Abstract
A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in Escherichia coli and the purified enzymes exhibited temperature optima of between 40–50°C. Substrate-profiling studies revealed that the characterised enzymes were ‘true’ carboxylesterases based on their preferences for short to medium chain length p-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches.
References
1.
Altschul SF, Madden TS, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–3402.
[PubMed]
2.
Arima, K, Woodley J: Computational methods for understanding bacterial and archeal genomes; in Xu Y, Gogatern P (eds): Metagenomics, chapt 14. London, Imperial College Press, 2008, pp 345–350.
3.
Arpigny KL, Jaeger KE: Bacterial lipolytic enzymes: classification and properties. J Biochem 1999;343:177–183.
4.
Bayer S, Kunerta A, Ballschnmitera M, Greiner-Stoeffelea T: Indication for a new lipolytic enzyme family: isolation and characterization of two esterases from a metagenomic library. J Mol Microbiol Biotechnol 2010;18:181–187.
[PubMed]
5.
Bell PJ, Sunna A, Gibbs MD, Curach NC, Nevalainen H, Bergquist OL: Prospecting for novel lipase genes using PCR. J Microbiol 2002;148:2283–2291.
6.
Bendten JD, Nielsen H, von Heijnie G, Brunak S: Improved prediction of signal peptide: SignalP 3.0. J Mol Biol 2004;340:783–795.
7.
Bornscheuer UT: Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 2002;26:73–81.
[PubMed]
8.
Bradford MM: A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
[PubMed]
9.
Chandrasekharaiah M, Thulasi A, Bagath M, Kumar DP, Santosh SS, Palanivel C, Jose VL, Sampath KT: Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite (Coptotermes formosanus) gut. BMB Rep 2011;44;52–75.
10.
Daniel RM, Danson MJ, Eisenthal RE, Lee CK, Petersen ME: The effect of temperature on enzyme activity: new insights and their implications. Extremophile 2008;12:51–59.
11.
Davis RW, Kamble ST, Prabhakaran SK: Characterization of general esterases in workers of the eastern subterranean termite. J Econ Entomol 1995;88:574–578.
12.
Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999;41:95–98.
13.
Hemingway J, Karunaratne SH: Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Med Vet Entomol 1998;12:1–12.
14.
Hu Y, Fu C, Huang Y, Yin Y, Cheng G, Lei F, Lu N, Li J, Ashforth EJ, Zhang L, Zhu B: Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment. FEMS Microbiol Ecol 2010;72:228–237.
15.
Jaeger KE, Dijkstra BW, Reetz MT: Bacterial biocatalysis: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 1999;53:315–351.
[PubMed]
16.
Jeon HJ, Kim JT, Lee HS, Kim SJ, Kang SG, Choi SH, Lee JH: Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment. Evid Based Complement Alternat Med 2011;2011:271419.
17.
Kambiranda DM, Asraful-Islam SM, Cho KM, Math RK, Lee YH, Kim H, Yun HD: Expression of esterase gene in yeast for organophosphates biodegradation. J Pestic Biochem Phys 2009;94:15–20.
18.
Kouker G, Jaeger KE: Specific and sensitive plate assay for bacterial lipases. J Appl Environ Microbiol 1987;53:211–213.
19.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685.
[PubMed]
20.
Lorenz P, Liebeton K, Niehaus F, Eck K: Screening novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequences space. J Curr Opin Biotechnol 2002;13:572–577.
21.
Marthur EJ, Toledo G, Green BD, Podar M, Richardson TB, Kulwiee M, Chang HW: A biodiversity-based approach to development of performance enzymes. J Ind Biotechnol 2005;4:283–287.
22.
Moreth K, Riester D, Hildmann C, Hempel R, Wegener D, Schober A, Schwienhorst A: An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme. J Biochem 2007;401:659–665.
23.
Nacke H, Christiane Will C, Herzog S, Nowka B, Engelhaupt M, Daniel R: Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity exploratories. FEMS Microbial Ecol 2011;78:188–201.
24.
Panda T, Gowrishankar BS: Production and application of esterases. J Appl Microbiol Biotechnol 2005;67:160–169.
25.
Rakotondravelo ML, Anderson TD, Charlton RE, Zhu YK: Sub-lethal effects of three pesticides on activities of selected target and detoxification enzymes in the Aquatic Midge, Chironomus tentans (Diptera: Chironomidae). J Arch Environ Contam Toxicol 2006;51:360–366.
26.
Rashamuse K, Ronneburg T, Hennessy F, Visser D, Van Heerden E, Piater L, Litthauer D, Moller C, Brady D: Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library. J Appl Microbiol 2009a;106:1532–1539.
27.
Rashamuse K, Sanyika W, Ronneburg T Brady D: A feruloyl esterase derived from a leachate metagenome library. BMB Rep 2012;45:13–18.
28.
Rashamuse KJ, Magomani V, Ronneburg T, Brady, D: A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin. J Appl Microbiol Biotechnol 2009b;83:491–500.
29.
Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR, Loiacone KA, Lynch BA, MacNeil IA, Minor C: Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. J Appl Environ Microbiol 2000;66:2541–2547.
[PubMed]
30.
Roodveldt C, Tawfik DS: Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily. J Biochem 2005;44:12728–12736.
31.
Ruvolo-Takasusuki MCR, Collet T: Characterization of Nasutitermes globiceps esterases. Biochem Genet 2000;38:367–375.
32.
Saitou N, Nei, M: The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–425.
[PubMed]
33.
Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual, ed 3. Cold Spring Harbour/NY, Cold Spring Harbor Laboratory Press, 2001.
34.
Sanyika TW, Rashamuse KJ, Hennessy F, Brady D: Luminal hindgut bacterial diversities of the grass and sugarcane feeding termite Trinervitermes trinervoides. Afr J Micro Res 2012;6:2639–2648.
35.
Sohaskey CD, Barbour AG: Spirochaeta aurantia has diacetyl chloramphenicol esterase activity. J Bacteriol 2000;182:1930–1934.
36.
Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;10:1093/molbev/msm1092.
37.
Villeneuve P, Muderhwa JM, Graille J, Haas MJ: Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J Mol Catal 2000;9:113–148.
38.
Wahler D, Reymond JL: Novel methods for biocatalyst screening. Curr Opin Chem Biol 2001;5:152–158.
39.
Wheeler MM, Tarver MR, Coy MR, Scharf ME: Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes. Arch Insect Biochem Physiol 2010;73:30–48.
© 2012 S. Karger AG, Basel
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
2012
You do not currently have access to this content.
