Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

ISSN: 2319-7706 Volume 4 Number 12 (2015) pp. 413-433 http://www.ijcmas.com

Original Research Article Identification and Characterization of Two Novel Thermostable and Thermoresistant Isolated from Rice Rhizosphere by Activity- Based on Metagenomic Screening

Elena Algar1, Doris Ribitsch2, Jose Antonio Lucas1, Beatriz Ramos-Solano1, Helmut Schwab2,3, F. Javier Gutierrez-Mañero1 and Ana Garcia-Villaraco1*

1Universidad San Pablo CEU, Department of Pharmaceutical & Health Sciences, Facultad Farmacia, Urb. Monteprincipe, Boadilla del Monte, 28668 Madrid, Spain 2acib Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz 3Graz University of Technology, Institute of Molecular Biotechnology, Petersgasse 14, 8010, Graz, Austria *Corresponding author

A B S T R A C T

A 72,000 recombinant phages metagenomic library was constructed from rice rhizosphere. An screening was performed and resulted in the identification K e y w o r d s of 6 positive esterase clones. Two of them, Ela1 and Ela2, were selected for a further characterization. Sequence analysis revealed that Ela1 exhibits a high Metagenomic, homology with proteins annotated as acetyl xylan esterase (AXE) and Ela2 with Esterases, SGNH . Both are carboxylic hydrolases, with a high Rhizosphere, stability, an alkaline optimum pH 8-9 and active at high temperatures (75°C). Lipolytic Additionally, a 16S rRNA library was performed in order to characterize the enzymes, biodiversity and biological diversity of the ecosystem source of this gene. It Biotechnology confirmed the predominance of thermophilic groups of bacteria matching with the esterases Ela1 and Ela2 annotation results and biochemical characterization. Thus, rice rhizosphere, which is a high-pressure selective ecosystem, arises as a very appropriate source of novel enzymes with a great potential for biotechnological and industrial applications.

Introduction Lipolytic enzymes attracting an enormous production of enantiopure pharmaceuticals, attention because of their biotechnological agrochemicals, and flavour compounds potential (Benjamin and Pandey 1998). They (Jaeger and Eggert 2002). constitute one of the most important group of biocatalysts for biotechnological Carboxylic ester hydrolases (EC 3.1.1.X) applications. Furthermore, novel generally known as lipolytic enzymes are a biotechnological applications have been diverse group of hydrolases that catalyze the successfully established using for the cleavage and formation of ester bonds synthesis of biopolymers and biodiesel, the

413 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

(Bornscheuer 2002). They include two techniques have been shown to provide a groups of enzymes, namely non-specific direct view of known or new protein esterases or carboxylesterases (EC 3.1.1.1) families and functionalities (Martínez- and lipases (EC 3.1.1.3), which have been Martínez et al. 2013). In this sense there is early, differentiated on the basis of their an increasingly greater number of studies specificity (Arpigny and Jaeger but to date only a few have used the 1999; Chahinian and Sarda 2009). rhizosphere as a source of esterases as is the case of Lee et al.(2010)and none have Lipolytic enzymes are widely distributed in specifically used the rhizosphere of rice. animals, plants and microorganisms. Many of them show a wide range of possible The rhizosphere is a selective environment, substrates and they also exhibit high source of specialized microorganisms. stereospecificity. These make them some of Besides, agronomic characteristics of rice the most versatile and important biocatalysts crop provide an environment with an in industrial biotechnology applications as increased selective pressure. All this makes the synthesis and hydrolysis of the rice rhizosphere potentially an excellent stereospecific compounds, including the source of new genes of interest. metabolic processing of drugs and antimicrobial agents (Bornscheuer 2002; Metagenomics will enable us to study the Jaeger and Eggert 2002; Panda and unknown ecological functions of microbial Gowrishankar 2005; Choi et al. 2013). communities, trying to unravel the biological functions that are present in a Lipases isolated from different sources have given environment or the type of organisms a wide range of properties depending on present therein. Additionally, by comparing their sources with respect to positional data from different environments, the specificity, fatty acid specificity, fundamental rules of microbial ecology, the thermostability, pH optimum, etc. (Huang mechanisms by which microorganisms adapt 1984). One could probably find a to environmental conditions or horizontal from nature that would be suitable for transfer phenomena may be studied. desired application. Moreover, identification of new genes and Esterases and lipases within the microbial proteins (new families of proteins) will be communities have particularly received possible and analysis of diversity within considerable attention, because they are known families may be carried out as basic widely distributed operating in most of knowledge studies. Identification of new environments where they have important genes and proteins is a relevant issue for physiological functions (McQueen and biotechnological applications. Functional Schottel 1987; Martínez-Martínez et al. metagenomics, which involves the 2013). Metagenomic approach based on heterologous expression of metagenomic expression is proposed as the most DNA in a surrogate host and activity-based advantageous compared to methods based screening, provides a means of discovering on independent culture for several reasons, genes, the function of which may not be such as sequencing errors (Gomez-Alvarez obvious from their sequence (Torres-Cortés et al. 2009) and the erroneous assignment of et al. 2011). substrate specificity (Fernández-Arrojo et al. 2010). In contrast, the activity-directed In view of the above, the aim of this work

414 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 was to carry out a metagenomic analysis in grid were united. At the end, 3 samples were an ecosystem such as the rizosphere to obtained. Rhizosphere soils were transported characterize genetic and functionally the to the laboratory at 4ºC and sieved (2mm rhizosphere microbial communities and on pore) before DNA was extracted within the the other hand, to discover new genes with following 48 hours. interest in agriculture and industry. DNA Extraction This study presents a metagenomic approach in rice cropping rhizosphere with two main DNA was extracted from 5 g soil samples, objectives: 1) Characterization of microbial using a PowerMax® Soil DNA Isolation Kit communities in rice rhizosphere through a (MoBio) and subsequently cleaned and structural analysis with 16S rRNA gene concentrated on Amicon® Ultra Centrifugal library and 2) Search of new lipolytic Filter Units (Millipore, Billerica, MA, enzymes: a function-based screening on a USA). DNA from the 3 replicates was 10kb phage library for esterases was pooled together. This DNA was used for carried out; a biochemical characterization 16S rRNA and metagenomic libraries of two esterases was performed in in order construction. to evaluate their potential for biotechnological applications. 16S rRNA Gene Library Construction

Materials and Methods Extraction of DNA was done as indicated above. Amplifications of 16S rRNA gene Soil Description and Sampling fragments were carried out using the primers 16sF (AGA GTT TGA TCC TGG CTC AG) The soil used to construct the metagenomic and 16sR (AAG GAG GTG ATC CAG library was a loamy soil from an agricultural CCG CA [52]. The 25 µl PCR mixtures field with a long history of rice cropping (10 consisted of: Ultratools PCR buffer 10X years) located in Vegas Altas Badajoz with MgCl2 (1X), dNTP Mix Ultratools (Spain) (38º 57´ 0´´ N, 5º 51´ 0´´ W) (each dNTP 250µM), Ultratools DNA (CASAT, www.casat.es). Sampling was polymerase (1,4 units), primer concentration conducted in July 2009 in tillering stage. (0.6µM, each). PCR products were purified Rice tiller is a specialized grain-bearing using the QIAquick PCR purification kit branch that is formed on the non-elongated (Qiagen) and then ligated into the pCR 4- basal internode and grows independently of TOPO vector (Invitrogen). Escherichia coli the mother stem (culm) by means of its own strain DH5 (Invitrogen) was then adventitious roots. transformed with the ligation products. One hundred clones were randomly picked and One hectare was marked within the suspended in tubes containing 6 ml LB and production area. Then it was surface grown for 24 hours at 37ºC. Plasmid DNA gridded, grids were numbered from 1 to 100 extraction was performed with the QIAprep and three grids (3 replicates) were selected Spin Miniprep kit (Qiagen) and fragment through a random number program. Three inserts were sequenced on a Applied plants (3 repetitions) of rice (Oryza sativa Biosystems 3730xl DNA Analyzer. var. Thaibonnet) were sampled in each grid. The soil closely adhered to roots Sequences were visualized with Sequence (rhizosphere soil) from the 3 plants of each Scanner software v1.0. and editing was

415 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 performed using the software Clone mg L-1 tetracycline and 50 mg L-1 Manager Professional Suite v6.0. Sequence kanamycin for strain and plasmid alignment was carried out on the server maintenance. MAFFT v6.0 (http://mafft.cbrc.jp/ alignment/software/) and annotated with The extracted metagenomic DNA was BLASTN 2.2.29. (Basic Local Alignment partially digested with Sau3A and subjected Search Tool, Altschul et al.(1997)in the to electrophoresis. DNA fragments of 9 10 National Center for Biotechnology kb in size were isolated from the gel and Information (NCBI: concentrated on Amicon® Ultra Centrifugal http://www.ncbi.nlm.nih.gov/) and Filter Units (Millipore, Billerica, MA, Ribosomal Database Project Release 10 USA). DNA (150 ng) was ligated to lambda (RDP: http://rdp.cme.msu.edu/) databases. vector, according to the recommended protocols. Library titre was determined by Biodiversity and richness estimation mixing various dilutions of the packaged (biological diversity) were carried out with ligation with E. coli XL1-Blue cells, DOTUR-1.53, which provided estimation of according to the manufacturer s instructions, biodiversity using the Shannon-Wiener and counted the plaques formed. index and of richness using Chao index. Distance considered between OTUs was The ZAP Express vector is designed to 0.03 in both cases. A phylogenetic tree with allow simple, efficient in vivo excision and the 16S rRNA soil bacteria partial sequences recircularization of any cloned insert of all the clones was inferred with contained within the lambda phage vector, MEGA5(Tamura et al. 2011)from aligned to form a phagemid (pBCK-CMV plasmid). sequences in MAFFT v6.0. Thus, it is possible to convert a lambda phage (Ferrer et al. 2005). The sequences reported in this work are available in the NCBI database under the The screening for lipolytic enzymes in this accession numbers, JQ30089-JQ300438. metagenomic library was performed directly on the lysis plaques produced by the phages. Metagenomic Library Construction and A total number of 15,000 phages were Activity-Based Screening plated to be screened. The esterase activity was revealed by the addition naphthyl Extraction of DNA was done as indicated and fast-blue (SIGMA). In this test, a above. Escherichia coli strain XL1-Blue 0.2 mg ml-1 naphthyl acetate in acetone and MRF was used for library construction and a 0.8 mg ml-1fast-blue in dimethylsulfoxide strain XLOLR was used for expression of solution were prepared. These two are added the lipolytic activity from phagemids to 0.5 % agarose (800 l of substrate per 45 (Stratagene). The pBK-CMV and the ZAP ml of agarose) and poured into the plates as Express vector digested with BamHI an overlayer on the colony or colonies (Stratagene) were used to construct the soil whose activity is to be tested. When metagenomic library. hydrolyzed, the naphthyl acetate reacts with the fast-blue, resulting in a brown precipitate Escherichia coli cells were routinely which indicates the phage lysis plaque cultured in LB medium at 37°C, and plated where esterase activity is occurring (Ferrer in LB medium contained 1.5% Agar. When et al. 2004). appropriate, media were amended with 10

416 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Sequencing and Gene Annotation of ligation with T4 DNA- (Fermentas, Positive Clones with Esterase Activity Germany) were performed in accordance to the manufacturer s instructions. Plasmid pBK-CMV phagemids were excised from Mini Kit from Qiagen (Germany) was used the two esterase positive selected phages to prepare plasmid DNA. Plasmids and using the coinfection with helper phage DNA fragments were purified by Qiagen according to Stratagene protocols. Then, DNA purification kits (Qiagen, Germany). pBK-CMV phagemids DNAs clones pBK- pET26b(+) (Novagen) was used to subclone CMVela1 and pBK-CMVela2 were the gene encoding for esterase activities and extracted and purified by using a Speedtools E. coli BL21 Gold (DE3) (Stratagene) was plasmid DNA purification kit (Biotools) used for heterologous expression. E. coli according to the manufacturer s instructions. strains were grown at 37°C in Luria-Bertani The insert DNA was sequenced using (LB) broth or on LB agar supplemented with universal primers and primer walking the appropriate antibiotics (kanamycin, 40- procedure as custom service by SECUGEN 50 g ml-1; tetracycline, 12.5 g ml-1). S.L. (Spain). ORFs in the assembled sequences were predicted by Vector NTI The following primer pairs were used to Suite 10 (Invitrogen, USA) and a BLASTX amplify ela1 and ela2 genes from the search performed against the NR database to corresponding phagemids: Ela1.fw (5´- access annotation information. Translated ATATACATATGGCTCTCTTTGACAAA protein sequences of ORFs coding for CC ACTGGA -3´) / Ela1.rev (5´-GCCGC hydrolases and their nearest neighbors were AAGCTTCCCCCAGAGATCGCCCAG- determined by BLASTP searches against the 3´) and Ela2.fw (5´-ATATACATA NCBI-NR database. Structural classification TGACCCGCACTCGACG- 3´) / Ela2.rev of protein domain were predicted by (5´-ATATA CATATGG CCGCGCAGC SUPERFAMILY database located at ATACC- 3´). Primers were designed to http://supfam.org/SUPERFAMILY/hmm.ht introduce NdeI and HindIII sites ml(Hasan et al. 2006).The signal peptide (underlined) in both cases and to remove predictions were conducted using SignalP ela2 signal peptide sequence. The PCR was 4.1 Server located at performed in 30 cycles, with the http://www.cbs.dtu.dk/services/SignalP/ corresponding phagemid DNA as template, (Petersen et al. 2011). 0.5 M of each primer, 0.2 mM dNTP s, 5 units HotStarTaq® DNA Polymerase Sequences of DNA inserts encoding for Ela1 (Quiagen) and 1× reaction buffer provided and Ela2 genes and several hypothetical by the supplier. NdeI and HindIII digested proteins were deposited in GenBank with PCR-products were purified and cloned into the following accession numbers, the expression vector pET26b(+) (Novagen) KC977252 and KF700947, respectively. introducing a C-terminal His Tag® sequence. Expression vectors harbouring General Recombinant DNA Techniques, esterase genes were transformed into E. coli Gene Cloning and Expression BL21-Gold (DE3) (Novagen). The nucleotide sequence was confirmed by DNA DNA manipulations were performed by sequencing (LGC genomics). standard methods. Digestion of DNA with restriction (New England The expression was done in shaking flasks. Biolabs, USA), dephosphorylation with Plasmids pET26b(+) harbouring ela1 and alkaline (Roche, Germany) and ela2 respectively, were freshly transformed 417 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 in E. coli BL21 and transformants were used activity of the purified protein, p- to inoculate 30 ml LB broth supplemented nitrophenyl (p-NP) of acetate (p- with 40 g ml-1 kanamycin. Cultures were NPA) and butyrate (p-NPB) and -naphthyl grown overnight at 37 °C on a rotary shaker acetate (Sigma-Aldrich) were employed as (130 rpm). Then, cultures composed of 200 substrates. The amount of p-nitrophenol or ml LB supplemented with 40 g ml- 1-Naphthol released by esterase-catalyzed 1kanamycin were inoculated to obtain an hydrolysis were used to determine the optical density (600 nm) of 0.1. The cultures catalytic activity. The reaction mixture for were grown at 37 °C and 130 rpm until an p-NP esters was done as follows. Two optical density of 0.6 was reached. Then the hundred L of the substrate solution cultures were incubated at 20, 25, 28 and 37 prepared in 50 mM Tris HCl buffer pH 7 °C, respectively and induced with IPTG at a and 10% DMSO was mixed with 20 L of final concentration of 0.05 mM. The induced enzyme solution. The product (p- cultures were incubated with shaking at 130 nitrophenol) was measured at 405 nm at rpm for 22 h and samples at 0, 3, 6, 8 and 22 25°C for 5 min. The reaction mixture for - hours were collected during the induction naphthyl acetate consisted of 0.6 mg ml- time. Finally, protein expression was 1Fast Blue RR (Sigma-Aldrich) dissolved in analyzed by SDS-PAGE, in 12% 50 mM Tris HCl buffer, pH 7, 2% DMSO polyacrylamide gels, and visualized by and 1% Triton X-100. Two hundred L of Coomassie Brilliant Blue staining. the substrate solution was mixed with 50 L of enzyme solution and the product (1- Protein Purification naphthol) was measured at 450 nm at 25°C for 10 min. A blank was measured using Induced cells were harvested by buffer instead of enzyme solution. One unit centrifugation at 2150 g and 4 °C for 30 of enzyme activity was defined as the min. Cell pellet was disrupted by sonication amount of enzyme needed to release 1 mol under ice cooling (cell disruptor, p-NP or 1-Naphthol per min under the BRANSON Ultrasonics) and cell debris was above-mentioned conditions. A Spectromax removed by centrifugation at 26300 g for 30 Plus 384 plate reader (Molecular Devices) min and 4 °C. Purification of the was used to follow the reaction unless 6xHistidine-tag enzymes from soluble otherwise indicated. All measurements were fraction was performed with Ni-NTA performed in triplicate and repeated three Sepharose Column (IBA BioTAGnology, times. The values were the mean of the data. Germany). The elution buffer was exchanged with 100 mM Tris HCl pH 7 by Kinetic Parameters and Thermostability the use of PD-10 desalting columns (GE Healthcare). The protein concentration was Kinetic parameters were determined under measured using the Bradford protein assay the standard conditions described above. (Bradford 1976). Esterase activity was determined with different substrate concentrations ranging Enzyme Characterization from 0.05 mM to 8 mM for p-NP esters and from 0.15 mM to 3.3 mM for -Naphthyl The purified Ela1 and Ela2 were subjected acetate. Values of Km, Vmax, kcat and kcat/Km to a series of biochemical analyses, were calculated from Hanes-Woolf plots in including specific activity, optimum pH, conditions described above using the optimum ionic strength, optimum SigmaPlot 11.0 software. temperature and thermostability. To measure 418 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Thermostability was investigated as follows. from the structural study indicated that the Purified enzymes were incubated in 100 mM ecosystem had a high biodiversity and high Tris HCl buffer pH 7 at 4, 25, 37 and 50 °C biological diversity (Shannon-Wiener = 4.36 at different time intervals to determine bits). The taxonomic classification of the remaining enzyme activity by the standard sequences (Figure 1) showed a assay with p-NPA. Enzyme activity was predominance of Gram-negative bacteria, monitored until a sharp decrease was being the Proteobacteria group the most detected. The Kinetic parameters Km, Vmax, abundant, which is frequent in soil. It is kcat and kcat/Km were also calculated. striking the relatively high presence of Acidobacteria phylum bacteria. Although Optima for Temperature, Ionic Strength this group is frequently found in soils, little and pH is known about them since they are non- culturable bacteria. Among the Gram The effect of temperature on enzyme positive bacteria, the Firmicutes phylum was activity and the optimum ionic strength was present but not very numerous and the investigated using 5.1 mM p-NPA as Actinobacteria phylum was abundant. substrate under the standard conditions described before with the following There was also presence of the phylum modifications. The final volume reaction Chloroflexi (represented almost exclusively was 1 ml and the substrate solution was by the family Anaerolineaceae), the phylum previously conditioned at different Verrucomicrobia and the family temperatures by incubating 4 min, then the Planctomycetaceae. reaction was conducted at the different temperatures ranging from 10 to 75 °C. A Construction of a Metagenomic Library Cary Series UV-Vis Spectrophotometer and Esterase Activity Screening (Agilent Technologies) was used to follow the reaction. The optimum ionic strength A metagenomic library (10 kb inserts) was was studied under the standard conditions by constructed with DNA extracted from rice adding different concentrations of NaCl (0- rizosphere (Badajoz, Spain) in tillering. A 0.9 M) into the standard reaction mixture. total of 72,000 recombinant phages were obtained using the lambda ZAP-expressing The effect of pH on enzyme activity was phagemid system and subsequently determined using 3.3 mM -Naphthyl amplified. The amplified library was acetate as substrate at 25 °C in the following subjected to functional screening for novel buffers: 50 mM citrate buffer (pH 4-6), 50 esterase enzymes (Reyes-Duarte et al. mM Tris-HCl (pH 7-8), 50 mM Glycine- 2012). Approximately 15,000 phages were NaOH buffer (pH 9-10). The molar screened and 6 esterase positive clones were extinction coefficients of 1-Naphthol were identified, purified and excised into pBK- corrected for pH variation. CMV phagemids (Mølgaard et al. 2000; Ferrer et al. 2009). Results and Discussion Identification of Esterase Genes and Structural Characterization of Microbial Sequence Analysis Communities with 16S rRNA Gene Library Two esterase positive clones were randomly selected for a further characterization. Analyses performed with the sequences Inserts of the selected clones called pBK- 419 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

CMVela1 and pBK-CMVela2 were YP_003321945; identity 63%) being a completely sequenced by primer walking thermophile bacteria closely related to the using universal primers. Both inserts were phyla Chloroflexi and Thermomicrobia lower than 10 kb in size (7.5 and 8.6 kb, (Botero et al. 2004).Moreover, Ela1 respectively). Subsequent BLASTX analysis exhibited similarity with a thermostable using the NCBI non-redundant protein acetyl esterase from Thermotoga maritima database revealed 3 and 4 open reading (accession no. 3M81_A; identity 60%) frames (ORF) in the 7.5 and 8.6 kb inserts, (Martínez-Martínez et al. 2013) and acetyl respectively. In both cases one of the xylan esterases from others thermophile identified ORF showed similarity with a and/or thermo-resistant bacteria as carboxylic ester . In the case of the Dictyoglomus thermophilum (accession 7.5 kb DNA fragment, the ORF consisted of no.YP_002251796; identity 61%); 987 nucleotides and encoded a protein of Thermobispora bispora DSM 43833 328 amino acids (named Ela1). The 8.6 kb (accession no.YP_003652570; identity DNA fragment contained an ORF of 783 55%); and bacterium with xylan degrading nucleotides and encoded a protein of 260 enzymes such as Acidothermus amino acids (named Ela2), moreover, a cellulolyticus 11B (accession Signal P4.1 analysis showed up acleavage no.YP_873803; identity 56%) and site between amino acid positions 24 and 25. Cellulomonas flavigena DSM 20109 (accession no. YP_003636033;identity 55%) Analysis of the deduced amino acid (Morris et al. 1998; Barabote et al. 2009; sequence revealed that Ela1 belongs to Abt et al. 2010; Liolios et al. 2010). alpha/beta-hydrolases superfamily and to the Alignment between Ela1 and Ela2 showed a acetyl xylan esterase-like . 14% of matching (according to Clone The common sequence Gly-X-Ser-X-Gly Manager 6.0, Align Plus 4.10. Method: found in many esterases, lipases, and serine FastScan- Max Qual). proteases (Brenner 1988) was located as Gly-Gly-Ser-Gln-Gly in the sequence from On the other hand, the analysis of the positions 186 to 190. Moreover a peptidase deduced amino acid sequence of Ela2 S9 domain was detected. A phylogenetic revealed that this protein belongs to SGNH tree was constructed with ten retrieved hydrolase superfamily and specifically to the protein sequences from the NCBI database platelet-activating factor-acetylhydrolase that showed high similarity with Ela1 (PAF-AH) like subfamily. Moreover an (Figure 2). This analysis showed that Ela1 similar to the typical Ser-His-Asp exhibited the higher similarity with several (Glu) triad from other serine hydrolases proteins annotated as acetyl xylan esterase- (Ser84-Asp233-His236) and the oxyanion like protein from bacteria of the phylum hole that characterizes these enzymes were Chloroflexi as Caldilinea aerophila predicted. Members of the SGNH-hydrolase (accession no. YP_005442439; identity superfamily facilitate the hydrolysis of a 73%); Roseiflexus sp (accession no. broad range of substrates, including YP_001275160; identity 66%);Anaerolinea polysaccharides, lysophospholipids, and thermophile (accession no. YP_004175118; other ester containing compounds (Mølgaard identity 64%);Herpetosiphon aurantiacus et al. 2000; Lo et al. 2003), giving SGNH (accession no.YP_001543662; identity hydrolase great potential in both basic 63%). Ela1 also showed high homology research and industrial applications (Bae et with an acetyl xylan esterase from al. 2013). Thermobaculum terrenum (accession no. 420 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

A phylogenetic tree was also constructed couples of primers for subcloning Ela1 and with protein sequences that showed high Ela2 into pET26b (+) were designed and similarity with Ela2 (Figure 3). In this case, ligation products were transformed into the analysis revealed that Ela2 showed E.coli BL21 for expression assays. The similarity with different classes of proteins, expression was strongest at 25 °C for both showing the highest similarity with several enzymes. In figure 4 the monitoring of the proteins annotated as GDSL family lipolytic expression of Ela1 and Ela2 in the soluble protein as the case of Pirellula staleyi DSM fraction at 0, 3, 6, 8, 22 hours after induction 6068 (accession no. YP_003370957.1; with IPTG and purification result is shown. identity 72%), Rhodopirellula maiorica For Ela1, high expression was obtained at 6 (accession no. WP_008697892.1; identity hours after induction and this expression 59%), Pedosphaera parvula (accession no. was maintained up to 22 hours. However, in WP_007416179.1; identity 44%), the case of Ela2, the expression was very Candidatus Nitrospira defluvii (accession low throughout the trial. no. YP_003799892.1; identity 50%), Marinomonas sp. MWYL1(accession no. The molecular mass of Ela1 and Ela2 were YP_001339539.1; identity 47%), and predicted to be 37.9 and 27.8 kDa, Pseudoalteromonas atlantica T6c(accession respectively; on the basis of the amino acid no. YP_663334.1; identity 45%). Also sequence using ProtParam tool (Swiss proteins annotated as 1-alkyl-2- Institute of Bioinformatics) and the mobility acetylglycerophosphocholine esterase on the SDS polyacrylamide gels (Fig. 4), showed a high similarity with Ela2, as the consistent with the molecular masses case of Planctomyces limnophilus DSM obtained for an acetyl xylan esterase from 3776 (accession no. YP_003630933.1; Bacillus subtilis (Tian et al. 2014) and for identity 65%), Glaciecola mesophila the recombinant protein FNE, classified as (accession no. WP_006991740.1; identity SGNH hydrolase, from Fervidobacterium 46%), and with N-acetylglucosamine-6- nodosum (Yu et al. 2010). from Blastopirellula marina and Verrucomicrobium spinosum (accession no. Kinetic Parameters WP_002649964.1, identity 48%; accession no.WP_009962309.1; identity 48%, Enzymes Ela1 and Ela2 hydrolyzed acyl respectively). All this proteins showed a esters.However, with p-NPB as substrate, SGNH hydrolase-type esterase domain but neither enzyme showed any activity (Table the molecular functions of these 1). Ela1 exhibited specific activities of 110.3 homologous proteins were inferred from and 72.9 µmolmin-1mg-1 proteinfor p-NPA electronic annotation and there is no more and -Naphthyl acetate, respectively. information in the literature about its Conversely, Ela2 exhibited a higher specific demonstrated structure or functions. activity for -Naphthyl acetate (86.5 U mg- 1protein) than p-NPA (40.7 U mg-1protein). Cloning and Heterologous Expression The Km and Vmax values were also determined from the Hanes-Woolfplot and Once the encoding esterases genes were subsequently kcat and kcat/Km parameters identified and their possible roles predicted, were calculated (Table 1) supporting a high we proceeded to the subcloning of both affinity toward p-NPA and -Naphthyl genes and its heterologous expression to acetate for Ela1 and Ela2, respectively. finally perform a biochemical characterization of the purified enzymes. So 421

Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Table.1 Specific Activities and kinetic Parameters of Ela1 and Ela2a

Specific activity V K max k k /K [µmol min 1 m [µmol cat cat m [mM] [1 s-1] [s-1mM-1] mg 1)] min 1] Ela1 p-NPA 110.3 ± 3.3 0.4 ± 0.04 93.3 ± 2.8 69.4 173.4 p-NPB 0 0 0 0 0 -Naphthyl acetate 72.9 ± 2.4 0.7 ± 0.06 61.8 ± 2.0 45.9 65.6 Ela 2 p-NPA 40.7 ± 1.1 0.3 ± 0.01 4.1 ± 0.05 19.0 62.6 p-NPB 0 0 0 0 0 -Naphthyl acetate 86.5 ± 1.8 0.2 ± 0.01 8.7 ± 0.1 40.3 201.5 a.Measurements were taken under standard conditions (pH 7 and 25° C). Ela1 data correspond with data taken after 24 hours of incubation at 25 °C. Kinetic measurements for each substrate were repeated three times and the values are given ± SE for specific activity, Km and Vmax. Kcat and Kcat/Km parameters were calculated by using the previous averages

Figure.1

Phylogenetic tree performed with the 16S rRNA sequences. The evolutionary distances were inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 500. The tree was conducted with MEGA5 [48]. Phylogenetic classification (taxonomic levels) was obtained in Ribosomal Database Project (http://rdp.cme.msu.edu).

422

Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Figure.2

Evolutionary relationships of Ela1 and homologous proteins obtained from NCBI database. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The tree was conducted with MEGA5 [48]

Figure.3

Evolutionary relationships of Ela2 and homologous proteins obtained from NCBI database. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The tree was conducted with MEGA5 [48]

423

Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Figure.4

SDS-PAGE analysis (4-12%) of esterases Ela1 (A) and Ela2 (B) expressed in E. coli BL21-Gold(DE3) and purified by affinity chromatography. Key: M, pre-stained protein molecular weight marker SM0671 (Fermentas); Lanes 1-5: cleared lysates from induced cells after 0 h, 3 h, 6 h, 8 h and 22 h; Lane 6: purified esterase

Figure.5

(A) (B) 100 100 ) )

% 80 % 80 ( ( y y t t i i v v i i

t 60 4 °C t 60 4 °C c c a 25°C a 25°C e e v v i i

t 40 37 °C t 40 37 °C a a l l

e 50 °C e 50 °C R R 20 20

0 0 0 24 48 72 96 168 0 24 48 72 96 168 Incubation time (h) Incubation time (h) Stability of (A) Ela1 and (B) Ela2. Purified enzyme was first incubated for 0-168 hours under 4, 25 and 37 °C and for 0-48 hours at 50 °C. Then, the residual activity was measured using p-NPA as the substrate under standard conditions (pH 7 and 25° C) and repeated three times. The highest activity was set as 100%. Values are given ± SE Figure.6

(A) 100 (B) 100 90 90 80 80 ) ) % %

( 70 ( 70 y y t t i i

v 60 v 60 i i t t c c

a 50 a 50 e e v v i i t 40 t 40 a a l l

e 30 e 30 R R 20 20 10 10 0 0 10 15 20 25 30 35 40 45 50 55 60 65 70 75 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Temperature ( C) Temperature ( C) Optimum temperature of (A) Ela1 and (B) Ela2. Enzyme activity was measured under various temperatures (10- 75°C). Measuremets were taken in 50 mM Tris HCl buffer pH 7 using p-NPA as substrate and repeated three times. The highest activity was set as 100%. Values are given ± SE

424 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Figure.7

(A) 100 (B) 100

80 80 ) ) % % ( ( y y t t i 60 i 60 v v i i t t c c a a e e

v 40 v 40 i i t t a a l l e e R R 20 20

0 0 4 5 6 7 8 9 10 4 5 6 7 8 9 10 pH pH Optimum pH of (A) Ela1 and (B) Ela2. Enzyme activity was measured under various at pH (4-10). Measurements were taken at 25 °C using -Naphthyl acetate as substrate and repeated three times. The highest activity was set as 100%. Values are given ± SE

Figure.8

(A) 120 (B) 120

100 100 ) ) % % ( (

y 80 y 80 t t i i v v i i t t

c 60 c 60 a a e e v v i i t 40 t 40 a a l l e e R R 20 20

0 0 0 0,15 0,3 0,45 0,6 0,75 0,9 0 0,15 0,3 0,45 0,6 0,75 0,9 NaCl [M] NaCl [M] Optimum ionic strength of (A) Ela1 and (B) Ela2. Esterase activity was measured under standard conditions (pH 7 and 25° C) with 0-1 M NaCl using p-NPA as the substrate and repeated three times. The highest activity was set as 100%. Values are given ± SE

Influence of Temperature, pH and Ionic On the other hand, at 4 and 25 °C was also Strength on Esterase Activity very stable presenting the 88 and 100% of the activity up to 96 hours, respectively. The stability at 4, 25 and 37 °Cduring 1 Furthermore, after 48 hours at 50 degrees, it week and at 50 °C for 48 hours were also still showed 64% activity. On the other assayed. Results are summarized in figure 5 hand, Ela2 also showed high stability but where the remaining specific activity during more moderate than Ela1. In this case, the the experiment is shown. Interestingly, Ela1 enzyme retained 90, 96, 100 and 71% of its displayed a higher activity after 24 hours of activity at 4, 25, 37 and 50 °C, respectively incubation in the case of all temperatures at 24 hours, and showed no other changes assayed suggesting that after purification a along the experiment. period of time for the correct folding is required. Both enzymes showed a high Both enzymes were active between 10 °C stability but Ela1 showed greater stability and 75°C (Figure 6) and showed similar than Ela2. Ela1 was highly stable at 37 °C as optimal temperatures (40-45 ºC). In the case 97% of its activity is retained at 168 hours. of Ela1, the optimal temperature for its

425 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 activity was 45°C (Figure 6 a) and it was presence of microorganisms belonging to 40°C for Ela2 (Figure 6 b). By contrast, the the phylum Planctomycetes such as lowest activity for Ela1 was at 10°C and for Pirulella, Planctomyces and Ela2 at 75°C. Ela1 retained values of up to Verrucomicrobium in thermophilic granular 76% while Ela2 retained only 51% of the sludges (Sekiguchi et al. 1998) and the activity in the most unfavorable temperature presence of lipolytic enzymes showing conditions. homology with proteins from Planctomyces, Rhodopirulella, Blastopirulella and Concerning optimum pH, purified Ela1 and Pirulella in activated sludge of a swine Ela2 also displayed activity between pH 6 wastewater (Liaw et al. 2010). and 9, with an optimal activity at pH 9 and 8, respectively (Figure 7). Ela1 showed Identification of Esterase Genes and higher tolerance to pH than Ela2. The Sequence Analysis enzyme activity was found to be over 65%, at pH 7 and 8 for Ela2 while Ela1 Analysis of the deduced amino acid maintained it at pH 9. sequence revealed that Ela1 belongs to alpha/beta-hydrolases superfamily and to the Finally, the optimum ionic strength was acetyl xylan esterase-like protein family. assayed and, again, similar results were Moreover, a phylogenetic tree was obtained for both enzymes. They were constructed with ten retrieved protein active from 0 to 0.9 M NaCl being the sequences from the NCBI database that enzyme activity over 80% up to 0.6 M in all showed high similarity with Ela1 (Figure 2). cases (Figure 8). This analysis showed that Ela1 exhibited the Structural Characterization of Microbial higher similarity with several proteins Communities with 16S rRNA Gene annotated as acetyl xylan esterase-like Library protein from bacteria of the phylum Chloroflexi as Caldilinea aerophila, Besides the presence of bacterial groups Roseiflexus sp, Anaerolinea thermophile typically found in soil, it is also interesting Herpetosiphon aurantiacus and to note the presence of some other bacterial Thermobaculum terrenum. Moreover, Ela1 groups which, although are not highly exhibited similarity with a thermostable abundant they are directly related to the acetyl esterase from Thermotoga maritima annotations found for the two esterase genes and acetyl xylan esterases from others isolated and studied in this work. This is the thermophile and/or thermo-resistant bacteria case of the phylum Chloroflexi (represented as Dictyoglomus thermophilum, almost exclusively by the family Thermobispora bisporaand bacterium with Anaerolineaceae), the phylum xylan degrading enzymes such as Verrucomicrobia and thefamily Acidothermus cellulolyticus and Planctomycetaceae. Many species from Cellulomonas flavigena, both included in the Chloroflexi and Planctomycetes phyla are Class Actinobacteria which was highly thermophilic (Sekiguchi et al. 1998) which represented in this rhizosphere according to is completely in accordance with the the microbial community structural analysis annotations found for esterases Ela1 and (Figure 1). Ela2 and with the thermostability shown by these two esterases. These data are These results suggest that Ela1 is a consistent with other studies that also report novelacetyl xylan esterase since all the 426 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 homologous proteins are annotated as such superfamily facilitate the hydrolysis of a protein (Figure 2) and because some of them broad range of substrates, including belong to microorganisms with polysaccharides, lysophospholipids, and demonstrated xylanolytic activity (Barabote other ester containing compounds (Mølgaard el al. 2009; Abt et al. 2010). Acetyl xylan et al. 2000; Lo et al. 2003), giving SGNH esterases are enzymes that hydrolyse the hydrolase great potential in both basic ester linkages of the acetyl groups of the research and industrial applications (Bae et xylose moieties of natural acetylated xylan al. 2013). from hardwood. These enzymes are one of the accessory enzymes, which are part of the The phylogenetic tree constructed with xylanolytic system, together with xylanases, protein sequences that showed high beta-xylosidases, alpha-arabino- similarity with Ela2 (Figure 3), revealed that furanosidases and methylglucuronidases; Ela2 showed similarity with different classes these are all required for the complete of proteins, showing the highest similarity hydrolysis of xylan. Xylan, next to cellulose, with several proteins annotated as GDSL is the most abundant renewable family lipolytic protein as the case of polysaccharide in nature. It is a major Pirellula staleyi, Rhodopirellula maiorica, hemicellulose component of plants and is Pedosphaera parvula, Candidatus located predominantly in the secondary cell Nitrospira defluvii, Marinomonas sp. walls of angiosperms and gymnosperms. Pseudoalteromonas atlantica. Also proteins The esterases involved in xylan breakdown annotated as 1-alkyl-2-acetyl- could be an important research tool in glycerophosphocholine esterase showed a studying and resolving cell wall structure high similarity with Ela 2, as the case of and mechanisms. Furthermore, these Planctomyces limnophilus; Glaciecola enzymes could be applied to the pulp and mesophilaand with N-acetylglucosamine-6- paper industry(McCleary and Matheson sulfatases from Blastopirellula marina and 1987; Christov and Prior 1993) as reported Verrucomicrobium spinosum. for xylanases from Dictyoglomus(Morris et al. 1998) or Cellulomonas (Abt et al. 2010). All this proteins showed a SGNH hydrolase- However, in all cases the acetyl xylan type esterase domain but the molecular esterase function is based in a sequence functions of these homologous proteins were prediction. inferred from electronic annotation and there is no more information in the literature The fact thatEla1 presents homology with about its demonstrated structure or the enzymes of the microorganisms functions.However, there is information in described above makes us think that the the literature, which relates Ela2 with a rhizosphere is also an ecosystem with a high A2 activitybecause thePAF- potential for the discovery of new glycosyl AH domain predicted for Ela2 is related hydrolases. with a function. This possible function is very interesting because The analysis of the deduced amino acid the discovery of the prokaryotic sequence of Ela2 revealed that this protein phospholipase A2 domain is relatively belongs to SGNH hydrolase superfamily and recent(Matoba et al. 2002).Phospholipase specifically to the platelet-activating factor- A2 hydrolyzes the 2-acyl ester bonds of 1,2- acetylhydrolase (PAF-AH) like subfamily. diacylglycero-3-phospholipids(Matoba et al. Members of the SGNH-hydrolase 2002; Sugiyama et al. 2002).Due to the

427 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433 versatility of this class of enzymes the removing the native signal peptide and potential functions of Ela2 are very diverse, fusing with pelB signal sequence. in a previous study (Sugiyama et al. 2002) an interesting possibility was proposed that Biochemical Characterization: Kinetic is how Streptomyces cells might produce the Parameters secreted PLA2 to take ecological priority among microorganisms living in a soil so Enzymes Ela1 and Ela2 hydrolyzed acyl PLA2 might function as a toxin to kill esters.However, with p-NPB as substrate, microorganisms having phosphatidylcholine neither enzyme showed any activity (Table as a membrane component (Sugiyama et al. 1). kcat and kcat/Km parameters calculated 2002). Moreover, the potential of these (Table 1) showed a high affinity toward p- esterase production genes is twofold, firstly NPA and -Naphthyl acetate for Ela1 and new esterase genes may have application in Ela2, respectively. These findings again are the industry, and also, an environmental consistent with the results obtained by Yu et application using these enzymes to break the al. (2010) and by Koseki et al.(2006) for a AHLs lactone ring and therefore disrupting SGNH hydrolase and an AXE, respectively, the quorum sensing of pathogenic bacteria in both cases, purified esterases displayed plant, and therefore, playing a key role in the greatest hydrolytic activity toward short plant fitness in natural environments and chain esters (C2). plant resistance to pathogens. Influence of Temperature, pH and Ionic Cloning and Heterologous Expression Strength on Esterase Activity Monitoring of the expression of Ela1 and Ela2 in the soluble fraction at 0, 3, 6, 8, 22 Both enzymes were active between 10 °C hours after induction with IPTG and and 75 °C (Figure 6) and showed similar purification showed that, for Ela1, high optimal temperatures (40-45 ºC). In the case expression was obtained at 6 hours after of Ela1, the optimal temperature for its induction with IPTG and this expression was activity was 45°C (Figure 6 a) and it was maintained up to 22 hours. However, in the 40°C for Ela2 (Figure 6 b). By contrast, the case of Ela2, the expression was very low lowest activity for Ela1 was at 10°C and for throughout the trial (Figure 4). In both cases Ela2 at 75°C. Results correlate well with the purification of the enzymes was those obtained in stability experiments satisfactory because although the (Figure 5), again, Ela1 showed higher concentration was lower for Ela2 this was activity than Ela2 at different temperatures enough for the further biochemical other than optimal temperature. Ela1 characterization. retained values of up to 76% while Ela2 retained only 51% of the activity in the most The results obtained for Ela2 may be unfavorable temperature conditions. All because it is a secreted protein and therefore, these results together are very promising although the signal peptide was eliminated since heat resistance and thermostability are in the subcloning process, the intracellular shown. Moreover, both enzymes, and expression may not be the most convenient. especially Ela1, might be adapted to a wide This assumption agrees with the recent work temperature range which is comparable to of Takemori et al. (2012) where periplasmic other esterases such as the QsdH from production of phospholipase A2 from Pseudoalteromonas byunsanensis that Streptomyces violaceoruber is optimized by exhibits activity ranging from 20°C to 60°C, reaching its optimum activity at 40°C 428 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

(Huang et al. 2012). These results are also in with QS-mediated signal molecules also agreement with the results of phylogenetic frequently occurs not only between bacteria analysis, which demonstrated that Ela1 and but also between bacteria and higher plants Ela2 showed homology with enzymes from (Braeken et al. 2008). Enzymatic AHL- heat-resistant microorganisms or identified degrading activities seem to be widespread from thermophilic granular sludges. and have been described in many species (Park et al. 2003; d´Angelo-Picardet al. Concerning optimum pH, purified Ela1 and 2005). Ela2 also displayed activity between pH 6 and 9, with an optimal activity at pH 9 and In summary, enzymes Ela1 and Ela2 show 8, respectively (Figure 7). These results an excellent biotechnological application suggest that Ela1 and Ela2 are alkaline potential because they are very stable, have enzymes as it was found for others heat resistance and are alkaline enzymes. esterasesisolated from plant rhizosphere soil Both could be used for many applications metagenome (Lee et al. 2010). Furthermore, with high interest in different industries after alkalophilic thermophiles have great a more comprehensive biochemical potential in detergent and leather industries characterization. Our results also show that (Hasan et al. 2006). rice rhizosphere, which is a high pressure ecosystem and therefore can be considered a Finally, the optimum ionic strength was great selective ecosystem, is very useful to assayed and both enzymes were active from isolate novel enzymes by functional 0 to 0.9 M NaCl being the enzyme activity metagenomics approaches. over 80% up to 0.6 M in all cases (Figure 8). Opposite results were found by Selvin et Acknowledgment al.(2012)where the recombinant lipase Lpc53E1 from the metagenome of a marine This research was supported by Ministerio sponge displayed increased levels of activity de Ciencia e Innovacion of Spanish upon exposure to increased concentrations Government AGL2006-13758-C05-02; of NaCl, probably associated to the higher AGL2009-13487-C04-04, BES-2007-15505, environmental pressure in the marine and Comunidad de Madrid CAM S- system. 0505/AMB/000321; S2009/AMB-1511. We thank Universidad CEU San Pablo and All this findings explain that microbial Banco Santander by awarding scholarship lipases are currently receiving considerable that allowed collaboration between the attention due to their diversity in catalytic university and tACIB. activity, high yield and low cost production, and relative ease of genetic manipulation References (Fan et al. 2011). Also, they have a great potential for agriculture since Abt, B., Foster, B., Lapidus, A., Clum, A., communication by quorum sensing (QS) Sun, H., Pukall, R., Lucas, S., Glavina between species, either synergistically or Del Rio, T., Nolan, M., Tice, H., competitively, may play an important role in Cheng, J.F., Pitluck, S., Liolios, K., the dynamics of these microbial Ivanova, N., Mavromatis, K., communities. In addition to the positive Ovchinnikova, G., Pati, A., Goodwin, interaction between the species, numerous L., Chen, A., Palaniappan, K., Land, reports have demonstrated that interference M., Hauser, L., Chang, Y.J., Jeffries,

429 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

C.D., Rohde, M., Göker, M., Woyke, Burr, M., Castenholz, R.W., Young, T., Bristow, J., Eisen, J.A., M. and McDermott, T.R. Markowitz, V., Hugenholtz, P., 2004.Thermobaculum terrenum gen. Kyrpides, N.C. and Klenk, H.P. 2010 nov., sp. nov.: a non-phototrophic Complete genome sequence of gram-positive thermophile Cellulomonas flavigena type strain representing an environmental clone 134. Genomic Sci. 3(1): 15-25. group related to the Chloroflexi (green Altschul, S.F., Madden, T.L., Schäffer, non-sulfur bacteria) and A.A., Zhang, J., Zhang, Z., Miller, W. Thermomicrobia.Arch and Lipman, D.J. 1997. Gapped Microbiol.181(4): 269-77. BLAST and PSI-BLAST: a new Bradford, M.M. 1976.A rapid and sensitive generation of protein database search method for the quantitation of programs. Nucleic Acids Res. 25: microgram quantities of protein 3389-3402. utilizing the principle of protein-dye Arpigny, J.L. and Jaeger, K.E. 1999. binding.Anal Biochem.72: 248 254. Bacterial lipolytic enzymes: Braeken, K., Daniels, R., Ndayizeye, M., classification and properties. Biochem Vanderleyden, J. and Michiels, J. J. 343(1): 177-183. 2008.Quorum Sensing in Bacteria- Bae, S.Y., Ryu, B.H., Jang, E., Kim, S. and Plant Interactions. InNautiyal C.S. and Kim, T.D. 2013.Characterization and Dion P (Eds.), Molecular Mechanisms immobilization of a novel SGNH of Plant and Microbe hydrolase (Est24) from Sinorhizobium Coexistence,Berlin Heidelberg: meliloti.Appl Microbiol Biotechnol. Springer-Verlag, pp. 265-289. 97(4): 1637-1647. Brenner, S. 1988. The molecular evolution Barabote, R.D., Xie, G., Leu, D.H., of genes and proteins: a tale of two Normand, P., Necsulea, A., Daubin, serines. Nature. 334: 528 530. V., Médigue, C., Adney, W.S., Xu, Chahinian, H. andSarda, L. 2009. X.C., Lapidus, A., Parales, R.E., Distinction between esterases and Detter, C., Pujic, P., Bruce, D., Lavire, lipases: comparative biochemical C., Challacombe, J.F., Brettin, T.S. properties of sequence-related and Berry, A.M. 2009. Complete carboxylesterases.Protein Pept Lett. genome of the cellulolytic 16(10): 1149-61. thermophile Acidothermus Choi, J.E., Kwon, M.A., Na, H.Y., Hahm, cellulolyticus 11B provides insights D.H. and Song, J.K. 2013. Isolation into its ecophysiological and and characterization of a metagenome- evolutionary adaptations. Genome derived thermoalkaliphilic esterase Res. 19(6): 1033-1043. with high stability over a broad pH Benjamin, S. and Pandey, A. 1998.Candida range. Extremophiles. 17(6):1013-21. rugosa lipases: molecular biology and Christov, L.P. and Prior, B.A. versatility in biotechnology. Yeast. 1993.Esterases of xylan-degrading 14: 1069 87. microorganisms: production, Bornscheuer, U.T. 2002.Microbial carboxyl properties, and significance. Enzyme esterases: classification properties and Microb Technol. 15(6):460-75. application in biocatalysis. FEMS d Angelo-Picard, C., Faure, D., Penot, I. and Microbiol Rev. 26: 73 81. Dessaux, Y. 2005. Diversity of N-acyl Botero, L.M., Brown, K.B., Brumefield, S., homoserine lactone producing and

430 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

degrading bacteria in soil and tobacco Known Structure. J Mol Biol. 313(4): rhizosphere. Environ Microbiol. 7: 903-919. 1796 1808. Hasan, F., Shah, A.A. and Hameed, A. Fan, X., Liu, X., Wang, K., Wang, S., 2006.Industrial applications of Huang, R. and Liu, Y. 2011.Highly microbial lipases.Enzyme Microb soluble expression and molecular Technol. 39: 235 251. characterization of an organic solvent- Huang, A.H.C. 1984. Studies on specificity stable and thermotolerant lipase of lipases.In Borgstrom B., and originating from the metagenome. J Brockmann H.L. (Eds.) Lipases, . Mol Catal B: Enzymatic. 72(3): 319- Elsevier, Amsterdam, pp. 419 442. 326. Huang, W., Lin, Y., Yi, S., Liu, P., Shen, J., Fernández-Arrojo, L., Guazzaroni, M.E., Shao, Z. and Liu, Z. 2012. QsdH, a López-Cortés, N., Beloqui, A. and Novel AHL Lactonase in the RND- Ferrer, M. 2010. Metagenomic era for Type Inner Membrane of Marine biocatalyst identification.Curr Pseudoalteromonas byunsanensis OpinBiotechnol.21: 725 733. Strain 1A01261.PLoS One. Ferrer, M., Chernikova, T.N., Timmis, K.N. 7(10):e46587. and Golyshin, P.N. 2004.Expression Jaeger, K.E. and Eggert, T. 2002. Lipases of a temperature-sensitive esterase in a for biotechnology. Curr Opin novel chaperone-based Escherichia Biotechnol. 3(4): 390 397. coli strain.Appl Environ Koseki, T., Miwa, Y., Akao, T., Akita, O. Microbiol.70:4499 4504. and Hashizume, K. 2006. An Ferrer, M., Golyshina, O.V., Chernikova, Aspergillus oryzae acetyl xylan T.N., Khachane, A.N., Reyes-Duarte, esterase: molecular cloning and D., Santos, V.A., Strompl, C., characteristics of recombinant enzyme Elborough, K., Jarvis, G., Neef, A., expressed in Pichia pastoris. J Yakimov, M.M., Timmis, K.N. and Biotechnol. 121: 381 389. Golyshin, P.N. 2005. Novel hydrolase Lee, M.H., Hong, K.S., Malhotra, S., Park, diversity retrieved from a metagenome J.H., Hwang, E.C., Choi, H.K., Kim, library of bovine rumen microflora. Y.S., Tao, W. and Lee, S.W. 2010.A Environ Microbiol. 7(12): 1996 2010. new esterase EstD2 isolated from Ferrer, M., Beloqui, A., Timmis, K.N. and plant rhizosphere soil metagenome. Golyshin, P.N. 2009.Metagenomics Appl Microbiol Biotechnol. 88(5): for Mining New Genetic Resources of 1125-34. Microbial Communities.J Mol Levisson, M., Han, G.W., Deller, M.C., Xu, Microbiol Biotechnol.16(1-2):109 Q., Biely, P., Hendriks, S., TenEyck, 123. L.F., Flensburg, C., Roversi, P., Gomez-Alvarez, V., Teal, T.K. and Schmidt, Miller, M.D., McMullan, D., von T.M. 2009.Systematic artifacts in Delft, F., Kreusch, A., Deacon, A.M., metagenomes from complex microbial van der Oost, J., Lesley, S.A., communities.ISME J. 3: 1314 1317. Elsliger, M.A., Kengen, S.W. and Gough, J., Karplus, K., Hughey, R. and Wilson, I.A. 2012. Functional and Chothia, C. 2001.Assignment of structural characterization of a Homology to Genome Sequences thermostable acetyl esterase from using a Library of Hidden Markov Thermotoga maritima.Proteins. 80(6): Models that Represent all Proteins of 1545-59.

431 Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

Liaw, R.B., Cheng, M.P., Wu, M.C. and Enzymic Analysis of Polysaccharide Lee, C.Y. 2010. Use of metagenomic Structure.Adv Carbohydr Chem approaches to isolate lipolytic genes Biochem. 44: 147-276. from activated sludge. Bioresour McQueen, D.A. and Schottel, J.L. 1987. Technol. 101(21):8323-329. Purification and characterization of a Liolios, K., Sikorski, J., Jando, M., Lapidus, novel extracellular esterase from A., Copeland, A., Glavina, T., Nolan, pathogenic Streptomyces scabies that M., Lucas, S., Tice, H., Cheng, J.F., is inducible by zinc. J Bacteriol.169: Han, C., Woyke, T., Goodwin, L., 1967 1971. Pitluck, S., Ivanova, N., Mavromatis, Mølgaard, A., Kauppinen, S. and Larsen, S. K., Mikhailova, N., Chertkov, O., 2000. Rhamnogalacturonan Kuske, C., Chen, A., Palaniappan, K., acetylesterase elucidates the structure Land, M., Hauser, L., Chang, Y.J., and function of a new family of Jeffries, C.D., Detter, J.C., Brettin, T., hydrolases. Structure. 8: 373 383. Rohde, M., Goker, M., Bristow, J., Morris, D.D., Gibbs, M.D., Chin, C.W., Eisen, J.A., Markowitz, V., Koh, M.H., Wong, K.K., Allison, Hugenholtz, P., Klenk, H.P. and R.W., Nelson, P.J. and Bergquist, P.L. Kyrpides, N.C. 2010. Complete 1998. Cloning of the xynB gene from genome of Thermobispora bispora Dictyoglomus thermophilum Rt46B.1 type strain (R51T). Stand Genomic and action of the gene product on kraft Sci. 2(3): 318-326. pulp.Appl Environ Microbiol. 64 (5): Lo, Y.C., Lin, S.C., Shaw, J.F. and Liaw, 1759 1765. Y.C. 2003. Crystal structure of Panda, T. and Gowrishankar, B.S. Escherichia coli 2005.Production and applications of I/protease I/lysophospholipase L1: esterases.Appl Microbiol Biotechnol. consensus sequence blocks constitute 67: 160 169. the catalytic center of SGNH- Park, S.Y., Lee, S.J., Oh, T.K., Oh, J.W., hydrolases through a conserved Koo, B.T., Yum, D.Y. and Lee, J.K. hydrogen bond network. J Mol Biol. 2003.AhlD, an N-acylhomoserine 330:539 551. lactonase in Arthrobacter sp., and Martínez-Martínez, M., Alcaide, M., predicted homologues in other Tchigvintsev, A., Reva, O., Polaina, bacteria.Microbiology. 149: 1541 J., Bargiela, R., Guazzaroni, M.E., 1550. Chicote, A., Canet, A., Valero, F., Petersen, T.N., Gunnar von Heijne, S.B. and Rico Eguizabal, E., Guerrero, C, Nielsen, H. 2011. SignalP 4.0: Yakunin, A.F. and Ferrer, M. discriminating signal peptides from 2013.Biochemical Diversity of transmembrane regions. Nature Carboxyl Esterases and Lipases from Method.8: 785-786. Lake Arreo (Spain): a Metagenomic Reyes-Duarte, D., Ferrer, M. and García- Approach. Appl Environ Microbiol. Arellano, H. 2012.Functional-based 79(12): 3553-3562. screening methods for lipases, Matoba, Y., Katsube, Y. and Sugiyama, M.. esterases, and in 2002. The crystal structure of metagenomic libraries.Methods Mol prokaryotic phospholipase A2. J Biol Biol. 861: 101 113. Chem. 277(22): 20059-20069. Sekiguchi, Y., Kamagata, Y., Syutsubo, K., McCleary, B.V. and Matheson, N.K. 1987. Ohashi, A., Harada, H. and Nakamura,

432

Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 413-433

K. 1998. Phylogenetic diversity of 2011.Characterization of novel mesophilic and thermophilic granular antibiotic resistance genes identified sludges determined by 16s rRNA gene by functional metagenomics on soil analysis. Microbiology.144: 2655- samples.Environ Microbiol.13(4): 2665. 1101 1114. Selvin, J., Kennedy, J., Lejon, D.P., Kiran, Ulrike, E., Rogall, T., Blocker, H., Emde, G.S. and Dobson, A.D. 2012. Isolation M. and Bottger, E.G. 1989. Isolation identification and biochemical and direct complete nucleotide characterization of a novel halo- determination of entire genes. tolerant lipase from the metagenome Characterization of a gene coding for of the marine sponge Haliclona 16S ribosomal RNA.Nucleic Acids simulans.Microb Cell Fact.11: 72. Res. 17: 7843 7853. Sugiyama, M., Ohtani, K., Izuhara, M., Yu, S., Zheng, B., Zhao, X. and Feng, Y. Koike, T., Suzuki, K., Imamura, S. 2010.Gene cloning and and Misaki, H. 2002.A novel characterization of a novel prokaryotic phospholipase thermophilic esterase from A2.Characterization, gene cloning, Fervidobacterium nodosum Rt17- and solution structure.J Biol Chem. B1.Acta Biochim Biophys Sin.42(4): 277(22): 20051-20058. 288 295. Takemori, D., Yoshino, K., Eba, C., Nakano, H. and Iwasaki, Y. 2012. Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli.Protein Expr Purif . 81(2): 145-50. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 28: 2731- 2739. Tian, Q., Song, P., Jiang, L., Li, S. and Huang, H. 2014.A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7- aminocephalosporanic acid.Appl Microbiol Biotechnol. 98(5): 2081- 2089. Torres-Cortés, G., Millán, V., Ramírez- Saad, H.C., Nisa-Martínez, R., Toro, N. and Martínez-Abarca, F.

433