A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology

Biotechnology for Biofuels

Liliane FC Ribeiro³  Rosymar C De Lucas³  Gabriela L Vitcosque³  Lucas F Ribeiro³  Richard J Ward⁵  Marcelo V Rubio⁵  Andre RL Damásio⁵  Fabio M Squina⁵  Rebecca C Gregory⁶  Paul H Walton⁶  João A Jorge²  Rolf A Prade¹  Marcos S Buckeridge⁴  Maria de Lourdes TM Polizeli² 

[1] Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, USA

[2] Biology Department of Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP, Av. Bandeirantes, 3900, Ribeirão Preto, SP 14040-901, Brazil

[3] Immunology and Biochemistry Department of Faculdade de Medicina de Ribeirão Preto - USP, Ribeirão Preto, SP, Brazil

[4] Institute of Biosciences, University of São Paulo, São Paulo, Brazil

[5] Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Campinas, SP, Brazil

[6] Department of Chemistry, The University of York, York, UK

关键词: Sugarcane bagasse;    Biomass degradation;    Thermostability;    Heterologous expression;    Glycosylation;    Malbranchea;    Xylanase;   

Others  :  1084764
DOI  :  10.1186/1754-6834-7-115

 received in 2014-02-28, accepted in 2014-07-15,  发布年份 2014

【 摘 要 】

Background

The search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.

Results

Xylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)₈ barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.

Conclusions

MpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

【 授权许可】

   
2014 Ribeiro et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Maheshwari R, Bharadwaj G, Bhat MK: Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 2000, 64:461-488.
• [2]Maalej I, Belhaj I, Masmoudi NF, Belghith H: Highly thermostable xylanase of the thermophilic fungus Talaromyces thermophilus: purification and characterization. Appl Biochem Biotechnol 2009, 158:200-212.
• [3]Turner P, Mamo G, Karlsson EN: Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 2007, 6:9-32.
• [4]Motta FAC, Santana M: A review of xylanase production by the fermentation of xylan: classification, characterization and applications. In Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization. Edited by Chandel AK, da Silva SS. Rijeka, Croatia: InTech; 2013.
• [5]Dimarogona M, Topakas E, Christakopoulos P, Chrysina ED: The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft. Acta Crystallogr D Biol Crystallogr 2012, 68:735-742.
• [6]Pereira MG, Guimaraes LHS, Furriel RPM, Polizeli MLTM, Terenzi HF, Jorge JA: Biochemical properties of an extracellular trehalase from Malbranchea pulchella var. sulfurea. J Microbiol 2011, 49:809-815.
• [7]Ribeiro LFC, Ribeiro LF, Jorge JA, Polizeli MLTM: Screening of filamentous fungi for xylanases and cellulases not inhibited by xylose and glucose. Br Biotechnol J 2013, 4:30-39.
• [8]Abu-Shady MR, El-Gindy AA, Saad RR, Ibrahim ZM: Production, partial purification and some properties of a thermostable alkaline protease from Malbranchea sulfurea and its compatibility with commercial detergents. Afr J Mycol Biotechnol 2001, 9(3):17-26.
• [9]Shoreit AAM, Ismail MA, Mohamed NH: Preliminary study on soil microbiota degrading plant latices and their protease and lipase enzymes. J Basic Appl Mycol (Egypt) 2013, 4:33-45.
• [10]Han P, Zhou P, Hu S, Yang S, Yan Q, Jiang Z: A novel multifunctional α-amylase from the thermophilic fungus Malbranchea cinnamomea: biochemical characterization and three-dimensional structure. Appl Biochem Biotechnol 2013, 170(2):420-435.
• [11]Matsuo M, Yasui T, Kobayashi T: Enzymatic properties of β-xylosidase from Malbranchea pulchella var. sulfurea No. 48. Agric Biol Chem 1977, 41(9):1601-1606.
• [12]Nuthakki S, Lomada D, Venkateswerlu G: An antifungal extracellular enzymes from Malbranchea chrysosporioidea. Int J Recent Sci Res 2014, 5(2):379-381.
• [13]Wu W, Schulein M, Kauppinen MS, Stringer MA: Xyloglucanase from Malbranchea. Official Gaz USA Pat Trademark Office Pat 2002. Us Patent: 6500658
• [14]Su X, Schmitz G, Zhang M, Mackie RI, Cann IK: Heterologous gene expression in filamentous fungi. Adv Appl Microbiol 2012, 81:1-61.
• [15]Segato F, Damasio ARL, Goncalves TA, De Lucas RC, Squina FM, Decker SR, Prade RA: High-yield secretion of multiple client proteins in Aspergillus. Enzyme Microb Technol 2012, 51:100-106.
• [16]Gomes DJ, Gomes J, Steiner W: Production of highly thermostable xylanase by a wild strain of thermophilic fungus Thermoascus aurantiacus and partial characterization of the enzyme. J Biotechnol 1994, 37:11-22.
• [17]Lafond M, Tauzin A, Desseaux V, Bonnin E, Ajandouz EH, Giardina T: GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production. Microb Cell Fact 2011, 10:20-28.
• [18]Matsuo M, Yasui T: Properties of xylanase of Malbranchea pulchella var. sulfurea No. 48. Agric Biol Chem 1985, 49(3):839-841.
• [19]Vafiadi C, Christakopoulos P, Topakas E: Purification, characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile. Process Biochem 2010, 45:419-424.
• [20]Mattinen ML, Linder M, Drakenberg T, Annila A: Solution structure of the cellulose-binding domain of endoglucanase I from Trichoderma reesei and its interaction with cello-oligosaccharides. Eur J Biochem 1998, 256:279-286.
• [21]Damaso MCT, De Castro AM, Castro RM, Andrade CMMC, Pereira N: Application of xylanase from Thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse. Appl Biochem Biotechnol 2004, 115:1003-1012.
• [22]Santos CR, Meza AN, Hoffmam ZB, Silva JC, Alvarez TM, Ruller R, Giesel GM, Verli H, Squina FM, Prade RA, Murakami MT: Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1. Biochem Biophys Res Commun 2010, 403:214-219.
• [23]Gorshkova TA, Kozlova LV, Mikshina PV: Spatial structure of plant cell wall polysaccharides and its functional significance. Biochemistry (Mosc) 2013, 78:836-853.
• [24]Kormelink FJM, Voragen AGJ: Degradation of different [(glucurono)arabino]xylans by a combination of purified xylan-degrading enzymes. Appl Microbiol Biotechnol 1993, 38:688-695.
• [25]De Souza AP, Leite DCC, Pattathil S, Hahn MG, Buckeridge MS: Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production. Bioenergy Res 2013, 6:564-579.
• [26]Limayem A, Ricke SC: Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Progr Energ Combust Sci 2012, 38:449-467.
• [27]Pandey A, Soccol CR, Mitchell D: New developments in solid state fermentation: I-bioprocesses and products. Process Biochem 2000, 35:1153-1169.
• [28]Rocha GJM, Silva FT, Curvelo AAS, Araújo GT: A fast and accurate method for determination of cellulose and polyoses by HPLC. In Proceedings of the. In Proceedings of the Fifth Brazilian Syimposium on the Chemistry of Lignins and Other Wood Components. Parana, Brazil: Parana, Brazil; 1997:3-8.
• [29]Barratt RWJG, Ogata WN: Wild-type and mutant stocks of Aspergillus nidulans. Genetics 1965, 52:233-246.
• [30]Selig MJ, Knoshaug EP, Decker SR, Baker JO, Himmel ME, Adney WS: Heterologous expression of Aspergillus niger beta-D-xylosidase (XlnD): characterization on lignocellulosic substrates. Appl Biochem Biotechnol 2008, 146:57-68.
• [31]Damasio ARD, Silva TM, Almeida FBD, Squina FM, Ribeiro DA, Leme AFP, Segato F, Prade RA, Jorge JA, Terenzi HF, Polizeli MLTM: Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application. Process Biochem 2011, 46:1236-1242.
• [32]Tilburn J, Scazzocchio C, Taylor GG, Zabickyzissman JH, Lockington RA, Davies RW: Transformation by integration in Aspergillus nidulans. Gene 1983, 26:205-221.
• [33]Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31:426-428.
• [34]Bradford MM: Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
• [35]Nakamura S, Nakai R, Wakabayashi K, Ishiguro Y, Aono R, Horikoshi K: Thermophilic alkaline xylanase from newly isolated alkaliphilic and thermophilic Bacillus sp. strain TAR-I. Biosci Biotechnol Biochem 1994, 58(1):78-81.
• [36]Leone FA, Baranauskas JA, Furriel RP, Borin IA: SigrafW: An easy-to-use program for fitting enzyme kinetic data. Biochem Mol Biol Educ 2005, 33(6):399-403.
• [37]Naran R, Pierce ML, Mort AJ: Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons. Plant J 2007, 50:95-107.
• [38]Hemsworth GR, Taylor EJ, Kim RQ, Gregory RC, Lewis SJ, Turkenburg JP, Parkin A, Davies GJ, Walton PH: The copper active site of CBM33 polysaccharide oxygenases. J Am Chem Soc 2013, 135:6069-6077.
• [39]Altschul SF, Madden TL, Schaffer AA, Zhang JH, 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.
• [40]Sali A, Potterton L, Yuan F, Vanvlijmen H, Karplus M: Evaluation of comparative protein modeling by MODELLER. Proteins 1995, 23:318-326.
• [41]Laskowski RA, Macarthur MW, Moss DS, Thornton JM: PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993, 26:283-291.
• [42]Raussens V, Ruysschaert JM, Goormaghtigh E: Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular dichroism spectra: a new scaling method. Anal Biochem 2003, 319(1):114-121.