Gutiérrez et al. 2009). However, this preparation lacks or synthetic polymers (Barba Cedillo et al. 2013b). In this sterol esterase activity and does not act on wood sterol esters context, the difficult distinction between lipases and sterol (Mustranta et al. 2001), yielding a paper of poor quality and esterases is a matter of controversy and, although it is some- physical characteristics from woods rich in these compounds thing merely formal, is sometimes confusing even for the sci- (Gutiérrez et al. 2001; Kokkonen et al. 2002, 2004). entists involved in this field. As will be demonstrated across Nevertheless, the use of crudes containing the sterol this paper, most enzymes with sterol esterase activity are ver- esterase/lipase secreted by O. piceae showed suitable results satile catalysts able to act on different substrates including at laboratory scale for degradation of triglycerides and sterol insoluble, long-chain fatty acid acylglicerols, which are the esters present in hardwood and softwood pulps, and was pat- standard substrates described for lipases. Here, we will deal ented to reduce pitch problems during papermaking process with enzymes with described sterol esterase activity, regard- (Calero-Rueda et al. 2002b). Similarly, mixtures of residual less of their categorization as sterol esterase or lipase in the substances from waxes and adhesives (mainly formed by es- original publication or by the provider company, in the case of ters), known as stickies, are deposited in recycled paper pulps, commercial catalysts. Many enzymes initially described as constituting a serious concern for these industries. The crude lipases for their ability to hydrolyze/synthesize triglycerides enzyme preparation from O. piceae demonstrated its hydro- can also act on sterol esters, as shown in further works in lytic effect on polyvinyl acetate, one of the compounds char- which these compounds were assayed as substrates. acteristic of these deposits, and its application for treatment of Probably, these two catalysts categories comprise a continuum recycled pulps and process waters was also proposed to palli- of enzymes whose substrate specificity range between ate stickies problems (Barba Cedillo et al. 2013b). those of true lipases, with activity only against insoluble Microbial sterol esterases can be used as clean catalysts for acylglycerols, and true sterol esterases, specific for sterol es- the synthesis of different kinds of sterol esters of industrial ters. This broad substrate specificity makes them attractive relevance, in cosmetics (Panitch 1997), or as additives in func- from an industrial and biotechnological perspective. tional foods. This is the case of the fatty acid esters of phytos- In this review, the information available on microbial sterol terols or phytostanols, currently commercialized as esterases has been gathered, taking into account their origin, nutraceuticals because of their ability to reduce blood choles- production and purification, heterologous expression, struc- terol levels (Plat and Mensink 2005), being more soluble, ture, stability, or substrate specificity, which are the main stable, and effective than the corresponding non-esterified ste- properties that make them attractive for different applications. rols and then generally preferred in the food industry (Weber Moreover, a comprehensive phylogenetic analysis on avail- et al. 2002; Villeneuve et al. 2005; Cantrill and Kawamura able sequences of cholesterol esterases has been done, includ- 2008). Several patents and research papers report the enzymat- ing putative sequences deduced from public genomes (Fig. 1). ic synthesis of phytosterol/stanol esters (Weber et al. 2002; Negishi et al. 2003; Norinobu et al. 2003; Basheer and Plat 2004; Villeneuve et al. 2005; Seo et al. 2006;Tökeetal.2007; Biotechnological relevance of microbial sterol Maeda et al. 2008; Søe and Jørgensen 2010; Barba Cedillo esterases et al. 2013a; Vaquero et al. 2015b) although at the present time the industrial production of these compounds is still per- As mentioned above, microbial sterol esterases are very inter- formed by chemical procedures due to the high cost of the esting catalysts for several biotechnological applications. The biocatalysts. Besides, lipases and esterases are used in laundry use of cholesterol esterase as diagnostic reagent for measuring products or detergents (Sugihara et al. 2002; Masaki et al. cholesterol in human blood serum (Allain et al. 1974) 2003) and were studied for surface modification of polyesters pioneered the industrial use of this kind of enzymes and, more used in the textile industry (Yoon et al. 2002; Gubitz and recently, non-clinical applications have also been postulated. Paulo 2003; Vertommen et al. 2005). Polyester (polyethylene For example, their use in the paper pulp industry was pro- terephthalate, PET) is a synthetic polymer that confers favor- posed for eliminating or decreasing lipidic accumulations able characteristics to textile products. However, it has several (denominated pitch) in pulps and process waters (Calero- undesirable properties: high tendency to pill, high glossiness, Rueda et al. 2002a; Kontkanen et al. 2006c). Wood extrac- it is difficult to dye, and resistant to removal of oil and grease tives, composed of lipophilic wood resin components (triglyc- stains (Abo et al. 1999; Yoon et al. 2002). Sterol esterases erides, sterol esters, resinic acids, free fatty acids, and sterols), have been studied for the surface modification of polyesters are known to produce problems during paper pulp production to improve these characteristics (Kontkanen et al. 2006b). and reduce paper quality (Gutiérrez et al. 2001). Commercial Some of the most commercially exploited cholesterol es- lipases, such as Resinase (Novozymes, Denmark), have been terases are produced by Pseudomonas species and by used to efficiently hydrolyze the triglycerides of pulp, increas- C. rugosa, although in the last case they are usually described ing operation stability and paper strength (Hata et al. 1996; and named as Bhigh substrate versatility lipases^ (Mancheño 2050 Appl Microbiol Biotechnol (2016) 100:2047–2061

Fig. 1 Phylogenetic analysis of different families of sterol esterases. The signal peptide prediction (SignalP 4.0), except the lysosomal, plant and tree was built with MEGA6 software, using MUSCLE for alignment and Archaea enzymes. The enzymes that have been expressed and Maximum-Likelihood for clustering. The name of each species is characterized are underlined preceded by the sequence accession number. All sequences have a et al. 2003). Several patents protect the production of certain amounts of the biocatalyst at low cost is mandatory, and for cholesterol esterases, demonstrating their interest. All these specific applications such as in the food industry, the recom- data have been summarized in Tables 1 and 2.Usually,heter- binant proteins must be produced in hosts BGenerally ologous expression of these enzymes for obtaining high Recognized as Safe^ (GRAS). Appl Microbiol Biotechnol (2016) 100:2047–2061 2051

Table 2 Microbial enzymes patented or commercialized as Patents and microorganism Applicant Purpose sterol esterases US4011138A P. fluorescens Kyowa Hakko Kirin 1975 Production US4052263 Nocardia cholesterolicum Eastman Kodak Company 1977 Production Masurekar and Goodhue 1977 US4343903A List of microorganisms Boehringer Mannheim Gmbh 1982 Production Beaucamp et al. 1982a US 4360596 A Pseudomonas sp. Boehringer Mannheim Gmbh 1982 Production Beaucamp et al. 1982b WO1993010224A1 P. cepacia Novo Nordisk 1993 Production Barfoed 1993

For specific purposes WO 1994023052 A1 Pseudomonas fragi Novo Nordisk 1994 Hydrolysis Barfoed 1994 EP0968268 A1Pseudomonas sp. The Procter & Gamble company Laundry products Vijayarani et al. 1998 Pitch reduction WO2000053843A1 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 care Masaki 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

Bacterial sterol esterases application is limited. In subsequent years and up to now, a few extracellular bacterial sterol esterases have been reported, and The first extracellular cholesterol esterase purified and charac- among them the number of fully characterized proteins is terized was obtained from the bacterium Pseudomonas small. Nevertheless, in the following sections we will go over fluorescens (Uwajima and Terada 1975). Later, the distribution the information recorded in the literature. of cholesterol esterase activity in about 500 microbial strains belonging to the kingdoms Bacteria and Fungi was examined Cholesterol esterases from the genus Pseudomonas (Uwajima and Terada 1976). The study revealed that cholester- ol esterase was produced by some Pseudomonas, Bacillus,and As mentioned above, Uwajima and Terada (1975)reportedfor Streptomyces strains. One of the P. fluorescens isolates resulted the first time on an enzyme with hydrolytic activity on cho- to be the most efficient, and enzyme production in this strain lesterol esters, secreted by P. fluorescens KY395. It was char- was later patented because of its high productivity and extra- acterized in native conditions as a 129-kDa protein with a cellular excretion (Table 2). Shortly thereafter, Harvie (1977) marked preference for long chain fatty acid cholesterol esters, reported the finding of a protein with cholesterol esterase activ- very stable at 55 °C, active in a wide range of pH (from 5 to ity from a Staphylococcus aureus strain. Although the informa- 12), and displaying also lipolytic activity. Later on, these au- tion available for the later is limited, it was described as a thors described two cholesterol esterase isoenzymes (I and II) protein with a molecular mass of 25.5 kDa with tendency to from the strain ATCC 21156 of the same microorganism aggregate under native conditions.Theenzymewaslabile,los- (Uwajima and Terada 1976), showing different isoelectric ing the activity easily, and hence its potential biotechnological point and Km value but the same molecular mass, pH stability, 2052 Appl Microbiol Biotechnol (2016) 100:2047–2061 and preference for long-chain fatty acid esters of cholesterol as particular subset of the α/β-hydrolase fold, with a disulfide the enzyme from strain KY395. They were thermally stable up bond, the catalytic triad (Ser87, Asp263, and His285), to 60 °C and were activated by the surfactant Triton X-100. and a Ca2+ binding-site in the largest of its three do- This detergent reduced their sedimentation coefficient, proba- mains. Interestingly, few years later, a protein from the bac- bly as a result of better solubilization. Both isoenzymes were terium Chromobacterium viscosum, with 100 % identical ami- more active on unsaturated fatty acid esters and the best sub- no acid sequence, was also crystallized (Lang et al. 1996), strate among the assayed was cholesteryl linoleate. They also showing a similar conformation and some differences, among proved to be absolutely specific against cholesterol esters, which the most relevant is the presence of a oxyanion hole that since they did not show lipase, , phospholi- seemstobeabsentintheB. glumae enzyme. The Ca2+ binding pase, or aliesterase activities. site is well described in this work, demonstrating that the ion, After these initial findings, cholesterol esterase activity has essential for enzyme activity, is six-coordinated, contacting been detected in commercial samples of the lipases from with four oxygen atoms from four residues and two water Pseudomonas mendocina (Lumafast, Genecor) (Gray et al. molecules. This Ca2+ site was too far from the active site, 1992), Pseudomonas cepacia (Novo Nordisk), and but could play an important stabilizing role. The presence of Pseudomonas pseudoalcalígenes (Svendsen et al. 1995). this ion is not a common feature of the structure of cholesterol Similarly, P. mendocina 3121 secretes a 30-kDa enzyme with esterases/lipases. It has only been described in the cholesterol esterase activity (Marcinkeviciene et al. 1994)that Pseudomonas family (Noble et al. 1993;Langetal.1996; is probably the same protein described later by Surinenaite Köffel et al. 2005) and in the human and pancreatic lipase et al. (2002) as a lipase, with the same molecular mass and (Hermoso et al. 1996), although the presence of these metal similar properties, although whose activity against sterol ions in Staphylococcus hyicus lipase has been suggested esters was not assayed. (Simons et al. 1999). It should be also mentioned that an Finally, Sugihara et al. (2002) described a cholesterol es- enzymatic cocktail commercialized as C. viscosum lipase, ini- terase from a strain of P. aeruginosa, with molecular mass tially produced by Asahi and now distributed by Merck around 53 kDa and an isoelectric point of 3.2. It tends to form Millipore, shows sterol esterase activity (Kontkanen aggregates in the culture filtrate. Its thermal and pH stability et al. 2004). As observed in Fig. 1,thelipasealready was in the same range as those from P. fluorescens and, sim- described in this species, which is likely responsible for ilarly to it, was inhibited by phenylmetanesulfonyl fluoride this activity in the commercial crude, clusters among the (PMSF), an inhibitor of serine hydrolases, and preferred γ-proteobacteria group of cholesterol esterases. long-chain fatty acid sterol esters as substrates. Nevertheless, A similar enzyme produced by B. cepacia (Svendsen et al. the enzyme from P. aeruginosa also hydrolyzed triglycerides 1995) is commercialized by Novo Nordisk (Barfoed 1994). Its of different length, cleaving the sn-2 ester bond faster than the structure in the open conformation was simultaneously pub- sn-1,3, and was activated by bile salts instead of by Triton lished by Schrag and Cygler (1993) that analyzed the protein X-100 as in P. fluorescens. provided by Genzyme without further purification, and Kim et al. (1997) that purified it from the crude sold by Amano. In Sterol esterases from the genus Burkholderia their respective reports, they confirmed the structural and se- quence similarity (84 % identity) of this enzyme with those As can be observed in Fig. 1, the sequences of the sterol from B. glumae and C. viscosum. The molecular basis of its esterases from this genus are phylogenetically related to enantioselectivity for some substrates was later analyzed, Pseudomonas enzymes. This is explained from the close sim- based on its crystal structure (Mezzetti et al. 2005). Then, ilarity of both genera: Burkholderia was created in 2001 several records deposited in the Protein Data Bank illustrate (Coenye et al. 2001) and all species currently included therein the structure of this protein (PDB: 3LIP, 1OIL, 2LIP, 1YS1, were formerly classified as Pseudomonas. These changes af- 1YS2). In addition, the studies by Pleiss et al. (1998) de- fecting the taxonomical classification of the producer species scribed a funnel-like shape for the substrate of may be misleading when enzymes from different genera are the B. cepacia enzyme. compared, since some of them appear in the literature with The first protein from Burkholderia described as a choles- their old names. This is the case of the lipases with sterol terol esterase was purified and partially characterized from esterase activity secreted by Pseudomonas (synonym B. cepacia ST200 (Takeda et al. 2006). The lipase activity of Burkholderia) glumae and P. (synonym Burkholderia) this enzyme was not tested, but it also had aryl esterase activ- cepacia. In this review, we will refer to them by their current ity and showed great stability to temperature (4–65 °C), pH names. (5.5–12), and organic solvents. Its molecular mass was around Noble et al. (1993) reported the first three-dimensional 37 kDa (SDS-PAGE), but aggregated in aqueous solution. structure of a bacterial lipase. The protein, from B. glumae, The analysis of its amino acidic sequence showed high iden- was crystallized in its closed conformation revealing a tity (87 %) with the well-studied lipase produced by other Appl Microbiol Biotechnol (2016) 100:2047–2061 2053 strains of B. cepacia and, on the basis of this homology, we Cholesterol esterase from Acinetobacter have elaborated a 3D model for the cholesterol esterase of B. cepacia ST200 (Fig. 2a, b), whose structure has not yet Du et al. (2010) screened the production of cholesterol esterase been reported. A Qmean of 0.94, very close to the highest activities by bacteria from environmental samples. The strain value, confirms the fidelity of our model. According to it, CHE4-1 from Acinetobacter sp., isolated from Panthera the amino acids involved in the catalytic machinery of this pardus feces, showed the highest activity growing in the pres- enzyme, Ser87, Asp264, and His286, and the residues of the ence of cholesteryl oleate as the only carbon source. The puri- oxyanion hole, Gln88 and Leu17, would be located exactly in fied enzyme, of 6.5 kDa molecular mass, is monomeric. Its the same positions as in the lipase template (Fig. 2a). In addi- activity was enhanced in the presence of Ca2+,Zn2+, and boric tion, the model is compatible with the presence of a six- acid, and it was sensitive to Ag and Hg salts, SDS, and DTT. coordinated Ca2+. Nevertheless, PMSF, which inhibits serine-hydrolases, did not

Fig. 2 Stereo view of the general structures of bacterial and fungal sphere. b Surface of the protein with the substrate-binding site occupied cholesterol esterases. a, b Homology model for the molecular structure by the inhibitor hexylphosphonic acid (R)-2-methyl-3-phenylpropyl of B. cepacia ST-200 cholesterol esterase. P.cepacia lipase (PDB: 1YS1), ester. c, d Crystal structure of C. rugosa Lip3 (C. cylindracea which showed 84 % sequence identity with the CHE of B. cepacia ST- cholesterol esterase, PDB:1CLE). The lid is colored in pink. c 200, was used as template. The lid is colored in light blue. a Representation of the α/β hydrolase fold and the most relevant residues Representation of the α/β hydrolase fold and the most relevant in the same color code that in a. d Surface of the protein with the substrate residues: the catalytic triad in red, the oxyanion hole in yellow, the (cholesteryl linoleate) inside the catalytic site, where the substrate is residues of the calcium-binding site in cyan, the calcium ion as a pink partially visible 2054 Appl Microbiol Biotechnol (2016) 100:2047–2061 affect its activity. These characteristics are markedly different esters, triglycerides, and phospholipids. It is noteworthy that from those of other microbial sterol esterases suggesting that reducing agents strongly reduced their activity, which can be the catalytic machinery of this cholesterol esterase could be related to the presence of two conserved Cys residues that different. The enzyme was stable in a pH range from 5.5 to could form a disulphide bridge, stabilizing the tertiary struc- 8, showing the optimal activity at pH 7, had an optimum tem- ture. Nevertheless, they were not affected by PMSF as in perature of 42 °C, and rapidly lost activity at 60 °C. Although it Acinetobacter sp. CHE4-1 (Xiang et al. 2006;Duetal. prefers long chain unsaturated fatty acid esters, it has high 2010). The absence of the GXSXG conserved sequence in activity to both long- and short-chain cholesterol esters, and the sterol esterases of actinomycetes suggests a unique cata- an outstanding activity on cholesteryl acetate. lytic mechanism and indicates that they are distantly related to the known lipases/esterases. Moreover, the lack of a con- Chlamydia trachomatis served His in the catalytic region points to the presence of a catalytic dyad instead of the usual triad and, according to these Chlamydia is a genus of intracellular pathogens of eukaryotic data, these enzymes should be classified in a different group host cells. They are auxotrophic for a variety of essential me- among bacterial sterol esterases (Xiang et al. 2007). This tabolites and obtain cholesterol and fatty acids from their host. agrees with the clustering of this group of enzymes presented Not many lipid metabolism enzymes have been identified in in Fig. 1 that shows the resemblance of the esterases from this genus. Strain CT149 of Chlamydia trachomatis secretes a Streptomyces and their low sequence homology with other protein that exhibits esterase activity in vitro and, when bacterial and eukaryotic cholesterol esterases. expressed in HeLa cells, hydrolyzes cholesteryl linoleate (Peters et al. 2012). The enzyme was annotated as a conserved putative hydrolase (CT149) but, after in silico analysis of its Fungal sterol esterases amino acidic sequence, we have identified two lipase/esterase GXSXG motifs, and a potential cholesterol recognition/ Different fungal species are producers of cholesterol esterases, interaction amino acid consensus (CRAC) sequence. The ami- mostly belonging to the C. rugosa-like lipase family, and no acidic sequence of this protein differs from that of other some of them have been characterized and proposed for bio- bacterial sterol esterases, grouping independently in the phy- technological applications. This shows com- logenetic tree (Fig. 1). mon structural features and frequently presents activity on a wide variety of substrates including triglycerides and aryl or Sterol esterases from Streptomyces sterol esters (Barriuso et al. 2013). The structures already re- solved within enzymes of this family are limited to the iso- The first cholesterol esterase activity from the actinomycete forms 1, 2, and 3 of C. rugosa lipase (PDBs: 1CRL, 1CLE, Streptomyces lavendulae H-646-SY2 was reported by Kamei 1GZ7, 1LLF, 1LPP, 1LPO, 1LPM, 1LPN, 1TRH, 1LPS) and et al. (1977). The same authors designed a new purification the O. piceae sterol esterase (PDBs: 4BE4, 4BE9 and 4UPD). method based on affinity to palmitoyl cellulose, describing the Structurally, its members possess the α/β hydrolase fold, in isolation a 12-kDa protein with lipolytic activity (Kamei et al. general with 11-stranded mixed β-sheets and 16 α-helices. 1979). This enzyme was sequenced and characterized, show- The catalytic machinery includes the residues of the catalytic ing to have a molecular mass of 22 kDa under denaturing triad (Ser, His, and Glu) and the oxyanion hole. The nucleo- conditions, similar to that deduced from its nucleotide se- philic serine is located in a very sharp turn (Fig. 2c), the nu- quence (Nishimura and Sugiyama 1994). More recently, cho- cleophilic elbow, composed of the conserved residues GESAG lesterol esterase activity was detected in several species of this as cholesterol esterases from mammalians, while the sterol genus, leading to the isolation, characterization, and sequenc- esterases from the yeast S. cerevisiae and the bacterium ing of three novel enzymes from Streptomyces sp. X9, P. a eru gi no sa contain GFSQG and GHS(H/Q)G sequences, Streptomyces avermitilis JCM5070, and Streptomyces griseus respectively (Pleiss et al. 2000;Köffeletal.2005). These en- IFO13350 (Xiang et al. 2006, 2007). The molecular masses of zymes are also characterized for having a substrate-binding site the purified proteins were 23.6, 19.7, and 25.7 kDa, respec- located in a long (25–30 Å) internal tunnel (Fig. 2d) formed by tively, although under native conditions the measured values aromatic and aliphatic residues, configuring a highly hydro- were 163, 120, and 153 kDa, corroborating their oligomeriza- phobic area along this region (Ghosh et al. 1995;Pleissetal. tion tendency. The addition of Triton X-100 increased their 1998;Mancheñoetal.2003; Gutiérrez-Fernández et al. 2014). activity, probably because this detergent promotes protein sol- The access to the active site is covered by an amphipathic α- ubilization as has also been reported by Vaquero et al. (2015a, helix that serves as a lid, fixed by a disulfide bond. The phy- b). The three enzymes displayed a wide range of substrate logenetic tree in Fig. 1 shows that the sequences of C. rugosa- specificity, hydrolyzing cholesterol esters of different chain like proteins group together and with the known fungal se- length, with preference for cholesteryl linoleate, p-nitrophenyl quences, forming an independent family. Appl Microbiol Biotechnol (2016) 100:2047–2061 2055

Small amounts of sterol esterase activity have also Nevertheless, altered isoenzyme profiles have been obtained been reported in crude enzymes from Rhizopus oryzae by growing C. rugosa on different substrates or using (Kontkanen et al. 2004) and in Lipase B from Geotrichum deregulated mutants derived from the producer wild-type candidum (Charton and Macrae 1992). strain (Ferrer et al. 2001).

Sterol esterases from C. rugosa Sterol esterase of M. albomyces C. rugosa is a non-sporogenic, imperfect hemiascomycete that secretes a variety of closely related enzymes. The genes for The strain VTT D-96490 of this thermophilic fungus, isolated five isoenzymes (lip1 to lip5) were identified and sequenced from composting soil samples, secretes a very hydrophobic and exhibit high identity (77–88 %), encoding 534 amino sterol esterase (Kontkanen et al. 2006c) with a molecular mass acid-proteins with molecular masses around 60 kDa that differ of 64 kDa estimated by SDS-PAGE. However, gel filtration in their predicted isoelectric point and degree of glycosylation chromatography analysis showed a single peak around (Lotti et al. 1994). The isoenzymes Lip1-Lip4 have been pu- 238 kDa, suggesting that it is tetrameric in solution. Its iso- rified, characterized (Kaiser et al. 1994;Rúaetal.1998; electric point was 4.5 and the glycosylation degree is about Pernas et al. 2000;Tangetal.2001), and expressed in various 5 %. As other sterol esterases of the C. rugosa-like family, this heterologous systems, showing that Pichia pastoris was the enzyme has broad substrate specificity and it is also active best host (Lee et al. 2002, 2007; Chang et al. 2006a, b; Ferrer against olive oil and aryl esters. With lipase substrates, the et al. 2009). As already mentioned, the crystal structures of enzyme worked better at neutral pH, while for sterol esters several C. rugosa isoforms have been reported: Lip1, both in the activity was optimal at values around 5–5.5, which are its open (Grochulski et al. 1993) and closed conformations lower than those reported for other fungal sterol esterases, (Grochulski et al. 1994), the closed form of Lip2 (Mancheño usually in the range 6–8 (Okawa and Yamaguchi 1977; et al. 2003), and Lip3 complexed with cholesteryl linoleate in Calero-Rueda et al. 2002b). Trying to improve the enzyme a dimeric arrangement (Ghosh et al. 1995). The presence of yields of the natural producer, it was cloned and expressed dimers in solution has only been reported for Lip3 (Pernas in two eukaryotic heterologous hosts (Kontkanen et al. et al. 2000, 2009), although higher aggregates have been de- 2006a). Its expression in P. pastoris GS115 (AOX promoter) tected in Lip2 (Ferrer et al. 2009). The comparative analysis generated extremely low extracellular sterol esterase activity, of these structures revealed that the main differences con- even lower than the achieved for the native enzyme, probably cerned their hydrophobic profiles in the flap and because it was not completely secreted. T. reeseii was used as substrate-binding pocket regions, which could contribute alternative host (cellobiohydrolase promoter) producing sim- to their different specificity towards triglycerides and ilar yields than M. albomyces, but the activity was bound to cholesterol esters. The isoenzymes Lip1–Lip4 display activity the mycelium or exists as aggregates. The high content of against cholesterol esters at different extent, but Lip2 presents hydrophobic amino acid residues (41.1 %) of this esterase the highest cholesterol esterase activity (Tenkanen et al. 2002; might propitiate its aggregation, and Triton X-100 was neces- Mancheño et al. 2003;Shawetal.2009). Revising the results sary to recover the enzyme. The production of the recombi- from different authors, the catalytic activity against sterol es- nant form in T. reeseii was further optimized in a laboratory ters of the C. rugosa isoenzymes can be ordered as bioreactor, and the properties of the pure protein analyzed Lip2>Lip4≈Lip3>Lip1. According to this, a high hydropho- (Kontkanen et al. 2006b). The enzyme was similar to the bic content in these two regions favors the catalysis of sterol native one but seemed to be dimeric, taking into account esters (Mancheño et al. 2003). In addition, all of them hydro- its molecular mass after gel filtration chromatography. It lyze triacylglycerides and p-nitrophenyl esters (López et al. was less stable under different conditions, showing 2004; Lee et al. 2007). This promiscuity has also been report- slightly lower activities on some of the assayed sub- ed for the sterol esterases produced by O. piceae (Calero- strates. The amino acid sequence deduced from the ste1 gene Rueda et al. 2002b)andMelanocarpus albomyces had high identity with C. rugosa-like lipase isoforms (Kontkanen et al. 2006c), and for lipases with similar charac- (Kontkanen et al. 2006a) and clusters with them in the phylo- teristics from Fusarium solani (anamorph of Nectria genetic tree depicted in Fig. 1. haematococca)andTrichoderma reesei (Vaquero et al. The effect of the recombinant enzyme from T. reseei on the 2015b), whose characteristics will be explained in subsequent properties of paper sheets and PET fabric was assayed, finding sections. that the strength and hydrophilicity of the paper increased due Commercial cocktails of C. rugosa contain different pro- to the hydrolysis of triglycerides and sterol esters. The polarity portions of the isoforms secreted by this yeast. The three iso- and dyeing capacity of PET also increased significantly by enzymes usually detected are Lip1, Lip2, and Lip3, although hydrolyzing the ester bonds in the polyester backbone Lip1 and Lip3 are the main activities (López et al. 2004). (Kontkanen et al. 2006b). 2056 Appl Microbiol Biotechnol (2016) 100:2047–2061

Sterol esterase of O. piceae O. piceae sterol esterase was produced in low yields in the GRAS yeast S. cerevisiae. This recombinant enzyme had an The ascomycete O. piceae is a wood saprophyte that causes intermediate aggregation state between the protein expressed the Bsap staining^ and produces important losses in the forest- in P. pastoris and the native one, and similar catalytic efficien- ry industry. The sterol esterase secreted by strain CECT 20416 cy (Vaquero et al. 2015a). of this dimorphic fungus was purified in a single step by The resolution of the molecular structure of the deglyco- hydrophobic interaction chromatography. It was described as sylated O. piceae enzyme produced in P. pastoris,inbothits a glycoprotein with molecular mass around 56.5 kDa, 8 % N- open and closed conformations, gave very interesting infor- linked carbohydrates (calculated after enzymatic deglycosyl- mation (Gutiérrez-Fernández et al. 2014). As described for ation), and pI of 3.3, which formed multi-aggregates in native other sterol esterases and lipases, in the closed form the am- conditions (Calero-Rueda et al. 2002b). Its biochemical char- phiphilic lid limits the access to the active site. Displacement acterization proved that this enzyme hydrolyzed sterol and p- of this element from its original position allows the entry of nitrophenol esters as well as triglycerides of different fatty substrates to the active site. They are housed in a large and acid chain-length, and then it was classified as a broad sub- straight tunnel-shaped substrate’s pocket rich in hydrophobic strate specificity sterol esterase. The protein maintained 50 % amino acids, ending in an area close to the surface, which activity after 24 h at acidic pH levels, but its stability was not could facilitate the hydrolysis of a great variety of substrates. good over pH 8 or 60 °C (Calero-Rueda et al. 2002b). The The open form of OPE is organized as a functional homodi- sequencing and molecular characterization of this enzyme and mer, leaving a large cavity between the two monomers. its comparison with related proteins indicated around 40 % Analytical ultracentrifugation experiments showed that the identity with C. rugosa lipase isoforms (Calero-Rueda et al. transition from monomer to dimer occurred in the presence 2009). Its catalytic efficiency showed to be much better than of a substrate or inhibitor (Gutiérrez-Fernández et al. 2014). those from several commercial lipases and sterol esterases, The active form of the Lip3 from C. rugosa is organized as a either fungal or bacterial, on all the substrates assayed tight homodimer with small cavities entering into the active (Table 3). In view of its good catalytic properties, the sterol sites, whereas the dimer of the recombinant O. piceae sterol esterase was expressed in different heterologous hosts. The esterase shows a packman-like structure with a very large highest yields were achieved in P. pastoris that secreted a opening. Considering these features, this enzyme could allow 75-kDa protein with 28 % of N-linked carbohydrates and both the entrance of large substrates and the quick release of higher stability at basic pH than the native form (Barba the reaction products, explaining its higher efficiency on tri- Cedillo et al. 2012). The recombinant enzyme showed im- glycerides and cholesterol esters when compared under the proved catalytic efficiency when compared to the native pro- same reaction conditions with commercial sterol esterases tein. The reason for this was attributed to the strategy used for (Table 3) (Calero-Rueda et al. 2009) or with other enzymes protein expression, since the recombinant form incorporated from the C. rugosa-like family (Vaquero et al. 2015b). The 6–8 extra amino acids in its N-terminus, which affected its efficiency of crude preparations of this enzyme has been eval- aggregation behavior. As a result, a more soluble protein uated in applications of biotechnological interest. Hydrolysis was secreted, which was corroborated from the finding that and synthesis reactions were performed at low temperatures only monomers and dimers were detected by analytical ultra- and in the absence of detergents or cofactors, comparing the centrifugation (Barba Cedillo et al. 2012). Recently, the results with those obtained for other sterol esterases. In

Table 3 Comparison of the specific activities (U/mg protein) Organism Activity pNPB pNPP TB TO CB CO CE on aryl esters, cholesterol esters, and triglycerides of commercial A. oryzae Lip 0.6 1.3 74.9 18.3 0.1 0.0 0.1 enzymes and the non-commercial C. antarctica Lip 2.2 0.0 22.3 0.2 0.2 0.2 0.2 sterol esterase/lipase of O. piceae 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 of Genapol X-100 (1 % for p-nitrophenol esters and 5 % for the rest of substrates). The abbreviations Lip and Che correspond 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 Appl Microbiol Biotechnol (2016) 100:2047–2061 2057

general, the recombinant O. piceae enzyme produced in during 24 h, and the T50 value (temperature in which the en- P. pastoris gave the best results under the assayed conditions, zyme maintained 50 % residual activity) was 46 °C. The sterol appearing as a very promising catalyst for these applications esterase from N. haematoccoca hydrolyzed triglycerides and (Calero-Rueda et al. 2004; Barba Cedillo et al. 2013a, b; p-nitrophenol and cholesterol esters, showing broad substrate Vaquero et al. 2015b). specificity, as other enzymes of the family C. rugosa-like. Nevertheless, this enzyme was not the best when compared Sterol esterases from Fusarium with other hypothetical enzymes expressed in P. pastoris and with the sterol esterase from O. piceae (Vaquero et al. 2015b). A search for long chain fatty acid sterol ester hydrolases in soil microorganisms led to the isolation of five Fusarium Sterol esterases from Trichoderma sp. strains, which released high activity levels to the culture me- dium (Okawa and Yamaguchi 1977). Specifically, one of The strain AS59 from Trichoderma sp. was also selected from them, identified as Fusarium oxysporum, gave the best activ- a screening of soil samples, aimed to find microorganisms that ity yield. Cholesterol esterase was induced with several types produced sterol esterases (Maeda et al. 2008). The purified of oils, and although lipase substrates were not assayed, it extracellular cholesterol esterase had pI of 4.3 and its molec- seems feasible that these enzymes present also lipase activity. ular mass measured by MALDI-TOF was 58 kDa, a value Gel filtration chromatography of the pure enzyme gave a peak very similar to that detected by chromatography, indicating that eluted with the void volume, indicating that the protein that aggregated protein forms are not present. After 18 h in- aggregated in solution. However, no estimate was made of the cubation, the enzyme maintained 75 % residual activity in the molecular mass of the active protein in this study. The esterase pH range 4–8, and the T50 value was 40 °C. The enzyme was remained active in the pH range 4–10 and at 50 °C, losing the able to hydrolyze cholesteryl and aryl esters, and triglycerides, activity at 60 °C. Detergents as Triton-X100 and Adekatol showing preference for the short-chain fatty acid substrates (nonionic surfactant) markedly activated the enzyme, and (highest activity for cholesteryl butyrate) and for the position also did sodium deoxycholate and sodium cholate although sn-1,3 of glycerol. Then, its specificity differs from those of to a lesser extent. In terms of substrate specificity, the enzyme other cholesterol esterases that usually hydrolyze better hydrolyzed cholesterol linoleate and oleate faster than other longer-chain esters such as cholesteryl linoleate. In addition, substrates of smaller fatty acid chain. the enzyme was activated by high concentrations of bile salts Madhosingh and Orr (1981) described in this fungus two (40 mM) and was inhibited by PMSF, suggesting its serine intracellular cholesterol esterases and one extracellular. The hydrolase character. As mentioned before, there is not avail- later showed a molecular mass of 75 kDa and the same sub- able information on the sequence or structure of this esterase, strate specificity and activation by Triton X-100 as the enzyme and therefore it is impossible to determine whether it belongs described above. Moreover, the pH range for optimal hydro- to the C. rugosa-like family. lase activities ranged from 4.3 to 8.6. Since no information is Finally, a hypothetic sterol esterase from T. reesei was also available regarding the sequence or structure of these en- identified as a result of in silico genome mining (Barriuso zymes, we cannot ascertain if they belong to the C. rugosa- et al. 2013). Its sequence analysis showed 56 and 59 % iden- like family or not, despite they fulfill several of the character- tity with O. piceae and M. albomyces sterol esterases, respec- istic properties of this family. tively. After analyzing the presence of conserved structural The sequence of a hypothetical cholesterol esterase/lipase motifs, it was ascribed to the C. rugosa-like family although from Nectria haematoccoca (teleomorph of Fusarium solani) it lacked the conserved N-glycosylation site characteristic of wasrecentlyidentifiedbyinsilicogenomemining(Barriuso members of this family. The enzyme was cloned, produced in et al. 2013). This protein showed 55 and 61 % sequence iden- P. pastoris, purified, and characterized as a glycoprotein with tity with the sterol esterases from O. piceae and M. albomyces, a molecular mass around 63 kDa (SDS-PAGE) and 10 % N- respectively; hence, it was classified into the C. rugosa-like linked carbohydrates (Vaquero et al. 2015b). Sedimentation family (abH03.01) (Fig. 1). After being cloned, this enzyme velocity experiments confirmed the coexistence of monomers was produced in P. pastoris, and its biochemical and catalytic and aggregated forms with higher sedimentation coefficient. properties studied (Vaquero et al. 2015b). The purified The enzyme retained more than 60 % residual activity at pH enzyme has a molecular mass of 65 kDa, calculated 4–9after72handhadT50 value of 42 °C after 24 h incuba- by PAGE-SDS, and 6.5 % N-linked carbohydrate content. tion, measured in the interval between 30 and 60 °C. The Analytic ultracentrifugation studies and gel filtration chroma- sterol esterase from T. reesei hydrolyzes p-nitrophenyl esters, tography revealed the presence of monomers, dimers, and big- triglycerides, and cholesterol esters. It showed similar efficien- ger multimeric species. The protein was stable in a pH range of cy than O. piceae esterase on esters of short chain fatty acids 4–11, preserving around 50 % activity at pH 11 after 72 h. although it was less efficient towards long chain fatty acids Temperature stability in the range 30–60 °C was measured esters (Vaquero et al. 2015b). 2058 Appl Microbiol Biotechnol (2016) 100:2047–2061

Sterol esterase from Phoma glomerata C. rugosa are commercially available. However, the sterol esterase from O. piceae is more versatile and shows the best The ascomycete P. glomerata is a ubiquitous conidial fungus catalytic performance when compared with commercial li- (Domsch et al. 1993). Extracellular lipolytic activity was ini- pases and sterol esterases in laboratory tests in both hydrolysis tially detected in its culture supernatants (Pollero et al. 1997), and synthesis reactions. and the enzyme responsible for this activity was purified, A deeper knowledge of the sequence and structure of this showing a molecular mass around 75 kDa (Pollero et al. type of enzymes could permit the development of structure- 2001). Nevertheless, this sterol esterase was not further stud- function studies and rational design approaches, aimed to im- ied and, as in other cases, the lack of information precludes prove or orient enzymatic activity towards a specific goal. making sequence or phylogenetic analysis to ascertain its po- Currently, the outcomes from genome sequencing and tential relationship with the C. rugosa-like family. metagenomic analysis provide a growing amount of gene and protein sequences. In this context, genome mining of hy- pothetical enzymes and sequence-based functional predictions Conclusions for a specific activity could be a way to follow in the imme- diate future. Nevertheless, to ascertain the verisimilitude of In spite of the importance of microbial sterol esterases as bio- these predictions, a true protein must always be expressed technological tools and at industrial scale, there is little infor- and purified to test its real activity. mation about the properties and characteristics of many of these biocatalysts. Intracellular cholesterol esterases are Acknowledgments This work was funded by the Spanish projects indispensible enzymes for the lipolytic metabolism of living BIO2012-36372, RTC-2014-1777-3 and S2013/MAE-2907. M.E. organisms but, at first glance, this activity is not frequently Vaquero gratefully acknowledges an FPU fellowship from MINECO. J. Barriuso thanks the financial support from the JAE-DOC CSIC program. found extracellularly, as deduced from the results of studies tackling the screening of a wide amount of microbial isolates. Compliance with ethical standards Many of these enzymes are extremely versatile, combining several esterase activities, although displaying preferences Conflict of interest The authors declare that they have not competing for some types of substrates, and these characteristics, together interests. with their stability, make them attractive for biotechnological Ethical statement This article does not contain any studies with ani- and industrial applications. Some cholesterol esterases, such mals performed by any of the authors. as those produced by Pseudomonas species, Schizophyllum commune,andC. rugosa are already commercialized. As a general rule, the characteristics of microbial sterol esterases fit within the features of serine hydrolases. They References are very hydrophobic proteins and tend to aggregate in water solution. Apart from these characters, shared by all sterol es- Abo M, Andersen BK, Borch K, Damgaard B (1999) A method of terases, those produced by different bacteria hardly have any- treating polyester fabrics. Patent No. WO 1999001604 A1 thing else in common. A diversity of individual traits can be Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of total serum-cholesterol. Clin Chem 20:470–475 found among them, as the peculiar catalytic mechanism sug- Appel W (1986) Chymotrypsin: molecular and catalytic properties. Clin gested for Acinetobacter esterase, the catalytic dyad of en- Biochem 19:317–322 zymes from actinomycetes, the Ca2+ requirement for the pro- Barba Cedillo V, Plou FJ, Martínez MJ (2012) Recombinant sterol ester- teins produced by some Pseudomonas and Burkholderia iso- ase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris. Microb Cell Fact 11:73 lates, or the strict cholesterol esterase character of the enzyme Barba Cedillo V, Prieto A, Martínez AT, Martínez MJ (2013a) from P. fluorescens ATCC 21156. The reported molecular Procedimiento de acilación para la obtención de compuestos de masses for the monomeric forms of bacterial sterol esterases interés alimenticio y/o farmacéutico utilizando esterol esterasas are quite variable, ranging from around 50 to 6.5 kDa, and fúngicas. Patent (International) PCT/ ES 2395582 B1 they are usually smaller than those of their fungal counter- Barba Cedillo V, Prieto A, Martínez MJ (2013b) Potential of Ophiostoma piceae sterol esterase for biotechnologically relevant hydrolysis re- parts. In contrast, the information on true or hypothetical yeast actions. Bioengineered 4:249–253 and fungal sterol esterases suggest that they form a quite ho- Barfoed M (1994) A method of hydrolysing cholesterol sters by mogeneous group, which allows their inclusion into the using a Pseudomonas fragi cholesterol esterase. Patent No. C. rugosa-like family. These enzymes do not require cofactors WO1994023052 A1 and show wide substrate specificity, although their catalytic Barriuso J, Prieto A, Martínez MJ (2013) Fungal genomes mining to discover novel sterol esterases and lipases as catalysts. BMC efficiency is diverse. Some of them are known to be glyco- Genomics 14:712–719 proteins with variable amount of N-glycidic chains and mo- Basheer S, Plat D (2004) Enzymatic modification of sterols using sterol- lecular mass around 60 kDa. Mixtures of isoenzymes from specific lipase. Patent No. US 2004/0105931 A1 Appl Microbiol Biotechnol (2016) 100:2047–2061 2059

Brockerhoff H, Jensen RG (1974) Lipolytic enzymes. Academic Press, Gutiérrez A, del Río JC, Martínez AT (2009) Microbial and enzymatic New York control of pitch in the pulp and paper industry. Appl Microbiol Calero-Rueda O, Gutiérrez A, del Río JC, Muñoz MC, Plou FJ, Martínez Biotechnol 82:1005–1018 ÁT, Martínez MJ (2002a) Method for the enzymatic control of pitch Gutiérrez-Fernández J, Vaquero ME, Prieto A, Barriuso J, Martínez MJ, deposits formed during paper pulp production using an esterase that Hermoso JA (2014) Crystal structures of Ophiostoma piceae sterol hydrolyses triglycerides and sterol esters. Patent No. WO 02/075045 esterase: structural insights into activation mechanism and A1R1 release. J Struct Biol 187:215–222 Calero-Rueda O, Plou FJ, Ballesteros A, Martínez AT, Martínez MJ Harvie NR (1977) Cholesteryl de-esterifying enzyme from (2002b) Production, isolation and characterization of a sterol ester- Staphylococcus aureus: separation from alpha toxin, purification, ase from Ophiostoma piceae. BBA Proteins Proteomics 1599:28–35 and some properties. Infect Immun 15:863–870 Calero-Rueda O, Gutiérrez A, del Río JC, Prieto A, Plou FJ, Ballesteros Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial A, Martínez AT, Martínez MJ (2004) Hydrolysis of sterol esters by lipases. Enzyme Microb Technol 39:235–251 an esterase from Ophiostoma piceae: application for pitch Hata K, Matsukura M, Taneda H, Fujita Y (1996) Mill-scale application control in pulping of Eucalyptus globulus wood. Intern J of enzymatic pitch control during paper production. In: Viikari L, Biotechnol 6:367–375 Jeffries TW (eds) Enzymes for pulp and paper processing. ACS, Calero-Rueda O, Barba V, Rodriguez E, Plou F, Martínez AT, Martínez Washington, pp 280–296 MJ (2009) Study of a sterol esterase secreted by Ophiostoma piceae: Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C, Fontecilla Camps sequence, model and biochemical properties. Biochim Biophys Acta JC (1996) Lipase activation by nonionic detergents the crystal struc- 1794:1099–1106 ture of the porcine lipase-colipase-tetraethylene glycol monooctyl – Cantrill R, Kawamura Y (2008) Phytosterols, phytostanols and their es- ether complex. J Biol Chem 271:18007 18016 ters: chemical and technical assessment for the 69th Joint FAO/ Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for – WHO Expert Committee on Food Additives (JECFA) biotechnology. Trends Biotechnol 16:396 403 Chang SW, Lee GC, Shaw JF (2006a) Codon optimization of Candida Juniper BE, Jeffree CE (1983) Plant surfaces. Eduard Arnold, Baltimore rugosa lip1 gene for improving expression in Pichia pastoris and Kaiser R, Erman M, Duax WL, Ghosh D, Jörnwall H (1994) Monomeric biochemical characterization of the purified recombinant LIP1 li- and dimeric forms of cholesterol esterase from Candida – pase. J Agr Food Chem 54:815–822 cylindracea.FEBSLett337:123 127 Chang SW, Lee GC, Shaw JF (2006b) Efficient production of active Kamei T, Suzuki H, Matsuzaki M, Otani T, Kondo H, Nakamura S (1977) recombinant Candida rugosa LIP3 lipase in Pichia pastoris and Cholesterol esterase produced by Streptomyces lavendulae.Chem – biochemical characterization of purified enzyme. J Agr Food Pharm Bull 25:3190 3197 Chem 54:5831–5838 Kamei T, Suzuki H, Asano K, Matsuzaki M, Nakamura S (1979) Charton E, Macrae AR (1992) Substrate specificities of lipases A and B Cholesterol esterase produced by Streptomyces lavendulae II. Purification and properties as a lipolytic enzyme. Chem Pharm from Geotrichum candidum CMICC 335426. Biochim Biophys – Acta 1123:59–64 Bull 27:1704 1707 Kim KK, Song HK, Shin DH, Hwang KY, Suh SW (1997) The crystal Coenye T, Vandamme P, Govan JRW, Lipuma JJ (2001) Taxonomy and structure of a triacylglycerol lipase from Pseudomonas cepacia re- identification of the Burkholderia cepacia complex. J Clin veals a highly open conformation in the absence of a bound inhib- Microbiol 39:3427–3436 itor. Structure 5:173–185 Domsch KH, Gams W, Anderson T-H (1993) Compendium of soil fungi. Köffel R, Tiwari R, Falquet L, Schneiter R (2005) The Saccharomyces IHW-Verlag, Eching, Germany cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes en- Du L, Huo Y, Ge F, Yu J, Li W, Cheng G, Yong B, Zeng L, Huang M code a novel family of membrane-anchored lipases that are required (2010) Purification and characterization of a novel extracellular cho- for steryl ester hydrolysis. Mol Cell Biol 25:1655–1668 lesterol esterase from Acinetobacter sp. J Basic Microb 50:S30–S36 Kokkonen P, Korpela A, Sundberg A, Holmbom B (2002) Effects of Ferrer P, Montesinos JL, Valero F, Sola C (2001) Production of native and different types of lipophilic extractives on paper properties. Nord — recombinant lipases by Candida rugosa a review. Appl Biochem Pulp Pap Res J 17:382–386 – Biotechnol 95:221 255 Kokkonen P, Fardim P, Holmbom B (2004) Surface distribution of ex- Ferrer P, Alarcón M, Ramón R, Benaiges MD, Valero F (2009) tractives on TMP handsheets analyzed by ESCA, ATR-IR, ToF- Recombinant Candida rugosa LIP2 expression in Pichia SIMS and ESEM. Nord Pulp Pap Res J 19:318–324 pastoris under the control of the AOX1 promoter. Biochem Kontkanen H, Tenkanen M, Fagerström R, Reinikainen T (2004) – Eng J 46:271 277 Characterisation of steryl esterase activities in commercial lipase Ghosh D, Wawrzak Z, Pletnev VZ, Li N, Kaiser R, Pangborn W, Jörnvall preparations. J Biotechnol 108:51–59 H, Erman M, Duax WL (1995) Structure of uncomplexed and Kontkanen H, Reinikainen T, Saloheimo M (2006a) Cloning and expres- linoleate-bound Candida cylindracea cholesterol esterase. sion of a Melanocarpus albomyces steryl esterase gene in Pichia – Structure 3:279 288 pastoris and Trichoderma reesei. Biotechnol Bioeng 94:407–415 Gray GL, Poulose AJ, Power SD (1992) Novel hydrolase and method of Kontkanen H, Saloheimo M, Pere J, Miettinen-Oinonen A, Reinikainen T production. Patent No. EP0268452 A2 (2006b) Characterization of Melanocarpus albomyces steryl ester- Grochulski P, Li YG, Schrag JD, Bouthillier F, Smith P, Harrison D, ase produced in Trichoderma reesei and modification of fibre prod- Rubin B, Cygler M (1993) Insights into interfacial activation from ucts with the enzyme. Appl Microbiol Biotechnol 72:696–704 an open structure of Candida rugosa lipase. J Biol Chem 268: Kontkanen H, Tenkanen M, Reinikainen T (2006c) Purification and char- 12843–12847 acterisation of a novel steryl esterase from Melanocarpus Grochulski P, Li Y, Schrag JD, Cygler M (1994) Two conformational albomyces. Enzyme Microb Technol 39:265–273 states of Candida rugosa lipase. Protein Sci 3:82–91 LangD,HofmannB,HaalckL,HechtHJ,SpenerF,SchmidRD, Gubitz GM, Paulo AC (2003) New substrates for reliable enzymes: en- Schomburg D (1996) Crystal structure of a bacterial lipase from zymatic modification of polymers. Curr Opin Biotech 14:577–582 Chromobacterium viscosum ATCC 6918 refined at 1.6 angstrom Gutiérrez A, del Río JC, Martínez MJ, Martínez AT (2001) The biotech- resolution. J Mol Biol 259:704–717 nological control of pitch in paper pulp manufacturing. Trends Lee GC, Lee LC, Sava V, Shaw JF (2002) Multiple mutagenesis of non- Biotechnol 19:340–348 universal serine codons of the Candida rugosa lip2geneand 2060 Appl Microbiol Biotechnol (2016) 100:2047–2061

biochemical characterization of purified recombinant LIP2 lipase Pernas MA, Pastrana L, Fucinos P, Rua ML (2009) Regulation of the overexpressed in Pichia pastoris. Biochem J 366:603–611 interfacial activation within the Candida rugosa lipase family. J Lee LC, Chen YT, Yen CC, Chiang TCY, Tang SJ, Lee GC, Shaw JF Phys Org Chem 22:508–514 (2007) Altering the substrate specificity of Candida rugosa LIP4 by Peters J, Onguri V, Nishimoto SK, Marion TN, Byrne GI (2012) The engineering the substrate-binding sites. J Agric Food Chem 55: Chlamydia trachomatis CT149 protein exhibits esterase activity 5103–5108 in vitro and catalyzes cholesteryl ester hydrolysis when expressed Levisson M, van der Oost J, Kengen SW (2009) Carboxylic ester hydro- in HeLa cells. Microbes Infect 14:1196–1204 lases from hyperthermophiles. Extremophiles 13:567–581 Plat J, Mensink RP (2005) Plant stanol and sterol esters in the control of López N, Pernas MA, Pastrana LM, Sánchez A, Rúa ML (2004) blood cholesterol levels: mechanism and safety aspects. Am J Reactivity of pure Candida rugosa lipase isoenzymes (Lip1, Lip2, Cardiol 96:15–22 and Lip3) in aqueous and organic media. Influence of the isoenzy- Pleiss J, Fischer M, Schimd RD (1998) Anatomy of lipase binding sites: matic profile on the lipase performance in organic media. Biotechnol the scissile fatty acid binding site. Chem Phys Lipids 93:67–80 Progr 20:65–73 Pleiss J, Fischer M, Peiker M, Thiele C, Schmid RD (2000) Lipase engi- Lotti M, Tramontano A, Longhi S, Fusetti F, Brocca S, Pizzi E, neering database: understanding and exploiting sequence-structure- Alberghina L (1994) Variability within the Candida rugosa lipases function relationships. J Mol Catal B Enzym 10:491–508 family. Protein Eng 7:531–535 Pollero R, Caspar M, Cabello M (1997) Lipolytic activity in free and Madhosingh C, Orr W (1981) Sterol ester hydrolase in Fusarium immobilized cells of Phoma glomerata. J Am Oil Chem Soc 74: oxysporum. Lipids 16:125–132 451–454 Maeda A, Mizuno T, Bunya M, Sugihara S, Nakayama D, Tsunasawa S, Pollero RJ, Gaspar ML, Cabello M (2001) Extracellular lipolytic activity Hirota Y, Sugihara A (2008) Characterization of novel cholesterol in Phoma glomerata. World J Microb Biot 17:805–809 esterase from Trichoderma sp. AS59 with high ability to synthesize Rúa ML, Díaz-Mauriño T, Fernández VM, Otero C, Ballesteros A (1993) steryl esters. J Biosci Bioeng 105:341–349 Purification and characterization of two distinct lipases from Mancheño JM, Pernas MA, Martínez MJ, Ochoa B, Rua ML, Hermoso Candida cylindracea. Biochim Biophys Acta 1156:181–189 JA (2003) Structural insights into the lipase/esterase behavior in the Rúa ML, Atomi H, Schmidt-Dannert C, Schmid RD (1998) High-level Candida rugosa lipases family: crystal structure of the lipase 2 iso- expression of the thermoalkalophilic lipase from Bacillus enzyme at 1.97 Å resolution. J Mol Biol 332:1059–1069 thermocatenulatus in Escherichia coli. Appl Microbiol Biotechnol Marcinkeviciene LY, Bakhmatova IV, Brazenas GR, Baratova LA, 49:405–410 Revina LP (1994) Purification and properties of cholesterol esterase Rudd EA, Brockman HL (1984) Pancreatic carboxyl ester lipase (choles- from Pseudomonas mendocina 3121. Biochemistry-Moscow 59: terol esterase). In: Borgström B, Brockman HL (eds) Lipases. 473–478 Elsevier Science Publishers, Amsterdam, pp 185–204 Mas E, Lombardo D (1994) Pancreatic cholesteryl esterase in health and Schrag JD, Cygler M (1993) 1.8 Å Refined structure of the lipase from disease. In: Mackness MI, Clerc M (eds) Esterases, lipases and Geotrichum candidum. J Mol Biol 230:575–591 . From structure to clinical significance. Plenum Seo N, Kaneko S, Sato F, Norinobu S, Mankura M (2006) Process for Press, New York, pp 75–81 producing edible sterol fatty acid esters. Patent No. US6989456 B2 Masaki I, Yusuke N, Sadanori O (2003) Enzyme-containing detergent. Shaw JF, Lee GC, Tang SJ (2009) Recombinant Candida rugosa lipases. Patent No. WO 2003066792 A1 Patent No. US 20090053795A1 Mezzetti A, Schrag JD, Cheong CS, Kazlauskas RJ (2005) Mirror-image Simons JWFA, van Kampen MD, Ubarretxena-Belandia I, Cox RC, dos packing in enantiomer discrimination: molecular basis for the Santos CMA, Egmond MR, Verheij HM (1999) Identification of a enantioselectivity of B. cepacia lipase toward 2-methyl-3-phenyl- calcium binding site in Staphylococcus hyicus lipase: generation of 1-propanol. Chem Biol 12:427–437 calcium-independent variants. Biochemistry-US 38:2–10 Mukherjee M (2003) Human digestive and metabolic lipases: a brief Søe JB, Jørgensen TL (2010) Method for producing phytosterol/ review. J Mol Catal B-Enzym 22:369–376 phytostanol phospholipid esters. Patent No. WO2010109441 A1 Mustranta A, Buchert J, Spetz P, Holmbom B (2001) Treatment of me- Sugihara A, Shimada Y, Nomura A, Terai T, Imayasu M, Nagai Y, Nagao chanical pulp and process waters with lipases. Nord Pulp Paper Res T, Watanabe Y,Tominaga Y (2002) Purification and characterization J 16:125–129 of a novel cholesterol esterase from Pseudomonas aeruginosa,with Negishi S, Hidaka I, Takahashi I, Kunita S (2003) Transesterification of its application to cleaning lipid-stained contact lenses. Biosci phytosterol and edible oil by lipase powder at high temperature. J Biotech Bioch 66:2347–2355 Am Oil Chem Soc 80:905–907 Surinenaite B, Bendikiene V,Juodka B, Bachmatova I, Marcinkevichiene Nishimura M, Sugiyama M (1994) Cloning and sequence analysis of a L (2002) Characterization and physicochemical properties of a li- Streptomyces cholesterol esterase gene. Appl Microbiol Biotechnol pase from Pseudomonas mendocina 3121–1. Biotechnol Appl Bioc 41:419–424 36:47–55 Noble MEM, Cleasby A, Johnson LN, Egmond MR, Frenken LGJ Svendsen A, Borch K, Barfoed M, Nielsen TB, Gormsen E, Patkar SA (1993) The crystal structure of triacylglycerol lipase from (1995) Biochemical properties of cloned lipases from the Pseudomonas glumae reveals a partially redundant catalytic aspar- Pseudomonas family. BBA Lipid Lipid Met 1259:9–17 tate. FEBS Lett 331:123–128 Takeda Y, Aono R, Doukyu N (2006) Purification, characterization, and Norinobu S, Seo N, Sato F, Kaneko S, Mankura M (2003) Process for molecular cloning of organic-solvent-tolerant cholesterol esterase producing dietary sterol fatty acid esters. Patent No. US 6660491 B2 from cyclohexane-tolerant Burkholderia cepacia strain ST-200. Okawa Y, Yamaguchi T (1977) Studies on sterol-ester hydrolase from Extremophiles 10:269–277 Fusarium oxysporum I partial purification and properties. J Tang SJ, Shaw JF, Sun KH, Sun GH, Chang TY,Lin CK, Lo YC, Lee GC Biochem 81:1209–1215 (2001) Recombinant expression and characterization of the Candida Panitch M (1997) Antiperspirant deodorant compositions. Patent No. rugosa LIP4 lipase in Pichia pastoris: comparison of glycosilation, US5635165 A activity, and stability. Arch Biochem Biophys 387:93–98 Pernas MA, Lopez C, Pastrana L, Rua ML (2000) Purification and char- Tenkanen M, Kontkanen H, Isoniemi R, Spetz P, Holmbom B (2002) acterization of Lip2 and Lip3 isoenzymes from a Candida rugosa Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa. pilot-plant scale fed-batch fermentation. J Biotechnol 84:163–174 Appl Microbiol Biotechnol 60:120–127 Appl Microbiol Biotechnol (2016) 100:2047–2061 2061

Töke ER, Nagy V, Recseg K, Szakacs G, Poppe L (2007) Production and Villeneuve P, Turon F, Caro Y, Escoffier R, Baréa B, Barouh B, Lago R, application of novel sterol esterases from Aspergillus strains by solid Piombo G, Pina M (2005) Lipase-catalyzed synthesis of canola phy- state fermentation. J Am Oil Chem Soc 84:907–915 tosterols oleate esters as cholesterol lowering agents. Enzyme Uwajima T, Terada O (1975) Studies on sterol-metabolism by microor- Microb Tech 37:150–155 ganisms III. Purification and properties of extracellular cholesterol Weber N, Weitkamp P, Mukherjee KD (2001) Steryl and stanyl esters of ester hydrolase of Pseudomonas fluorescens. Agr Biol Chem Tokyo fatty acids by solvent-free esterification and transesterification in 39:1511–1512 vacuo using lipases from Rhizomucor miehei, Candida antarctica, Uwajima T, Terada O (1976) Studies on sterol metabolism by mi- and Carica papaya. J Agric Food Chem 49:5210–5216 croorganisms V. Purification and properties of cholesterol es- Weber N, Weitkamp P, Mukherjee KD (2002) Cholesterol-lowering food terase from Pseudomonas fluorescens. Agr Biol Chem Tokyo additives: lipase-catalysed preparation of phytosterol and 40:1957–1964 phytostanol esters. Food Res Int 35:177–181 Vaquero ME, Barriuso J, Medrano F, Prieto A, Martinez MJ (2015a) Xiang HY, Takaya N, Hoshino T (2006) Novel cholesterol esterase se- Heterologous expression of a fungal sterol esterase/lipase in differ- creted by Streptomyces persists during aqueous long-term storage. J ent hosts: effect on solubility, glycosylation and production. J Biosci Biosci Bioeng 101:19–25 Bioeng 129:637–643 Xiang H, Masuo S, Hoshino T, Takaya N (2007) Novel family of choles- Vaquero ME, Prieto A, Barriuso J, Martinez MJ (2015b) Expression terol esterases produced by actinomycetes bacteria. BBA-Proteins and properties of three novel fungal lipases/sterol esterases pre- Proteom 1774:112–120 dicted in silico: comparison with other enzymes of the Candida Yoon MY, Kellis J, Poulose AJ (2002) Enzymatic modification of poly- rugosa-like family. Appl Microbiol Biotechnol. doi:10.1007/ ester. AATCC Rev 2:33–36 s00253-015-6890-9 Zorn H, Bouws H, Takenberg M, Nimtz M, Getzlaff R, Breithaupt DE, Vertommen MAME, Nierstrasz VA, van der Veer M, Warmoeskerken Berger RG (2005) An extracellular carboxylesterase from the basid- MMCG (2005) Enzymatic surface modification of poly(ethylene iomycete Pleurotus sapidus hydrolyses xanthophyll esters. Biol terephthalate). J Biotechnol 120:376–386 Chem 386:435–440