Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) from Aspergillus niveus

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Damasio, Andre R. L. ^[1] ; Ribeiro, Liliane F. C. ^[1] ; Ribeiro, Lucas F. ^[1] ; Furtado, Gilvan P. ^[1] ; Segato, Fernando ^[2] ; Almeida, Fausto B. R. ^[3] ; Crivellari, Augusto C. ^[4] ; Buckeridge, Marcos S. ^{[2, 4]} ; Souza, Tatiana A. C. B. ^[5] ; Murakami, Mario T. ^[5] ; Ward, Richard J. ^[6] ; Prade, Rolf A. ^[7] ; Polizeli, Maria L. T. M. ^[8]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Bioquim & Imunol, FMRP, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Ctr Nacl Pesquisas Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol CTBE, Campinas, SP - Brazil

^[3] Univ Sao Paulo, Dept Biol Celular & Mol & Bioagentes Patogen, FMRP, BR-14040901 Ribeirao Preto, SP - Brazil

^[4] Univ Sao Paulo, Inst Biociencias, Dept Bot, Lab Fisiol Ecol Plantas, BR-14040901 Ribeirao Preto, SP - Brazil

^[5] Ctr Nacl Pesquisas Energia & Mat, Lab Nacl Biociencias, Campinas, SP - Brazil

^[6] Univ Sao Paulo, Dept Quim, FFCLRP USP, BR-14040901 Ribeirao Preto, SP - Brazil

^[7] Oklahoma State Univ, Dept Microbiol & Mol Genet, Stillwater, OK 74078 - USA

^[8] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, BR-14040901 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1824, n. 3, p. 461-467, MAR 2012.
• Citações Web of Science: 24

Resumo

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-beta-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 degrees C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan beta-1,3 or beta-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in beta-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 degrees C and 81.3 degrees C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 degrees C. the enzymatic assays demonstrated that XegA is more active in its monomeric state. (c) 2012 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 10/52322-3 - Bioprospecção de fungos filamentosos produtores de holoenzimas com aplicação em biorefinaria
• Beneficiário: Maria de Lourdes Teixeira de Moraes Polizeli
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOTA - Regular