title: A designed bifunctional laccase/β-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse.
abstract: A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by ∼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP-USP,  Universidade de São Paulo, São Paulo,  Brazil.
authors.forename: G P
authors.initials: GP
authors.lastname: Furtado
authors.forename: L F
authors.initials: LF
authors.lastname: Ribeiro
authors.forename: M R
authors.initials: MR
authors.lastname: Lourenzoni
authors.forename: R J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031786:Saccharum / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D002482:Cellulose; C027433:bagasse; D042845:Laccase; D044644:Endo-1,3(4)-beta-Glucanase