Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera

Abstract

Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger

“…Both the N-and C-terminal fusions of C2 improved the Xyn thermostability, indicating that the C2 is a thermostabilizing CBM distinct from those binding only to the substrate (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Mai-Gisondi et al 2015;Mangala et al 2003). In addition, the N-terminal C2 improved enzyme T opt by 2°C.…”

“…These modules were later recognized as CBMs (Dias et al 2004;Sunna et al 2000), and the thermostabilizing effects of CBM deletions were regarded as lacking discrete linker peptides separating from the catalytic domains (Dias et al 2004;Sunna et al 2000). The V max and K m values on oat spelt xylan were 1.55 and 2.03 lmol/min, and 6.98 and 7.76 mg/ml for the enzyme C2-Xyn, respectively made (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Liu et al 2011Liu et al , 2012aMai-Gisondi et al 2015;Mangala et al 2003;Tang et al 2014;Voutilainen et al 2014;Ye et al 2011), and fusion seems better for elucidating modular functions. 3 Kinetics of the C2-Xyn.…”

“…T opt and t 1/2 are related but different parameters with the former indicating enzyme adaptivity to a higher temperature and the later, enzyme longevity at a certain temperature. Other CBM fusions also improved catalytic activities of the related enzymes (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Li et al 2015;Mai-Gisondi et al 2015;Mangala et al 2003;Ye et al 2011). 3; Table 1).…”

“…Presently, the terminus is intuitively selected consisting with a CBM natural terminal location (Furtado et al 2015;Li and Shao 2006;Liu et al 2011Liu et al , 2012bMai-Gisondi et al 2015;Ye et al 2011). Presently, the terminus is intuitively selected consisting with a CBM natural terminal location (Furtado et al 2015;Li and Shao 2006;Liu et al 2011Liu et al , 2012bMai-Gisondi et al 2015;Ye et al 2011).…”

Criação de uma enzima multifuncional feruloil esterase/acetil-xilano esterase por desenho racional

“…Nesse contexto, a criação de enzimas bifuncionais que atuam em materiais lignocelulósicos tem demonstrado ser uma opção prática para a melhoria de diversos processos envolvidos (FAN; YUAN, 2010;FAN et al, 2009;FONSECA-MALDONADO et al, 2014;LOURENZONI, 2013;FURTADO et al, 2015;RIBEIRO et al, 2011a;XUE et al, 2009). …”