Skip to main content
SpringerLink
Log in

Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays

  • Protocol
  • First Online:
Protein Downstream Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2178))

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 to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383

    Article  CAS  Google Scholar 

  2. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  CAS  Google Scholar 

  3. 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

    Article  CAS  Google Scholar 

  4. 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

    Article  CAS  Google Scholar 

  5. 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

    Article  CAS  Google Scholar 

  6. 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

    Article  CAS  Google Scholar 

  7. 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

    Article  CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  9. 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

    Article  CAS  Google Scholar 

  10. 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

    Article  CAS  Google Scholar 

  11. 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

    Article  CAS  Google Scholar 

  12. 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

    Article  CAS  Google Scholar 

  13. 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

    Article  CAS  Google Scholar 

  14. 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

    Article  CAS  Google Scholar 

  15. 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

    Article  Google Scholar 

  16. 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

    Article  Google Scholar 

  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

    Article  Google Scholar 

  18. 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

    Article  CAS  Google Scholar 

  19. 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

    Article  CAS  Google Scholar 

  20. 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

    Article  CAS  Google Scholar 

  21. 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

    Article  CAS  Google Scholar 

  22. 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

    Article  CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  24. 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

    Article  CAS  Google Scholar 

  25. 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

    Article  CAS  Google Scholar 

  26. 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

    Article  CAS  Google Scholar 

  27. 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

    Article  CAS  Google Scholar 

  28. 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

    Article  CAS  Google Scholar 

  29. 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

    Article  CAS  Google Scholar 

  30. Miller RB, Karn RC (1980) A rapid spectrophotometric method for the determination of esterase activity. J Biochem Biophys Methods 3(6):345–354

    Article  CAS  Google Scholar 

  31. 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

    Article  CAS  Google Scholar 

  32. 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

    Article  CAS  Google Scholar 

  33. 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

    Article  CAS  Google Scholar 

  34. 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

    Article  CAS  Google Scholar 

  35. 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

    Article  Google Scholar 

  36. 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

    Article  CAS  Google Scholar 

  37. 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

    Article  CAS  Google Scholar 

  38. Johannes C, Majcherczyk A (2000) Laccase activity tests and laccase inhibitors. J Biotechnol 78(2):193–199

    Article  CAS  Google Scholar 

  39. 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

    Article  CAS  Google Scholar 

  40. 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

    Article  CAS  Google Scholar 

  41. 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

    Article  CAS  Google Scholar 

  42. 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

    Article  CAS  Google Scholar 

  43. 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

    Article  Google Scholar 

  44. 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

    Article  CAS  Google Scholar 

  45. Mai-Gisondi G, Master ER (2017) Colorimetric detection of acetyl xylan esterase activities. Methods Mol Biol 1588:45–57

    Article  CAS  Google Scholar 

  46. 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

    Article  CAS  Google Scholar 

  47. 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

    Article  CAS  Google Scholar 

  48. Anderson JA (1934) The use of tributyrin agar in dairy bacteriology. J Bacteriol 27:69

    CAS  Google Scholar 

  49. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  50. 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

    CAS  Google Scholar 

  51. 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

    Article  CAS  Google Scholar 

  52. 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

    Article  Google Scholar 

  53. 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

    Article  Google Scholar 

  54. 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

    Article  CAS  Google Scholar 

  55. 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

    Chapter  Google Scholar 

  56. Donaghy J, Kelly PF, McKay AM (1998) Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli. Appl Microbiol Biotechnol 50:257–260

    Article  CAS  Google Scholar 

  57. 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

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece

    Anthi Karnaouri, Anastasia Zerva & Evangelos Topakas

  2. Biochemical Process Engineering, Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, Luleå, Sweden

    Paul Christakopoulos & Evangelos Topakas

Authors
  1. Anthi Karnaouri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anastasia Zerva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul Christakopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Evangelos Topakas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Evangelos Topakas .

Editor information

Editors and Affiliations

  1. Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens, Greece

    Nikolaos E. Labrou

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

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

Publish with us

Policies and ethics

Search

Navigation