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Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

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Abstract

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.

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References

  • Andreaus J, Campos R, Gubitz G, Cavaco-Paulo A (2000) Influence of cellulases on indigo backstaining. Text Res J 70:628–632

    Article  CAS  Google Scholar 

  • Berkeley MJ, Broome CE (1875) Enumeration of the fungi of Ceylon. Part II. J Linn Soc Bot 14:29–141

    Article  Google Scholar 

  • Berlin A, Maximenko V, Gilkes N, Saddler J (2007) Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 97:287–296

    Article  PubMed  CAS  Google Scholar 

  • Boesewinkel HJ (1982) Cylindrocladiella, a new genus to accommodate Cylindrocladium parvum and other small-spored species of Cylindrocladium. Can J Bot 60:2288–2294

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding.Anal Biochem 72:248–254

    Google Scholar 

  • Bradner JR, Sidhu RK, Gillings M, Nevalainen KMH (1999a) Hemicellulase activity of antarctic microfungi. J Appl Microbiol 87:366–370

    Article  PubMed  CAS  Google Scholar 

  • Bradner JR, Gillings M, Nevalainen KMH (1999b) Qualitative assessment of hydrolytic activities in antarctic microfungi grown at different temperatures on solid media. World J Microbiol Biotechnol 15:131–132

    Article  Google Scholar 

  • Britton HT, Robinson RA (1931) Universal buffer solutions and the dissociation constant for veronal. J Chem Soc 458:1456–1462

    Article  Google Scholar 

  • Bull AT (2004) Biotechnology, the art of exploiting biology. In: Bull AT (ed) Microbial diversity and bioprospecting. ASM Press, Washington, pp 3–12

    Google Scholar 

  • Cai L, Jeewon R, Hyde KD (2006) Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology. Mycol Res 110:137–150

    Article  PubMed  CAS  Google Scholar 

  • Castro-Ochoa LD, Rodríguez-Gómez C, Valerio-Alfaro G, Ros RO (2005) Screening, purification and characterization of the thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzyme Microb Technol 37:648–654

    Article  CAS  Google Scholar 

  • Choi N-S, Yoon K-S, Lee J-Y, Han K-Y, Kim S-H (2001) Comparison of three substrates (casein, fibrin, and gelatin) in zymographic gel. J Biochem Mol Biol 34:531–536

    CAS  Google Scholar 

  • Comfort DA, Chhabra SR, Conners SB, Chou CJ, Epting KL, Johnson MR, Jones KL, Sehgal AC, Kelly RM (2004) Strategic biocatalysis with hyperthermophilic enzymes. Green Chem 6:459–465

    Article  CAS  Google Scholar 

  • Cupp-Enyard C (2008) Sigma’s non-specific protease activity assay-casein as substrate. Journal of Visualised Experiments 19. http://jove.com/index/Details.stp?ID=899, doi:10.3791/899. Accessed 17 April 2009

  • De Notaris G (1867) Nuove reclute per la pirenomicetologia italica. Comment SocCrittogamol Ital 2:477–492

    Google Scholar 

  • Dhawan S, Kaur J (2007) Microbial mannanases: An overview of production and applications. Crit Rev Biotechnol 27:197–216

    Article  PubMed  CAS  Google Scholar 

  • Dodd SL, Lieckfeldt E, Samuels GJ (2003) Hypocrea atroviridis sp. nov., the teleomorph of Trichoderma atroviride. Mycologia 95:27–40

    Article  PubMed  Google Scholar 

  • Dogaris I, Vakontios G, Kalogeris E, Mamma D, Kekos D (2009) Induction of cellulases and hemicellulases from Neurospora crassa under solid-state cultivation for bioconversion of sorghum bagasse into ethanol. Ind Crops Prod 29:404–411

    Article  CAS  Google Scholar 

  • Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJ, Yao J, Ward M (2003) Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278:31988–31997

    Article  PubMed  Google Scholar 

  • Friedrich J, Kern S (2003) Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus. J Mol Catal B Enzym 21:35–37

    Article  CAS  Google Scholar 

  • Garcia D, Stchigel AM, Cano J, Guarro J, Hawksworth DL (2004) A synopsis and re-circumscription of Neurospora (syn. Gelasinospora) based on ultrastructural and 28 S rDNA sequence data. Mycol Res 108:1119–1142

    Article  PubMed  CAS  Google Scholar 

  • Gibbs MD, Reeves RA, Bergquist PL (1995) Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate. Appl Environ Microbiol 61:4403–4408

    PubMed  CAS  Google Scholar 

  • Gradišar H, Kern S, Friedrich J (2000) Keratinase of Doratomyces microsporus. Appl Microbiol Biotechnol 53:196–200

    Article  PubMed  Google Scholar 

  • Herpoël-Gimbert I, Margeot A, Dolla A, Jan G, Mollé D, Lignon S, Mathis H, Sigoillot J-C, Monot F, Asther M (2008) Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol Biofuels 1:18

    Article  PubMed  Google Scholar 

  • Joseph B, Ramteke PW, Thomas G, Shrivastava N (2007) Standard review. Cold-active microbial lipases: a versatile tool for industrial applications. Biotechnol Mol Biol Rev 2:39–48

    Google Scholar 

  • Karhunen T, Mäntylä A, Nevalainen KMH, Suominen PL (1993) High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet 241:515–522

    Article  PubMed  CAS  Google Scholar 

  • Karsten PA (1892) Finlands mögelsvampar. (Hyphomycetes fennici). Bidrag till Kännedom af Finlands Natur och Folk 51:343–534

    Google Scholar 

  • Kaur J, Chadha BS, Kumar BA, Saini HS (2007) Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922. Bioresour Technol 98:74–81

    Article  PubMed  CAS  Google Scholar 

  • Khan RS, Krug JC (1989) New species of Gelasinospora. Mycologia 81:226–233

    Article  Google Scholar 

  • Kovács K, Megyeri L, Szakacs G, Kubicek CP, Galbe M, Zacchi G (2008) Trichoderma atroviride mutants with enhanced production of cellulase and β-glucosidase on pretreated willow. Enzyme Microb Technol 43:48–55

    Article  Google Scholar 

  • Kovács K, Szakacs G, Zacchi G (2009) Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride. Bioresour Technol 100:1350–1357

    Article  PubMed  Google Scholar 

  • Krug JC, Benny GL, Keller HW (2004) Coprophilous fungi. In: Mueller GM, Bills GF, Foster MS (eds) Biodiversity of fungi: Inventory and monitoring methods. Elsevier Academic, San Diego, pp 467–500

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Le Crom S, Schackwitz W, Pennacchio L, Magnuson JK, Culley DE, Collett JR, Martin J, Druzhinina IS, Mathis H, Monot F, Seiboth B, Cherry B, Rey M, Berka R, Kubicek CP, Baker SE, Margeot A (2009) Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing. Proc Natl Acad Sci USA 106:16151–16156

    Article  PubMed  Google Scholar 

  • Link JHF (1809) Observationes in ordines plantarum naturales. Ges Naturf Freunde Berlin Mag 3:3–42

    Google Scholar 

  • Mandels M, Reese ET (1957) Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol 73:269–278

    Article  PubMed  CAS  Google Scholar 

  • Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EGJ, Grigoriev IV, Harris P, Jackson M, Kubicek CP, Han CS, Ho I, Larrondo LF, Lopez de Leon A, Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B, Salamov AA, Schmoll M, Terry A, Thaye N, Westerholm-Parvinen A, Schoch CL, Yao J, Barbote R, Nelson MA, Detter C, Bruce D, Kuske CR, Xie G, Richardson P, Rokhsar DS, Lucas SM, Rubin EM, Dunn-Coleman N, Ward M, Brettin TS (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 26:553–560

    Article  PubMed  CAS  Google Scholar 

  • Meyer V (2008) Genetic engineering of filamentous fungi- Progress, obstacles and future trends. Biotechnol Adv 26:177–185

    Article  PubMed  CAS  Google Scholar 

  • Montenecourt BS, Eveleigh DE (1979) Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. In: Brown R, Jurasek L (eds) Hydrolysis of cellulose: mechanisms of enzymatic and acid catalysis. Advances in chemistry series ,vol 181. American Chemical Society, Washington DC, pp 289–301

    Chapter  Google Scholar 

  • Morton FJ, Smith G (1963) The genera Scopulariopsis Bainier, Microascus Zukal and Doratomyces Corda. Mycol Pap 86:1–96

    Google Scholar 

  • Nevalainen KMH, Te'o VSJ, Bergquist PL (2005) Heterologous protein expression in filamentous fungi. Trends Biotechnol 23:468–474

    Article  PubMed  CAS  Google Scholar 

  • Ouyang J, Yan M, Kong D, Xu L (2006) A complete protein pattern of cellulase and hemicellulase genes in the filamentous fungus Trichoderma reesei. Biotechnol J 1:1266–1274

    Article  PubMed  CAS  Google Scholar 

  • Penttilä M (1998) Heterologous protein production in Trichoderma. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor and Francis, London, pp 365–382

    Google Scholar 

  • Penttilä ME, Nevalainen H, Salminen E, Rättö M, Knowles J (1987) Versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164

    Article  PubMed  Google Scholar 

  • Peterson RA, Bradner JR, Roberts TH, Nevalainen KMH (2009a) Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates. Lett Appl Microbiol 48:218–225

    Article  PubMed  CAS  Google Scholar 

  • Peterson R, Grinyer J, Joss J, Khan A, Nevalainen H (2009b) Fungal proteins with mannanase activity identified directly from a Congo Red stained zymogram by mass spectrometry. J Microbiol Methods 79:374–377

    Article  PubMed  CAS  Google Scholar 

  • Ruiza C, Falcocchioa S, Xoxia E, Pastorb F, Diazb P, Sasoa L (2004) Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay. Biochim Biophys Acta 1672:184–191

    Google Scholar 

  • Samson RA (1974) Paecilomyces and some allied hyphomycetes. Stud Mycol 6:1–119

    Google Scholar 

  • Saxena RK, Malhotra B, Batra A (2004) Commercial importance of some fungal enzymes. In: Arora DK (ed) Handbook of fungal biotechnology, mycology, vol 20. Marcel Dekker, New York, pp 287–298

    Google Scholar 

  • Seidl V, Gamauf C, Druzhinina I, Seiboth B, Hartl L, Kubicek C (2008) The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome. BMC Genomics 9:327

    Article  PubMed  Google Scholar 

  • Sheir-Neiss G, Montenecourt BS (1984) Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl Microbiol Biotechnol 20:46–53

    Article  CAS  Google Scholar 

  • Simmons EG (1977) Classification of some cellulase-producing Trichoderma species. In: Bigelow HE, Simmons EG (eds) Second International Mycological Congress, Abstracts, vol 2. University of South Florida, Tampa, p 618

    Google Scholar 

  • Sonia K, Chadha B, Badhan A, Saini M, Bhat M (2008) Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi. World J Microbiol Biotechnol 24:599–604

    Article  CAS  Google Scholar 

  • Turner NJ (2003) Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol 21:474–478

    Article  PubMed  CAS  Google Scholar 

  • Upadhyay MK, Sharma R, Pandey AK, Rajak RC (2005) An improved zymographic method for detection of amylolytic enzymes of fungi on polyacrylamide gels. Mycologist 19:138–140

    Article  Google Scholar 

  • von Schlechtendal DFL (1824) Cryptogamia. Flora Berolinensis, 2. Ferdinand Dümmler, Berolini, pp 1–284

    Google Scholar 

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Acknowledgement

This work was supported by a Research Excellence Scholarship to R.P. in the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.

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  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia

    Robyn Peterson, Jasmine Grinyer & Helena Nevalainen

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  1. Robyn Peterson
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Correspondence to Robyn Peterson.

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Peterson, R., Grinyer, J. & Nevalainen, H. Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest. Mycol Progress 10, 207–218 (2011). https://doi.org/10.1007/s11557-010-0690-5

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