Biochemical and structural characterization of a beta-1,3-1,4-glucanase from Bacillus subtilis 168

Author(s)

Furtado, Gilvan Pessoa ^[1] ; Ribeiro, Lucas Ferreira ^[1] ; Santos, Camila Ramos ^[2] ; Tonoli, Celisa Caldana ^[2] ; de Souza, Angelica Rodrigues ^[2] ; Oliveira, Renata Rocha ^[2] ; Murakami, Mario Tyago ^[2] ; Ward, Richard John ^[3]

Affiliation

^[1] Univ Sao Paulo, Dept Bioquim & Imunol, FMRP USP, BR-14049 Ribeirao Preto, SP - Brazil

^[2] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias, Campinas, SP - Brazil

^[3] Univ Sao Paulo, Dept Quim, FFCLRP USP, BR-14049 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: Process Biochemistry; v. 46, n. 5, p. 1202-1206, MAY 2011.
• Web of Science Citations: 29

Abstract

beta-1,3-1,4-Glucanases (E.C. 3.2.1.73) hydrolyze linked beta-D-glucans, such as lichenan and barley beta-glucan. Recombinant beta-1,3-1,4-glucanase from Bacillus subtilis expressed in Escherichia coil and purified by Ni-NTA chromatography exhibited optimum activity at 50 degrees C and pH 6.0. The catalytic half-life at 60 degrees C decreased from 90 to 5 min when the enzyme was incubated in the presence and absence of Ca(2+) respectively. The kinetic parameters of lichenan hydrolysis were 2695, 3.1 and 1220 for V(max) (mu mol/min/mg), K(m) (mg mL(-1)) and K(cat) (s(-1)), respectively. Analysis by DLS, AUC and SAXS demonstrated the enzyme is monomeric in solution. Chemical denaturation monitored by ITFE and far-UV CD yielded Delta G(H2O) values of 9.6 and 9.1 kcal/mol, respectively, showing that the enzyme has intermediate stability when compared with other Bacillus beta-1,3-1,4-glucanases. The crystal structure shows the anti-parallel jelly-roll beta-sheet conserved in all GH16 beta-1,3-1,4-glucanases, with the amino acid differences between Bacillus sp. enzymes that are likely determinants of stability being distributed throughout the protein. (C) 2011 Elsevier Ltd. All rights reserved. (AU)

• FAPESP's process: 07/01615-8 - Rational design of a novel bifunctional laccase-xylanase enzyme from Bacillus subtilis for potential application in paper and cellulose industry
• Grantee: Lucas Ferreira Ribeiro
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 07/01623-0 - Rational design, construction and characterization of a bifunctional enzyme (laccase-cellulase) for biomass conversion to biofuel
• Grantee: Gilvan Pessoa Furtado
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 08/57908-6 - National Institute of Science and Technology of Bioethanol
• Grantee: Marcos Silveira Buckeridge
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 10/51890-8 - SMOLBnet 2.0: Structural studies of transcription factors involved in the regulation of hydrolytic enzyme genes and swollenin from Aspergillus niger and A. fumigatus
• Grantee: Mário Tyago Murakami
• Support Opportunities: Regular Research Grants