Abstract
In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383
Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
Robichon C, Luo J, Causey TB et al (2011) Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl Environ Microbiol 77:4634–4646. https://doi.org/10.1128/AEM.00119-11
Anbar M, Bayer EA (2012) Approaches for improving thermostability characteristics in cellulases. Methods Enzymol 510:261–271. https://doi.org/10.1016/B978-0-12-415931-0.00014-8
McCleary BV (1988) Soluble, dye-labeled polysaccharides for the assay of endohydrolases. Methods Enzymol 160:74–86. https://doi.org/10.1016/0076-6879(88)60108-X
Biely P, Mislovicova D, Toman R (1985) Soluble chromogenic substrates for the assay of endo-1,4-β-glucanase. Anal Biochem 144:142–146. https://doi.org/10.1016/0003-2697(85)90095-8
Thorn RG (1993) The use of cellulose azure agar as a crude assay of both cellulolytic and ligninolytic abilities of wood-inhibiting fungi. Proc Jpn Acad Ser B Phys Biol Sci 69:29–34. https://doi.org/10.2183/pjab.69.29
Karnaouri A, Topakas E, Paschos T et al (2013) Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila. PeerJ 1:e46. https://doi.org/10.7717/peerj.46
Gu C, Zheng F, Long L et al (2014) Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus. PLoS One 9(4):e93912. https://doi.org/10.1371/journal.pone.0093912
Zerva A, Christakopoulos P, Topakas E (2015) Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol. Enzyme Microb Technol 75–76:49–56. https://doi.org/10.1016/j.enzmictec.2015.04.012
Neddersen M, Elleuche S (2015) Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras. AMB Express 5(1):122. https://doi.org/10.1186/s13568-015-0122-7
Vianna Bernardi A, Kimie Yonamine D, Akira Uyemura S et al (2019) A thermostable Aspergillus fumigatus GH7 endoglucanase over-expressed in Pichia pastoris stimulates lignocellulosic biomass hydrolysis. Int J Mol Sci 20:9. https://doi.org/10.3390/ijms20092261
Scheirlinck T, Meutter J, Arnaut G et al (1990) Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum. Appl Microbiol Biotechnol 33:534–541
Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 4:777–780
Adesioye FA, Makhalanyane TP, Vikram S et al (2018) Structural characterization and directed evolution of a novel acetyl xylan esterase reveals thermostability determinants of the carbohydrate esterase 7 family. Appl Environ Microbiol 84:8. https://doi.org/10.1128/AEM.02695-17
Carrazco-Palafox J, Rivera-Chavira BE, Ramírez-Baca N et al (2018) Improved method for qualitative screening of lipolytic bacterial strains. Methods X 5:68–74. https://doi.org/10.1016/j.mex.2018.01.004
Katsimpouras C, Dimarogona M, Petropoulos P et al (2016) A thermostable GH26 endo-β-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation. Appl Microbiol Biotechnol 100(19):8385–8397. https://doi.org/10.1007/s00253-016-7609-2
Mattéotti C, Bauwens J, Brasseur C et al (2012) Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut (Reticulitermes santonensis). Protein Expr Purif 83(2):117–127. https://doi.org/10.1016/j.pep.2012.03.009
Zhou C, Xue Y, Ma Y (2018) Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10. Microb Cell Factories 17(1):124. https://doi.org/10.1186/s12934-018-0973-0
Kračun SK, Schückel J, Westereng B et al (2015) A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Biotechnol Biofuels 8:70. https://doi.org/10.1186/s13068-015-0250-y
Li LL, Taghavi S, McCorkle SM et al (2011) Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases. Biotechnol Biofuels 4:23. https://doi.org/10.1186/1754-6834-4-23
Huang Y, Busk PK, Lange L (2015) Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases. Enzym Microb Technol 73–74:9–19. https://doi.org/10.1016/j.enzmictec.2015.03.001
Manafi M (1996) Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. Int J Food Microbiol 31:45. https://doi.org/10.1016/0168-1605(96)00963-4
Cotson S, Holt SJ (1958) Studies in enzyme cytochemistry. IV. Kinetics of aerial oxidation of indoxyl and some of its halogen derivatives. Proc R Soc Lond B Biol Sci 148(933):506–519
Perry JD, Morris KA, James AL et al (2007) Evaluation of novel chromogenic substrates for the detection of bacterial b-glucosidase. Appl Microbiol 102(2):410–415. https://doi.org/10.1111/j.1365-2672.2006.03096.x
Arab-Jaziri F, Bissaro B, Dion M et al (2013) Engineering transglycosidase activity into a GH51 a-l-arabinofuranosidase. New Biotechnol 30:5. https://doi.org/10.1016/j.nbt.2013.04.002
Bissaro Β, Durand J, Biarnés X et al (2015) Molecular design of non-Leloir furanose-transferring enzymes from an α-l-arabinofuranosidase: a rationale for the engineering of evolved transglycosylases. ACS Catal 5(8):4598–4611. https://doi.org/10.1021/acscatal.5b00949
Meddeb-Mouelhi F, Moisan JK, Beauregard M (2014) A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity. Enzym Microb Technol 66:16–19. https://doi.org/10.1016/j.enzmictec.2014.07.004
Miller RB, Karn RC (1980) A rapid spectrophotometric method for the determination of esterase activity. J Biochem Biophys Methods 3(6):345–354
Leschot A, Tapia RA, Eyzaguirre J (2002) Efficient synthesis of 4-methylumbelliferyl dihydroferulate. Synth Commun 32:3219–3223. https://doi.org/10.1081/SCC-120013746
Dimarogona M, Topakas E, Olsson L et al (2012) Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile. Bioresour Technol 110:480–487. https://doi.org/10.1016/j.biortech.2012.01.116
Childs RE, Bardsley WG (1975) The steady-state kinetics of peroxidase with 2.2%-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem J 145:93–103. https://doi.org/10.1042/bj1450093
Srinivasan C, Dsouza TM, Boominathan K et al (1995) Demonstration of laccase in the white rot basidiomycete Phanerochaete chrysosporium BKM-F1767. Appl Environ Microbiol 61(12):4274–4277
Yang Q, Zhang M, Zhang M et al (2018) Characterization of a novel, cold-adapted, and thermostable laccase-like enzyme with high tolerance for organic solvents and salt and potent dye decolorization ability, derived from a marine metagenomic library. Front Microbiol 9:2998. https://doi.org/10.3389/fmicb.2018.02998
Garg N, Bieler N, Kenzom T et al (2012) Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase. BMC Biotechnol 12(1):1–12. https://doi.org/10.1186/1472-6750-12-75
Zerva A, Koutroufini E, Kostopoulou I et al (2019) A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone. New Biotechnol 49:10–18. https://doi.org/10.1016/j.nbt.2018.12.001
Johannes C, Majcherczyk A (2000) Laccase activity tests and laccase inhibitors. J Biotechnol 78(2):193–199
Sadhasivam S, Savitha S, Swaminathan K et al (2008) Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem 43(7):736–742. https://doi.org/10.1016/j.procbio.2008.02.017
Soden DM, O'Callaghan J, Dobson AD (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148(Pt 12):4003–4014. https://doi.org/10.1099/00221287-148-12-4003
Topakas E, Vafiadi C, Christakopoulos P (2007) Microbial production, characterization and applications of feruloyl esterases. Process Biochem 42:497–509. https://doi.org/10.1016/j.procbio.2007.01.007
Hassan S, Hugouvieux-Cotte-Pattat N (2011) Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid. J Bacteriol 193:963–970. https://doi.org/10.1128/JB.01239-10
Xu Z, He H, Zhang S et al (2017) Characterization of feruloyl esterases produced by the four Lactobacillus species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, isolated from ensiled corn Stover. Front Microbiol 8:941. https://doi.org/10.3389/fmicb.2017.00941
Moukouli M, Topakas E, Christakopoulos P (2008) Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum. Appl Microbiol Biotechnol 79:245–254. https://doi.org/10.1007/s00253-008-1432-3
Mai-Gisondi G, Master ER (2017) Colorimetric detection of acetyl xylan esterase activities. Methods Mol Biol 1588:45–57
Rosenberg M, Roegner V, Becker FF (1975) The quantitation of rat serum esterases by densitometry of acrylamide gels stained for enzyme activity. Anal Biochem 66(1):206–212
Blum DL, Li XL, Chen H et al (1999) Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl Environ Microbiol 65(9):3990–3995
Anderson JA (1934) The use of tributyrin agar in dairy bacteriology. J Bacteriol 27:69
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Gilkes NR, Langsford ML, Kilburn DG et al (1984) Mode of action and substrate specificities of cellulases from cloned bacterial genes. J Biol Chem 259:10455–10459
Kasana RC, Salwan R, Dhar H et al (2008) A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine. Curr Microbiol 57:503–507. https://doi.org/10.1007/s00284-008-9276-8
Casciello C, Tonin F, Berini F et al (2017) A valuable peroxidase activity from the novel species Nonomuraea gerenzanensis growing on alkali lignin. Biotechnol Rep 13:49–57. https://doi.org/10.1016/j.btre.2016.12.005
Falade AO, Eyisi O, Mabinya LV et al (2017) Peroxidase production and ligninolytic potentials of freshwater bacteria Raoultella ornithinolytica and Ensifer adhaerens. Biotechnol Rep (Amst) 16:12–17. https://doi.org/10.1016/j.btre.2017.10.001
Xu H, Guo MY, Gao YH et al (2017) Expression and characteristics of manganese peroxidase from Ganoderma lucidum in Pichia pastoris and its application in the degradation of four dyes and phenol. BMC Biotechnol 17(1):19. https://doi.org/10.1186/s12896-017-0338-5
Torres E, Ayala M, de Weert S et al (2010) Heterologous expression of peroxidases. In: Biocatalysis based on Heme peroxidases. Berlin Heidelberg, Springer, New York, pp 315–333
Donaghy J, Kelly PF, McKay AM (1998) Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli. Appl Microbiol Biotechnol 50:257–260
Westereng B, Loose JSM, Vaaje-Kolstad G et al (2018) Analytical tools for characterizing cellulose-active lytic polysaccharide monooxygenases (LPMOs). In: Lübeck M (ed) Cellulases. Methods in molecular biology, vol 1796. Humana Press, New York, NY
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Karnaouri, A., Zerva, A., Christakopoulos, P., Topakas, E. (2021). Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_30
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0775-6_30
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0774-9
Online ISBN: 978-1-0716-0775-6
eBook Packages: Springer Protocols