Artículos de revistas
The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan
Registro en:
Plos One. Public Library Of Science, v. 9, n. 9, p. - , 2014.
19326203
10.1371/journal.pone.0108393
2-s2.0-84907452148
Autor
Miotto L.S.
De Rezende C.A.
Bernardes A.
Serpa V.I.
Tsang A.
Polikarpov I.
Institución
Resumen
The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-b-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on b-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50uC on b-glucan. Under these conditions specific activity was 239.269.1 U mg21 and the half-life of the enzyme was 84.663.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using b-glucan as a substrate, the Km was 3.260.5 mg mL21 and the Vmax was 0.4160.02 mmol min21. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process. 9 9
Kuhad, R.C., Gupta, R., Singh, A., Microbial cellulases and their industrial applications (2011) Enzyme Res, 2011, p. 280696 Horn, S., Vaaje-Kolstad, G., Westereng, B., Vgh, E., Novel enzymes for the degradation of cellulose (2012) Biotechnol Biofuels, 5, p. 45 Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., The carbohydrate-active enzymes database (cazy): An expert resource for glycogenomics (2009) Nucleic Acids Res, 37, pp. D233-D238 Murphy, C., Powlowski, J., Wu, M., Butler, G., Tsang, A., Curation of characterized glycoside hydrolases of fungal origin (2011) Database (Oxford) 2011, , bar020 Sandgren, M., Shaw, A., Ropp, T.H., Wu, S., Bott, R., The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 Å resolution (2001) J Mol Biol, 308, pp. 295-310 Schülein, M., Enzymatic properties of cellulases from Humicola insolens (1997) J Biotechnol, 57, pp. 71-81 Birsan, C., Johnson, P., Joshi, M., Macleod, A., McIntosh, L., Mechanisms of cellulases and xylanases (1998) Biochem Soc Trans, 26, pp. 156-160 Goodell, B., Brown-rot fungal degradation of wood: Our evolving view (2003) Wood Deterioration and Preservation, ACS Symposium Series., pp. 97-118. , Goodell B, Darrel DN, Schultz TP, editors Floudas, D., Binder, M., Riley, R., Barry, K., Blanchette, R.A., The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes (2012) Science, 336, pp. 1715-1719 Cohen, R., Suzuki, M.R., Hammel, K.E., Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum (2005) Appl Environ Microbiol, 71, pp. 2412-2417 Aslanidis, C., Dejong, P., Ligation-independent cloning of pcr products (LIC-PCR) (1990) Nucleic Acids Res, 18, pp. 6069-6074 Storms, R., Zheng, Y., Li, H., Sillaots, S., Martinez-Perez, A., Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger (2005) Plasmid, 53, pp. 191-204 Debets, A.J.M., Bos, C.J., Isolation of small protoplasts from Aspergillus niger (1986) Fungal Genet Newsl, 33, p. 24 Käfer, E., Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations (1977) Adv Genet, 19, pp. 33-131 Wood, T.M., Preparation of crystalline, amorphous, and dyed cellulase substrates (1988) Methods in Enzymology, pp. 19-25. , Willis A, Wood STK, editors Academic Press Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar (1959) Anal Chem, 31, pp. 426-428 Wood, T.M., Bhat, K.M., Methods for measuring cellulase activities (1988) Methods in Enzymology., pp. 87-112. , Willis A, Wood STK, editors Academic Press Vincken, J.-P., Beldman, G., Agj, V., Substrate specificity of endoglucanases: What determines xyloglucanase activity? (1997) Carbohyd Res, 298, pp. 299-310 Pauly, M., Andersen, L.N., Kauppinen, S., Kofod, L.V., York, W.S., A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: Expression cloning in yeast, purification and characterization of the recombinant enzyme (1999) Glycobiology, 9, pp. 93-100 Grishutin, S.G., Gusakov, A.V., Dzedzyulya, E.I., Sinitsyn, A.P., A lichenase-like family 12 endo-(1→4)-β-glucanase from Aspergillus japonicus: Study of the substrate specificity and mode of action on β-glucans in comparison with other glycoside hydrolases (2006) Carbohyd Res, 341, pp. 218-229 Gloster, T.M., Ibatullin, F.M., Macauley, K., Eklöf, J.M., Roberts, S., Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12 (2007) J Biol Chem, 282, pp. 19177-19189 Shimokawa, T., Shibuya, H., Nojiri, M., Yoshida, S., Ishihara, M., Purification, molecular cloning, and enzymatic properties of a family 12 endoglucanase (EGII) from Fomitopsis palustris: Role of EG-II in larch holocellulose hydrolysis (2008) Appl Environ Microbiol, 74, pp. 5857-5861 Henriksson, G., Nutt, A., Henriksson, H., Pettersson, B., Ståhlberg, J., Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase (1999) Eur J Biochem, 259, pp. 88-95 Tambor, J.H., Ren, H., Ushinsky, S., Zheng, Y., Riemens, A., Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-β-glucanases that efficiently hydrolyse cellulosic substrates (2012) Appl Microbiol Biotechnol, 93, pp. 203-214 Bok, J.D., Yernool, D.A., Eveleigh, D.E., Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana (1998) Appl Environ Microbiol, 64, pp. 4774-4781 Bauer, M.W., Driskill, L.E., Callen, W., Snead, M.A., Mathur, E.J., An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes beta-1,4 bonds in mixed-linkage (1→3),(1→4)-beta-D-glucans and cellulose (1999) J Bacteriol, 181, pp. 284-290 Wicher, K.B., Abou-Hachem, M., Halldórsdóttir, S., Thorbjarnadóttir, S.H., Eggertsson, G., Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus (2001) Appl Microbiol Biotechnol, 55, pp. 578-584 Prates, E.T., Stankovic, I., Silveira, R.L., Liberato, M.V., Henrique-Silva, F., X-ray structure and molecular dynamics simulations of endoglucanase 3 from Trichoderma harzianum: Structural organization and substrate recognition by endoglucanases that lack cellulose binding module (2013) PLoS ONE, 8, p. e59069 Gama, F.M., Mota, M., Enzymatic hydrolysis of cellulose (i): Relationship between kinetics and physico-chemical parameters (1997) Biocatal Biotransfor, 15, pp. 221-236 Dourado, F., Gama, F.M., Chibowski, E., Mota, M., Characterization of cellulose surface free energy (1998) J Adhes Sci Technol, 12, pp. 1081-1090 Wood, P.J., Fulcher, R.G., Interaction of some dyes with cereal β-glucans (1978) Cereal Chem, 55, pp. 952-966 Buckeridge, M.S., Rayon, C., Urbanowicz, B., Tine, M.A.S., Carpita, N.C., Mixed linkage (1→3),(1→4)-β-d-glucans of grasses (2004) Cereal Chem, 81, pp. 115-127 Carpita, N.C., Cell wall development in maize coleoptiles (1984) Plant Physiol, 76, pp. 205-212 Wood, P.J., Evaluation of oat bran as a soluble fibre source Characterization of oat b-glucan and its effects on glycaemic response (1994) Carbohyd Polym, 25, pp. 331-336 Ragaee, S.M., Wood, P.J., Wang, Q., Tosh, S., Brummer, Y., Extractability, structure and molecular weight of b-glucan from Canadian rye (Secale cereale L.) Whole Meal (2008) Cereal Chem, 85, pp. 283-288 Tiwari, U., Cummins e Factors influencing b-glucan levels and molecular weight in cereal-based products (2009) Cereal Chem, 86, pp. 290-301 Sims, R.E., Mabee, W., Saddler, J.N., Taylor, M., An overview of second generation biofuel technologies (2010) Bioresour Technol, 101, pp. 1570-1580 Buckeridge, M.S., Goldman, G.H., (2011) Routes to Cellulosic Ethanol, 263p. , New York: Springer