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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 Esterases 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 esterase 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 hydrolases. Both enzymes are carboxylic ester 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 lipases 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 substrate 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 lipase 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/