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Combinatorial Chemistry & High Throughput Screening, 2016, 19, 605-615

REVIEW ARTICLE

ISSN: 1386-2073 eISSN: 1875-5402

Impact Factor: High Throughput Screening of Esterases, and in 1.041 Mutant and Metagenomic Libraries: A Review

BENTHAM SCIENCE

Carlina Peña-García1, Mónica Martínez-Martínez2, Dolores Reyes-Duarte*,3 and Manuel Ferrer*,2

1Posgrado en Ciencias Naturales e Ingeniería, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Av. Vasco de Quiroga 4871, Col. Santa Fe, Deleg, Cuajimalpa, 05348, D.F, México; 2Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Marie Curie 2, 28049 Madrid, Spain; 3Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Av. Vasco de Quiroga 4871, Col. Santa Fe, Deleg. Cuajimalpa, 05348, CDMX, México

Abstract: Nowadays, can be efficiently identified and screened from

metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive A R T I C L E H I S T O R Y screening process that only very few enzymes finally pass. Accessing to the highest possible and mutant diversity by different, but complementary approaches is Received: December 25, 2014 Dolores Reyes-Duarte Revised: June 8, 2015 increasingly important. The aim of this review is to deliver state-of-art status of Accepted: November 6, 2015 traditional and novel screening protocols for targeting lipases, esterases and phospholipases of

industrial relevance, and that can be applied at high throughput scale (HTS) for at least 200 distinct DOI: 10.2174/1386207319666151 5 8 110123927 substrates, at a speed of more than 10 – 10 clones/day. We also review fine-tuning sequence analysis pipelines and in silico tools, which can further improve enzyme selection by an unprecedent speed (up to 1030 enzymes). If the hit rate in an enzyme collection could be increased by HTS approaches, it can be expected that also the very further expensive and time-consuming enzyme optimization phase could be significantly shortened, as the processes of enzyme-candidate selection by such methods can be adapted to conditions most likely similar to the ones needed at industrial scale. Keywords: Biocatalysts, esterases, high throughput screening, lipases, metagenomic, phospholipases, protein engineering.

1. INTRODUCTION present there is a great necessity of suitable biocatalysts with high activity and stability, substrate selectivity and high Current challenges for biotechnology include looking for enantio-selectivity [8], that can be obtained either by green alternatives to chemical synthesis, which relies on the engineering enzymes that are available or by the discovery of use of environmentally damaging bulk organic solvents and new enzyme activities in environmental samples. Of energy-demanding high-pressure and high-temperature particular importance are biocatalysts like esterases, lipases processes [1]. In contrast, the emerging “white and phospholipases (herein referred as ELP biocatalysts), biotechnology” has the mission to largely implement green which together with other enzymes have a remarkable biocatalysis in place of conventional chemical synthesis [2, market potential [9]. Currently, about 5 billion USD turnover 3]. Biocatalysis gives higher specificity/enantio-selectivity are generated by the application of such enzymes in different and catalytic efficiency [4, 5] and can be performed at markets and world enzyme demand is forecast to rise 6.4 ambient temperature and pressure [6]. It is expected that up percent p.a. to 6.9 billion USD in 2017 to 40% of bulk chemical synthesis processes could be (www.rnrmarketresearch.com/world-enzymes-to-2017- substituted by biocatalysis by 2020 [7]. Therefore, at the market-report.html).

*Address correspondence to these authors at the Departamento de Procesos Theoretical considerations and experimental results from y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, previous studies indicate that it is possible to design new Av. Vasco de Quiroga 4871, Col. Santa Fe, Deleg, Cuajimalpa, 05348, ELP variants that have not existed in nature or that have not CDMX, México; Tel: 525558146500; Ext: 3872; needed to be identified in a random process, with improved E-mail: [email protected] catalytic performance for known substrates or with specific Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Marie Curie 2, 28049 Madrid, Spain; Tel: +34915854872; activities for new substrate molecules [10-14]. These Fax: +34915854760; E-mail: [email protected] enzymes would have sequences that would contain amino Combinatorial Chemistry & High Throughput Screening

1875-5402/16 $58.00+.00 © 2016 Bentham Science Publishers 606 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 Peña-García et al. acids that, when the chain is generated by recombinant method. Thus, in a recent example it has been demonstrated techniques, cause the chain to fold in such a way that its that the incidence decrease from 1:188 to 1:3,937 and quaternary structure gives rise to a new enzyme whose active 1:15,625 when using 1% (v/v) tributyrin, tricaprylin and site is specific to the reaction and substrate of interest for triolein as the indicator substrates, respectively, after which the screening method was designed. In this process, examining the same metagenomic clone library [19]. This known as enzyme engineering [11], new enzymes are implies that enzymes with character are less abundant generated and tested on the substrates of interest. Based on (in this experiment, 83-fold) than those with esterase the results, the process is repeated iteratively, under the character. Additionally, among the common substrates used required conditions, to produce an enzymatic activity that for ELP screen, methods using pH indicators resulted in a provides the desired catalytic yield. Technically improving higher number of incidence rate (1:29), followed to much the processes of high-throughput screening (HTS) for such lower extend by methods based on the utilization of molecules can provide significant competitive advantages for poly(DL-lactic acid) (1:13,334), tributyrin (1:15,478), α- the generation of new ELP variants through the engineering naphthyl acetate (1:19,925), polyethylene terephthalate process. Bibliographic records on HTS methods applied to (1:21,400), triolein or olive oil and rhodamine B (1:22,061), engineering protocols for ELP revealed that the production Tween-20 (1:26,496), methyl and ethyl ferulate (1:26,496), of readily screenable mutant libraries poses a minor risk, 5-bromo-4-chloro-3-indolylcaprylate (1:50,000) and with a rate of positive hits, that is of mutants with improved tricaprylin (1:68,279), in this order (Fig. 1B, inset). This performance as compared with the wild type, ranging from suggests, that in metagenomic experiments the substrate to 1:140 to 1:125,000 (Fig. 1A). Although, theoretical use might cause biases in the number of selected positives. considerations and experimental results from protein Moreover, among the detected enzymes in any metagenomic engineering studies indicate that it is possible to design such study, it should be highlighted that only 20% of them have new enzymes, this type of process currently leads to the properties making them useful for a biocatalytic process generation of new enzymes that do not appreciably improve [18], and therefore intense screen procotocols should be their industrial incorporation. It is anticipated that increasing applied in any metagenomic investigation if the final the power of screening capability will allow for substantial outcome is to find an enzyme fitting industrial performance. improvement in this process. As example, methods based on Note, that for such studies in some cases general ELP the screening of in silico (virtual) mutant libraries with a size substrates such as α-naphthyl acetate are first used to reduce up to 1080 variants, have been applied successfully [15]. the number of clones to work with, which are later tested The impressive power of environmental microbiology for with the substrate of interest in a small scale HTS assay; this has been applied to select (S)-ketoprofen specific activities improving the commercial potential of biotechnology has [20]. recently been greatly further strengthened by the advent of the “metagenomics” [1]. This technology provides, as With these antecedents the selection of a good method of compared to protein engineering, the capacity to discover screening the variants is of vital importance because enterily new proteins in microorganisms and their conventional assay methods are not suitable for many targets communities without the need to culture them as individual due to the nature of the substrates and products involved. species (which is technically very difficult) [2, 16, 17]. Therefore, HTS methods are essential for success because Instead, with metagenomics, the DNA of microbial they allow determining enzyme properties with high communities is directly harvested from the environment, accuracy and reproducibility [21] and facilitate the use the sequenced and bioinformatic analysis and/or expression is native biological substrate of a target. A great advantage of then performed in surrogate microbial hosts enabling HTS assays is the number of enzyme variants or clones screening for enzymatic activities of interest. This has containing environmental enzymes to be studied, less with already been shown to facilitate the discovery of new other methodologies such as gas chromatography and HPLC. enzymes with novel activities of industrial relevance [18]. Below a number of commonly and successful HTS methods for screening ELP biocatalysts are described. Note, 2. EFFICIENCY ANALYSIS OF HTS PROTOCOLS that actually at least 200 substrate molecules have been APPLIED TO METAGENOMIC LIBRARIES successfully applied in HTS assays for ELP biocatalysts, which can be grouped into 12 major HTS methods whose Own experiences with ’ collections coming main characteristics are herein reviewed. from several metagenomic libraries showed that only very few have substrate specificities, which allow their use in It should be firstly highlighted that assays on agar-plated biocatalysis. More than 95% of the found enzymes cannot be colonies, where the positive signal is visible by eye, typically used either due to their very narrow substrate specificity or enable the screening of >104 variants in a matter of days, but other missing properties, e.g. low solvent tolerance, are often limited in sensitivity; thus, a concentration of 2.5 – insufficient enantio-selectivity, etc. [18]. 10,000 mg/l of the substrate to be tested is commonly required for an optimal visualization [19, 22]. Soluble Bibliographic records on HTS methods applied to products diffuse away from the colony and hence, only very metagenomic library screen protocols for ELP revealed that active variants are detected. This problem can be solved by the production of readily screenable clone libraries also using colorimetric or fluorescence liquid-based assays in 96- poses a minor risk, with a rate of positive hits, that is of or 384-plate format, which possess higher sensitivity (from clones with the desired activity referred to the total number ng to few mg/l of substrate), but requires a clone array of clones screened, ranging from 1:11 to 1:193,200 (Fig. 4 system that typically enable also the screening of >10 1B). Among other factors affecting the screen result, the variants in a matter of days [18, 23, 24]. Cell sorting and incidence hit rate depends on the substrate used for the HTS HTS of Esterases, Lipases and Phospholipases in Metagenomic Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 607

200000 200000

A 83x 175000 15000 B 175000

150000 10000 150000 21x 125000 5000

0 125000 100000 Triolein Tributyrin 75000 Tricaprylin 100000 50000 75000 Incidence of positive clones 25000

50000 0 Incidence of positive clones

25000

Tween-20 Tributyrin X-caprylate Tricaprylin 0 Ferulate Triolein-Naphthyl - Olive oil acetate a Mutant libraries Metagenomic clone libraries Poly (DL-lactic acid)

Polyethylene terephthalate Acetates of tertiary

Fig. (1). Box plots of the incidence rate (the number of clones to be tested to find 1 positive clone) for reported studies in which metagenomic clone libraries or mutant libraries were subjected to HTS systems. Results are based on the average values for studies related to ELP enzymes. Panel B represents the box plots for HTS methods applied for the screening of ELP active clones in metagenomic clone libraries using different substrates; note that to avoid biases due to the type of libraries, only studies considering the screen of fosmid clones have been considered. Inset in Panel B, represents the incidence rate for a single metagenomic library after screening with HTS methods using tributyrin, tricaprylin and triolein as the indicator substrates and identification of clear zones on the indicator plates. microfluidic devices allow selection from libraries of >107 – nitrocefin [3-(2, 4 dinitrostyrl)-(6R,7R-7-(2-thienylacet- 108 different variants or clones per day in a quantitative amido)-ceph-3-em-4-, E-isomer)] which has manner [25]; in some cases, these techniques have allowed been successfully used to find one esterase/β-lactamase out the screening of up to 50,000 clones per second, or over 1 of 10,000 clones from a leachate metagenome library [38]; billion clones per day [26]. This approach has been ii) 5-bromo-4-chloro-3-indolylcaprylate (X-caprylate) whose successfully applied to screen enzymes acting against hydrolysis produces blue colonies and has been successfully tributyrin and p-nitrophenyl butyrate [24, 25]. Genetic traps applied to detect one pyrethroid hydrolyzing esterase out of allow successful screens with an average limit of detection of 93,000 clones from top soil samples (5 to 10 cm) from approximately 105 molecules per cell [27]. vegetable soil [39]; iii) α-naphtyl acetate or laurate and Fast Blue B or RR, where positive clones appeared due to the 3. METHODS FOR HTS formation of a brown precipitate [40], and which has been successfully applied as primary screening method to detect 3.1. Functional HTS Assay Screenings (S)-ketoprofen specific esterases [20] and dual esterases:meta-cleavage C-C esterases [41], as well as in 3.1.1. Halo Formation Method mutant library screens for esterase and lipase activities [42]; and iv) indoxyl acetate [40], where positive clones appeared One of the simplest and commonly used methods to due to the formation of a blue precipitate. screen ELP enzymes is that based on the formation of halo in Although, p-nitrophenyl (pNP) are commonly used agar plates. Substrates typically used in mutant and for characterization of ELP enzymes, they are rarely used for metagenomic clone library screens included the following: i) metagenomic library screens. However, they have been tributyrin, trioctanoin, olive oil or triolein (1% v/v), successfully applied in HTS systems in protein engineering emulsified with Gum Arabic (e.g. [19, 28-31]); ii) Tween-20 studies, e.g. Victoria Blue polyvinyl (PVA)-p- and Tween-80 (0.5 -1.0 % v/v) and CaCl2 [32, 33]; iii) 3 poly nitrophenyl-myristate (pNP-C14)-agarose (VPMA) plates (DL-lactic acid) solution (1% v/v) [34, 35]; iv) poly(ε- [14], pNP 2-phenylpropanoate [43], 2-methyldecanoic acid caprolactone) solution (1% v/v) [36]; v) polyethylene pNP ester [44], 4-nitrophenyl 2-methylheptanoate [45], pNP terephthalate [36]; vi) egg yolk [37]; vii) cutin fibers [36]. In acetate [46], pNP butyrate [47], and pNP laurate [48]. Note all cases, colonies that showed clear zones on the indicator that the first HTS assay was performed by Reetz and co- plates are isolated as positive clones. These methods are  workers [49] using optically pure (R)- or (S)-2-methyl more appropriate for HTS assays on agar plated colonies decanoate pNP esters to find enantio-selective variants of a rather than in liquid assays. lipase from Pseudomonas aeruginosa. 3.1.2. Chromogenic Assay Method Another HTS protocol is the one using nonchromogenic triglycerides for lipases (both trioctanoin and tributyrin) and A number of chromogenic substrates are typically used to esterases (only tributyrin), and p-nitrophenol as indicator. screen for ELP enzymes. They include, the following: i) 608 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 Peña-García et al.

This method is based on the proton exchange between the by in vivo imaging using the FRET analysis of two labelled released fatty acid and the indicator. β-Cyclodextrin is phospholipids. One intact molecule contains a DNP needed to improve linearity. This method has been very quencher molecule which interacts with 4,4-difluoro-3a,4a- useful for enzyme screening in the hydrolysis toward short diaza-s-indacene or Boron DiPyrromethene as a fluorophore, and medium triglycerides but not suitable for biochemical then the A2 (PLA2) splits the lipid molecule characterization [50]. releasing the quencher that produces a green fluorescence.

Improved methods using purified triacylglycerols A similar activity assay for PLA2 is the use of pyrenePG (TAGs) from different sources (Punica granatum- (1-palmitoyl-2-(10-pyrenedecanoyl)-phosphatidylglycerol). pomegranate seed oil or Tung oil) have been used coated on This method allows direct quantification of hydrolysis based microtiter plates for the continuous assay of lipase activity on the observed difference in fluorescent emission of pyrene measuring UV absorption spectra at 272 nm. These kind of aggregated in vesicles compared to monomeric pyrene oils contain fatty acids with a conjugated diene or triene sequestered by binding to excess bovine serum albumin which allow their UV absorption when they are released [51, (BSA) after lipolytic activity. This method is a sensitive tool 52]. for HTS chemical libraries for potent PLA2 inhibitors [64]. For the particular case of phospholipase HTS assays, a 3.1.4. Mass Spectrometry Method substrate mixture containing 0.1% phosphatidyl-2-naphtol (P2NAP) and 0.05% Fast Blue B salt has been successfully This technique detects a wide range of compounds based applied for screening mutant libraries [53]. P2NAP, an on mass to charge ratio so it allows the direct quantification artificial substrate, is hydrolyzed by phospholipases to of the substrate and/or product and reduces the interferences generate 2-naphthol, which spontaneously reacts with co- using a triple quadrupole mass spectrometry. Özbal and co- existing Fast Blue B salt via diazo-coupling to form workers [65] developed this method analyzing the inhibitors insoluble red-purple dye [54], and improved variants are (more than 10,000 per day) of the esterase, detected as a red-purple spots on membranes, well or agar using chromatography. plates. A method that uses 4-nitro-3-(octanoyloxy)-benzoic Manfred T. Reetz and his team [66] described a method acid as substrate and the colored product 4-nitro-3-hydroxy- based on electrospray ionization mass spectrometry which benzoic acid, produced by the action of phospholipases, and allows the determination of enantio-selectivity in about which can be measured at 425 nm, have also been 1,000 catalytic or stoichiometric asymmetric reactions per successfully used [55]. Another UV spectrophotometric day. The technique is based on the use of isotopically labeled method using a specific glycerophosphatidylcholine (PC) substrates in the form of pseudo-enantiomers or pseudo- esterified at the sn-1 and sn-2 positions, with α-eleostearic prochiral compounds. acid (an intrinsic chromophore) has been developed for measuring the PLA activity and to screen new PLA and/or 3.1.5. Adrenaline Assay Method PLA inhibitors from biological samples [56]. This assay is used to profile lipases and esterases. It 3.1.3. Fluorimetric Assay Method consists of adding a measured amount of sodium periodate to the reaction assay, which reacts with any 1,2-diol product Fluorogenic and chromogenic substrates like formed by the enzymatic reaction of the corresponding ester. umbelliferone and nitrophenol have been used to monitor The remaining sodium periodate is back-titrated by the enzymatic activities [57]. Among fluorescence-based assays, addition of an excess of adrenaline, which reacts one of the best well known is the one to check lipolytic quantitatively and instantaneously with sodium periodate activity on agar plates containing olive oil (1 %, v/v) [58, 59] obtaining a red colored dye adrenochrome [67]. for hydrolysis activity, or fatty acids and fatty alcohols for synthesis activity [60], both with the fluorescent dye 3.1.6. In Vivo Growth Selection Method rhodamine B (0.001 %, wt/vol). Plates are incubated for 12- 16 h at 37 ºC and then subjected to UV irradiation (350 nm) This method allows a cell-viability based ultra-high and photographed. Positive lipase clones appeared due to the throughput systems which allow identifying esterase variants formation of orange fluorescent halo. However, this method with altered enantio-selectivity; the method is based on the only allows detecting very active variants or clones at a high utilization of a mixture of pseudo-enantiomeric esters, one of substrate concentration (typically 1% v/v). them acting as a potential carbon source for growth and the opposite as a potential growth inhibitor [68]. In this study, Apart for the method described above, this kind of authors used two carboxylic esters (enantiomers) whose methods is very sensitive and reduces background signals. hydrolysis produced either glycerol (serving as carbon Commonly used HTS fluorescence assay for esterase and source) or 2,3-dibromopropanol (serving as toxic compound) lipases included those based on the utilization of (Fig. 2). Therefore, this method could be applied to any umbelliferyl [61] and resorufin esters, which have been used chemically synthesized ester containing glycerol- and 2,3- to identify enantio-selective mutants for the resolution of dibromopropanol-like moieties. chiral carboxylic acids [62]. The fluorescence resonance energy transfer (FRET) is a 3.1.7. Acetic Acid-Specific Assay Method technique that is based on the interaction of two This method is based on the hydrolysis of an acetate ester chromophores and it is used to assay bond-cleavage catalyzed by an esterase or lipase that releases acetic acid, reactions, such as proteolysis of peptides. Farber and co- which is used to determine the initial rate of the reaction [69, workers [63] developed a screening of phospholipase activity 70]. Acetic acid is catalyzed by acetyl-CoA-synthetase HTS of Esterases, Lipases and Phospholipases in Metagenomic Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 609

1

Br Br

Esterase Br O Br H2O 2 -(S)-PBA HO

O

Fig. (2). Graphical scheme of the HTS systems based on in vivo selection methods. Here, hydrolysis of reactive 1, by an esterase or lipase, generates the carbon source glycerol supporting bacterial growth; opposite, hydrolysis of reactive 2 releases 2,3-dibromopropanol resulting in cell death [modified from 68].

(ACS) to acetyl-CoA in the presence of ATP and coenzyme the apparent enantio-selectivity. Finally, it is necessary to A (CoA). Next, this molecule and oxaloacetate are converted use analytical methods, such as chiral GC or HPLC to by a citrate synthase (CS) to citrate. Oxaloacetate is determine conversion, kinetic parameters, and the true produced by a L-malate-dehydrogenase (L-MDH) catalyzed enantio-selectivity (Fig. 4). This method has been also oxidation of malate yielding NADH. Because the product successfully used for the identification of a metagenome- NADH has absorbance at 340 nm, the initial rate of acetate derived esterase with high enantio-selectivity in the kinetic hydrolysis can be measured spectrophotometrically resolution of arylaliphatic tertiary alcohols [23], where 35 following this enzyme cascade (Fig. 3). clones encoding esterases out of 10,000 tested were found active for the hydrolysis of the (+)-enantiomer of 1,1,1- O trifluoro-2-phenylbut-3-yn-2-yl acetate. Note that acetic acid O OH formation can be also detected by pH indicator methods, as the reduction of pH by acetic acid increases the protonated O Esterase/Lipase + H O + 2 OH R1 R2 indicator concentration, producing a net change of the color R1 R2 Acetic acid assay (see section 3.1.8.). 3.1.8. Activity Using pH Indicator Method A sensitive and efficient HTS colorimetric method for Citrate CoA ATP detection of esterase and lipase enzymes, based on pH changes, has been successfully used. The method is CS ACS applicable to any kind of ester, and is based on the Acetyl-CoA AMP + Pyrophosphate H2O production of carboxylic acid after ester hydrolysis. Thus, the reduction of pH increases the protonated indicator concentration (typically p-nitrophenol, phenol red or bromothymol blue), changing the color of the reaction: i) if the indicator is p-nitrophenol, the color changes from yellow NADH + H+ + Oxaloacetate L-Malate + NAD+ L-MDH to colorless [18]; ii) if phenol red, from red to yellow [22]; and iii) if bromothymol blue, from blue to yellow [72]. This Fig. (3). Cascade of reactions performed in the acid acetic-specific method was initially applied to determinate the activity and assay. As shown, the final outcome from the action of an esterase or enantio-selectivity of lipases and esterases [17] using a pH lipase is NADH, which can be measured at 340 nm [modified from indicator to follow enzymatic hydrolysis. If the initial rates 69]. of hydrolysis of separate pure enantiomers is measured in the presence of an achiral reference compound (resorufin tetradecanoate), then it is plausible to determine what is This assay can be used to differentiate active from called apparent enantio-selectivity. Here, hydrolysis of the inactive and enantio-selective enzyme variants. In the chiral substrates yields the p-nitrophenolate ion, monitored at activity test [71] the result is the relative activity of each 404 nm (yellow color) and the hydrolysis of the reference enzyme variant for the tested acetate ester; in the selectivity compound resorufin is detected at 572 nm (pink color). This test, it is necessary to calculate the initial reaction rates for is the reason why this method has been commonly known as each enantiomer separately, then quotient of the two rates is “Quick E method” [73] (Fig. 5). 610 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 Peña-García et al.

Transfer of active clones

Metagenomic Agar activity Expression plate. library test Induction with rhamnose for enzyme production.

(S)-acetate Acetate assay (R)-acetate Cell lysis. Transfer an equal ammount of each well into two wells of the assay plate.

Fig. (4). Graphical representation of HTS systems for selecting enantio-selective enzymes. Screening of metagenomic libraries or mutant clone libraries for altered enantio-selectivity is performed by adding separately (R )- and (S )-substrates to the same enzyme variant [modified from 74].

CH3

O - yellow O

O NO2 NO2 (R) or (S) -1

- R O O O O O O

O pink N N

Resorufin ester Positive hydrolysis of enantiomer (R) or (S) (reference) Yelow + pink= deep orange Negative hydrolysis of enantiomer (R) or (S): Pink Fig. (5). Quick E method. The hydrolysis of the chiral substrates yields the yellow p-nitrophenolate ion, monitored at 404 nm and the hydrolysis of the reference compound resorufin (pink color) is detected at 572 nm.

This methodology has been used in engineering studies bromothymol blue as pH indicator [24, 62]. For [74] as well as for pure cultures [72] and metagenomic clone metagenomic library screens, substrates included 1,1,1- library screens [18, 22, 40, 75], as it can be used in liquid trifluoro-2-phenylbut-3-yn-2-yl acetate [23], ethyl ferulate and solid assays with a wide range of short- to large chain [77], methyl ferulate [76], polyaromatic hydrocarbon esters. As examples, this method has been applied to screen (phenanthrene, anthracene, naphthalene, benzoyl, for stereo-selective hydrolytic enzymes using acetates of protocatechuate and phthalate) esters [23], as well as 86 secondary alcohols as model substrates [74] and about other different esters that included 25 halogenated alkyl and aryl hundred carboxylic esters [22], as well as to screen for esters, 34 alkyl esters, 12 aryl esters, 10 hydroxycinammic feruloyl esterases using methyl ferulate [76]. Moreover, esters, 2 epoxides, 1 lactone, and 2 carbohydrate esters, all of although olive oil screens are traditionally performed in them using p-nitrophenol or phenol red as pH indicators (for combination with the fluorescent dye rhodamine B on agar the complete list see ref. [18]). plates, 15 lipases able to hydrolyze olive oil as the indicator substrate have been identified in metagenomic clone libraries 3.1.9. Synthesis Activity Assay Method created from soil samples in chromogenic substrate plates Hydrolysis of a product of interest is typically used for prepared by using phenol red (0.01 %) along with 1 % olive screening ELP enzymes. However, these enzymes are oil as substrate [59]. typically used as biocatalysts in synthetic processes. Recent examples of substrates applied in HTS methods, Therefore, HTS methods in which such capability is directly in a 96-well microtiter plate, in protein engineering studies tested, under conditions most similar to those required at an included: i) (R)- or (S)- 1-(3′,4′-methylenedioxyphenyl)ethyl industrial level, are desired. Following on from this, a recent acetate [13] and (R)- and (S)-enantiomer of tetrahydrofuran- method based on the trans-esterification between an alcohol 3-yl acetate (THF-3-Ac) [12] using p-nitrophenol as pH and a vinyl ester of a carboxylic acid, has been developed indicator; ii) (R)- or (S)-methyl mandelate using [78]. The acetaldehyde generated during the reaction, reacts HTS of Esterases, Lipases and Phospholipases in Metagenomic Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 611 with a hydrazine to produce hydrazone which is quantified acid (ABTS), which can be quantified at 695 nm (Fig. 7). In by a fluorimetric measurement. This principle allows the this experiment triglycerides are converted by the lipase A of rapid identification of active enzymes using a broad range of Candida antarctica in the presence of ethanol to obtained the solvents [78]. corresponding ethyl ester. Alcohol not consumed is quantified by the alcohol oxidase (obtaining acetaldehyde 3.1.10. Microfluidic Device-Based Method and hydrogen peroxide) and the peroxidase. This HTS method includes specific previous steps including single cell compartmentalization, screening for 3.2. Methods of HTS Based on Sequence Screenings enzymatic activity, and recovery of improved mutants or The revolution in the high throughput DNA sequencing active clones using microfluidic devices [25, 26, 79]. The first step is to lyse cells to expose the intracellular expressed technologies has resulted in a significant reduction of the sequencing costs and thus led to an explosion of in silico enzyme to the compartmentalized substrate. The droplet data production and dramatic expansion of the databases retains the fluorescent product with the DNA plasmid that [82]. In contrast to that, the pipelines for functional protein encodes the enzyme and then the plasmid from selected analysis operate at much lower rates and throughputs, droplets is electroporated into Escherichia coli. The obtained opening the gap between the numbers of proteins predicted plasmids were also used as DNA templates for the generation of new libraries to facilitate additional round of in silico [83] and those experimentally characterized in the lab: the proportion of the later asymptotically approaches directed evolution. zero per cent [84]. There is a growing appreciation that this 3.1.11. HTS Systems Based on Artificial Genetic Circuits emerging gap between the high throughput metagenomic sequencing data and experimentally characterized proteins A genetic circuit termed Genetic Enzyme Screening has to be dealt with [84-86]. For example, there are a few System, which has been applied in enzyme screening from existing US NIH- and DOE-sponsored initiatives to mitigate diverse microbial metagenomes, has been recently this, e.g. through large NIH-funded Structural Proteomics developed. Basically it consists in action of an enzyme Consortia (supported since 2001; towards its substrate and the further accumulation of phenol http://www.beem.utoronto.ca; funded by Genome Canada), inside the cells which induce a cascade of genetic events or a more recent COMBREX initiative [84], which looks producing a cellular fluorescence signal. The crucial feature into the systematic characterization of proteins from a few of this approach can be used to screen a variety of enzymes dozen reference microorganisms, including the best-studied that produce a phenol compound from respective synthetic microbes E. coli and Helicobacter pylori that combined have phenyl-substrates, including lipases and esterases. The only 0.33% of their gene functions characterized. There is highly sensitive and quantitative capacity of this genetic tool therefore an urgent demand in fostering functional analysis together with flow cytometry techniques could provide a of enzymes and, among them, ELP enzymes; this will widely relevant toolkit for discovering enzymes from required extensive HTS effort with known and novel metagenomic libraries and engineering novel enzymes at a chemicals. single cell level [80] (Fig. 6). Actually a number of sophisticated bioinformatics tools, 3.1.12. Other Methods than can be considered as HTS methods, have been designed for an initial sequence data mining and to make a rapid pre- Müller and co-workers [81] developed a method to selection of candidate genes encoding ELP enzymes. For quantify acyltransferase activity based on the consumption of that, predicted protein-coding genes are filtered according to the alcohol as co-substrate. The alcohol is firstly oxidized their similarity with generalist protein databanks (UniProt, producing H2O2 that is reduced by a peroxidase that also NCBI NR), conserved domains from Pfam and Common oxidizes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic

Substrates TF on

TF off Flow cytometry Single cell assay Single cell sorting Enzyme library

Fig. (6). Genetic Enzyme Screening System. The circuit consists of: 1) The target enzyme and its substrate, both responsibles for an accumulation of a phenol compound in the cell; 2) this compound and the activation of its transcription factor (TF) trigger the expression of a reporter gene producing a cellular fluorescence signal that can be measured by flow cytometry [modified from 80]. 612 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 8 Peña-García et al.

Oxidase ELP = esterase-lipase-phospholipase Alcohol Aldehyde HTS = high throughput screening pNP = p-nitrophenol O2 + H2O H2O2 PBA = 3-phenyl butyric acid Peroxidase H2O P2NAP = phosphatidyl-2-naphtol DNP = dinitrophenols

ABTSred ABTSox CONFLICT OF INTEREST Fig. (7). Cascade of reactions performed in the HTS oxidase assay for detecting esterase and lipase activity based on the consumption The authors confirm that they do not have any conflicts of an alcohol used as co-substrate and the final release of ABTS, of interest. which can be monitored at 695 nm [modified from 81]. ACKNOWLEDGEMENTS Domains database (CDD), or specific updated enzyme resources such as the Carbohydrate-Active Enzyme [87] The authors gratefully acknowledge the financial support which may help identifying esterase acting on carbohydrate provided by the European Community project MAMBA substrates, and the lipase/esterase and related databases [88, (FP7-KBBE-2008-226977), MAGICPAH (FP7-KBBE- 89]. In a second step, for each type sequences it is possible to 2009-245226), ULIXES (FP7-KBBE-2010-266473), and obtain general features of the proteins (mass, pKa, motifs, KILLSPILL (FP7-KBBE-2012-312139), and by Mexican existence or absence of a secretion signal), and protein Council CONACYT CB-2008-01 (101784). This project has sequences can be analyzed in detail to determine domains or also received funding from the European Union’s Horizon motifs that are specific for the desired activity, or structurally 2020 research and innovation program [Blue Growth: classified by the active sites modeling and clustering Unlocking the potential of Seas and Oceans] under grant (ASMC) method [90, 91]. agreement No [634486]. This work was further funded by grants PCIN-2014-107 (within the ERA NET-IB2 program), HTS bioinformatics tools applied to the screen of BIO2011-25012 and BIO2014-54494-R from the Spanish sequence data, have been successful to screen epoxide and Ministry of Economy and Competitiveness. The authors haloalkane hydrolases and [88], esterases-lipases [18] and gratefully acknowledge the financial support provided by the carbohydrate esterases [92]. In addition, coupled high- European Regional Development Fund (ERDF). C.P-G. throughput functional screening, using HTS approaches, and thanks Doctoral Fellowship CONACYT no. 372746. We next generation sequencing for identification of enzymes in thank Rafael Bargiela for his excellent support for the metagenome bio-resources are increasingly important to preparation of Fig. (1). generate enzyme collections [93]. 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