The capability of endophytic fungi for production of hemicellulases and related enzymes

BMC Biotechnology

Diogo Robl³  Priscila da Silva Delabona³  Carla Montanari Mergel²  Juan Diego Rojas²  Patrícia dos Santos Costa³  Ida Chapaval Pimentel¹  Vania Aparecida Vicente¹  José Geraldo da Cruz Pradella³  Gabriel Padilla² 

[1] Departament of Basic Pathology, Federal University of Paraná (UFPR), Caixa Postal 19020, CEP 81531-980 Curitiba, PR, Brazil

[2] Institute of Biomedical Sciences, University of São Paulo (USP), Avenida Lineu Prestes 1374 CEP, 05508-900 São Paulo SP, Brazil

[3] Brazilian Bioethanol Science and Technology Laboratory – CTBE, Pólo II de Alta Tecnologia, Rua Giuseppe Maximo Scolfaro 10000, CEP 13083-970 Campinas, SP, Brazil

关键词: Accessory enzymes;    Hemicellulases;    Xylanase;    Endophytic fungi;   

Others  :  835168
DOI  :  10.1186/1472-6750-13-94

 received in 2013-05-20, accepted in 2013-09-12,  发布年份 2013

【 摘 要 】

Background

There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G.

Results

The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media.

Conclusions

The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

【 授权许可】

   
2013 Robl et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Berlin A, Maximenko V, Gilkes N, Saddler J: Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 2007, 97:287-296.
• [2]Gao D, Chundawat SPS, Krishnan C, BalanV DBE: Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol 2010, 101:2770-2781.
• [3]Gusakov AV, Salanovich TN, Antonov AI, Ustinov BB, Okunev ON, Burlingame R, Emalfarb M, Baez M, Sinitsyn AP: Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnol Bioeng 2007, 97:1028-1038.
• [4]Kovács K, Megyeri L, Szakacs G, Kubicek CP, Galbe M, Zacchi G: Trichoderma atroviride mutants with enhanced production of cellulase and β-glucosidase on pretreated willow. Enzyme Microb Technol 2008, 43:48-55.
• [5]Maijala P, Kango N, Szijarto N, Viikari L: Characterization of hemicellulases from thermophilic fungi. Antonie Van Leeuwenhoek 2012, 101:905-917.
• [6]Panno L, Bruno M, Voyron S, Anastasi A, Gnavi G, Miserere L, Varese GC: Diversity, ecological role and potential biotechnological applications of marine fungi associated to the seagrass Posidonia oceanica. N Biotechnol 2013. doi:10.1016/j.nbt.2013.01.010
• [7]Zabalgogeazcoa I, Oleaga A, Pérez-Sánchez R: Pathogenicity of endophytic entomopathogenic fungi to ornithodoros erraticus and ornithodoros moubata (acari: argasidae). Vet Parasitol 2008, 158:336-343.
• [8]Huang Z, Cai X, Shao C, She Z, Xia X, Chen Y, Yang J, Zhou S, Lin Y: Chemistry and weak antimicrobial activities of phomopsins produced by mangrove endophytic fungus Phomopsis sp. ZSU-H76. Phytochemistry 2008, 69:1604-1608.
• [9]Wipusaree N, Sihanonth P, Piapukiew J, Sangvanich P, Karnchanatat A: Purification and characterization of a xylanase from the endophytic fungus alternaria alternata isolated from the Thai medicinal plant, croton oblongifolius roxb. Afr J Microbiol Res 2011, 5:5697-5712.
• [10]Burke RM, Cairney JWG: Purification and characterization of a β-1,4-endoxylanase from the ericoid mycorrhizal fungus Hymenoscyphus ericae. New Phytol 1997, 35:345-352.
• [11]Sorgatto M, Guimarães NCA, Zanoelo FF, Marques MR, Peixoto-Nogueira SC, Giannesi GG: Purification and characterization of an extracellular xylanase produced by the endophytic fungus, Aspergillus terreus, grown in submerged fermentation. Afr J Biotechnol 2012, 11:8076-8084.
• [12]de Almeida MN, Guimarães VM, Bischoff KM, Falkoski DL, Pereira OL, Gonçalves DS, de Rezende ST: Cellulases and hemicellulases from endophytic acremonium species and its application on sugarcane bagasse hydrolysis. Appl Biochem Biotechnol 2012, 165:594-610.
• [13]Suto M, Takebayashi M, Saito K, Tanaka M, Yokota A, Tomita F: Endophytes as producers of xylanase. J Biosci Bioeng 2002, 93:88-90.
• [14]Harnpicharnchai P, Champreda V, Sornlake W, Eurwilaichitr L: A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr Purif 2009, 67:61-69.
• [15]Silva RLO, Luz JS, Silveira EB, Cavalcante UMT: Fungos endofíticos em annona spp.: isolamento, caracterização enzimática e promoção do crescimento em mudas de pinha (annona squamosa L.). Acta Bot Bras 2006, 20:649-655.
• [16]Luz JS, Silva RLO, Silveira EB, Cavalcante UMT: Atividade enzimática de fungos endofíticos e efeito na promoção do crescimento de mudas de maracujazeiro-amarelo. Caatinga 2006, 19:128-134.
• [17]Castro AM, Ferreira MC, da Cruz JC, Pedro KC, Carvalho DF, Leite SG, Pereira N: High-yield endoglucanase production by trichoderma harzianum IOC-3844 cultivated in pretreated sugarcane mill byproduct. Enzyme Res 2010, 2010:854526.
• [18]Sánchez-Ballesteros J, González V, Salazar O, Acero J, Portal MA, Julián M, Rubio V: Phylogenetic study of hypoxylon and related genera based on ribosomal ITS sequences. Mycologia 2000, 92:964-977.
• [19]Hsieh HM, Ju YM, Rogers JD: Molecular phylogeny of hypoxylon and closely related genera. Mycologia 2005, 97:844-865.
• [20]Chou HH, Wu WS: Phylogenetic analysis of internal transcribed spacer regions of the genus Alternaria, and the significance of filament-beaked conidia. Mycol Res 2002, 106:164-169.
• [21]Pryor BM, Bigelow DM: Molecular characterization of embellisia and nimbya species and their relationship to alternaria, ulocladium and stemphylium. Mycologia 2003, 95:1141-1154.
• [22]Andrew M, Peever TL, Pryor BM: An expanded multilocus phylogeny does not resolve morphological species within the small-spored alternaria species complex. Mycologia 2009, 101:95-109.
• [23]Silva VFN, Arruda PV, Felipe MGA, Gonçalves AR, Rocha GJM: Fermentation of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing. J Ind Microbiol Biotechnol 2011, 38:809-817.
• [24]De Vries RP, Visser J, de Graaff LH: CreA modulates the XlnR-induced expression on xylose of Aspergillus Niger genes involved in xylan degradation. Res Microbiol 1999, 150:281-285.
• [25]Delabona Pda S, Farinas CS, Lima DJ, Pradella JG: Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation. Bioresour Technol 2013, 132:401-405.
• [26]Souza AP, Leite DCC, Pattathil S, Hahn MG, Buckridge MS: Composition and structure of sugarcane cell wall polysaccharides: implications for seconf-generation bioethanol production. Bioenerg Res 2012. doi:10.1007/s12155-012-9268-1
• [27]Gao D, Uppugundla N, Chundawat SPS, Yu X, Hermanson S, Gowda K, Brumm P, Mead D, Balan V, Dale BE: Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides. Biotechnol Biofuels 2011, 4:5. BioMed Central Full Text
• [28]Gottschalk LMF, Oliveira RA, Bom EPS: Cellulases, xylanases, β-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse. Biochem Eng J 2010, 51:72-78.
• [29]Kumar R, Wyman CE: Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologie. Bioresour Technol 2009, 100:4203-4213.
• [30]Alvira P, Negro MJ, Ballesteros M: Effect of endoxylanase and a-L-arabinofuranosidase supplementation on the enzymatic hydrolysis of steam exploded wheat straw. Bioresour Technol 2011, 102:4552-4558.
• [31]Stone J, Polishook J, White-Jr F: Endophytic fungi. In Biodiversity of fungi: inventory and monitoring methods. 1st Edition. Edited by Foster M, Bills G, Mueller G. New York: Academic Press; 2004:241-270.
• [32]Gazis R, Chaverri P: Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru. Fungal Ecol 2010, 3:240-254.
• [33]Wei DL, Chang SC, Wei YH, Lin YW, Chuang CL, Jong SC: Production of cellulolytic enzymes from the Xylaria and Hypoxylon species of xylariaceae. World J Microbiol Biotechnol 1992, 8:141-146.
• [34]Thomma BPHJ: Alternaria spp.: from general saprophyte to specific parasite. Mol Plant Pathol 2003, 4:225-236.
• [35]Lupo S, Tiscornia S, Bettucci L: Endophytic fungi from flowers, capsules and seeds of Eucalyptus globules. Rev Iberoam Micol 2001, 18:38-41.
• [36]Kharwar RNG, Surendra KKA, Mishra A: A comparative study of endophytic and epiphytic fungal association with leaf of Eucalyptus citriodora Hook., and their antimicrobial activity. World J Microbiol Biotechnol 2010, 26:1941-1948.
• [37]Isshiki A, Akimitsu K, Nishio K, Tsukamoto M, Yamamoto H: Purification and characterization of an endopolygalacturonase from the rough lemon pathotype of Alternaria alternata, the cause of citrus brown spot disease. Physiol Mol Plant Pathol 1997, 51:155-167.
• [38]Sáenz-de-Santamaría M, Guisantes JA, Martínez J: Enzymatic activities of Alternaria alternata allergenic extracts and its major allergen (Alt a 1). Mycoses 2006, 49:288-292.
• [39]Ward OP, Qin WM, Dhanjoon J, Ye J, Singh A: Physiology and biotechnology of Aspergillus. Adv Appl Microbial 2005, 58:1-75.
• [40]Meijer M, Houbraken JAMP, Dalhuijsen S, Samson RA, Vries RP: Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus Niger and other black aspergilla. Stud Mycol 2011, 69:19-30.
• [41]Ilyas M, Kanti A, Jamal Y, Herdina AA: Biodiversity of endophytic fungi associated with uncaria Gambier roxb. (Rubiaceae) from west Sumatra. Biodiversitas 2009, 10:23-28.
• [42]Zhao K, Ping W, Li Q, Hao S, Zhao L, Gao T, Zhou D: Aspergillus Niger var. Taxi, a new species variant of taxol-producing fungus isolated from taxus cuspidate in China. J Appl Microbiol 2009, 107:1202-1207.
• [43]Jang Y, Lee J, Lee H, Lee S, Kim G, Kim J: Screening for xylanase and β-xylosidase production from wood-inhabiting Penicillium strains for potential use in biotechnological applications. Holzforschung 2012, 66:267-271.
• [44]Lee J, Jang Y, Lee H, Lee S, Kim G, Kim J: Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 2. Genus penicillium. Holzforschung 2011, 65:265-270.
• [45]Xia X, Lie TK, Qian X, Zheng Z, Huang Y, Shen Y: Species diversity, distribution, and genetic structure of endophytic and epiphytic Trichoderma associated with banana roots. Microb Ecol 2011, 61:619-625.
• [46]Ahamed A, Vermette P: Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem Eng J 2008, 40:399-407.
• [47]Ahamed A, Vermette P: Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production. Bioresour Technol 2009, 100:5979-5987.
• [48]Olsson L, Christensen TMIE, Hansen KP, Palmqvist EA: Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C – 30. Enzyme Microb Technol 2003, 33:612-619.
• [49]Moy M, Li HM, Sullivan R, White JF Jr, Belanger FC: Endophytic fungal beta-1,6-glucanase expression in the infected host grass. Plant Physiol 2002, 130:1298-1308.
• [50]Müller MM, Valjakka R, Suokko A, Hantula J: Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers. Mol Ecol 2001, 10:1801-1810.
• [51]Petrini O: Fungal endophyte of tree leaves. In Microbial ecology of leaves. Edited by Andrews JHSS. New York: Spring-verlag; 1991:179-197.
• [52]Kumaresan V, Suryanarayanan TS: Endophyte assemblages in young, mature and senescent of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Divers 2002, 9:81-91.
• [53]Lumyong S, Lumyong P, McKenzie EH, Hyde KD: Enzymatic activity of endophytic fungi of six native seedling species from Doi Suthep-Pui National Park, Thailand. Can J Microbiol 2002, 48:1109-1112.
• [54]Schulz B, Boyle C: The endophytic continuum. Mycol Res 2005, 109:661-686.
• [55]Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D: Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Standard biomass analytical proceduresAvailable http://www.nrel.gov/docs/gen/fy08/42623.pdf webcite Accessed 23 Fev 2013
• [56]Gouveia ER, Nascimento RT, Souto-Maior AM, Rocha GJM: Validação de metodologia para a caracterização química de bagaço de cana-de-açúcar. Quim Nova 2009, 32:1500-1503.
• [57]Rocha GJM, Gonçalves AR, Oliveira BR, Olivares EG, Rossel CEV: Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugar cane bagasse for bioethanol production. Ind Crops Prod 2012, 35:274-279.
• [58]Rodriguez-Zuniga UF, Farinas CS, Neto VB, Couri S, Crestana S: Aspergillus niger production of cellulases by solid-state fermentation. Pesqui Agropecu Bras 2011, 46:912-919.
• [59]Kasana RC, Salwan R, Dhar H, Dutt S, Gulati A: A rapid and easy method for the detection of microbial cellulases on agar plates using gram’s iodine. Curr Microbiol 2008, 57:503-507.
• [60]Saqib AAN, Whitney PJ: Esculin gel diffusion assay (EGDA): A simple and sensitive method for screening β-glucosidases. Enzyme Microb Technol 2006, 39:182-184.
• [61]Mandels M, Reese ET: Induction of cellulase in fungi by cellobiose. J Bacteriology 2001, 73:816-826.
• [62]Xiao Z, Storms R, Tsang A: Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 2004, 88:832-837.
• [63]Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31:426-428.
• [64]Barnett HC, Hunter BB: Illustrated genera of imperfect fungi. New York: Macmillan Publishing Company; 1999.
• [65]De Hoog GS, Guarro J, Gené J, Figueras MJ: Atlas of clinical fungi. Centraalbureau voor Schimmelcultures, Utrecht: Universitat Rovira I Virgili, Reus; 2000.
• [66]Larone DH: Medically important fungi: a guide to identification. Washington: ASM Press; 2002.
• [67]Kern MA, Blevins KS: Micologia médica. São Paulo: Premier; 1999.
• [68]AHG G v d e, de Hoog GS: Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol 1999, 43:151-162.
• [69]White TJ, Bruns T, Lee S, Taylor J: PCR protocols: a guide to methods and applications. In Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Edited by Innis MA, Gelfand DH, Sninsky JJ, White TJ. New York: Academic Press; 1990:315-322.
• [70]Glass NL, Donaldson GC: Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995, 61:1323-1330.
• [71]Staden R: The Staden sequence analysis package. Mol Biotechnol 1996, 5:233-241.
• [72]Tamura K, Dudley J, Nei M, Kumar S: MEGA 4: molecular evolutionary genetics analysis. Mol Biol Evol 2007, 24:1596-1599.
• [73]Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425.
• [74]Jukes TH, Cantor CR: Evolution of protein molecules. In Mammalian protein metabolism. Edited by Munro HN. New York: Academic Press; 1969:21-132.