Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance

Abstract

BackgroundThe Bacillus subtilis endo-β-1,4-glucanase (BsCel5A) hydrolyzes β-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds β-1,4-linked glucan.MethodsHere we investigate the BsCel5A β-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having β-1,3-1,4 glucan affinity.ResultsThe BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley β-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with β-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with β-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the β-1,3-1,4 glucan. ConclusionsThese results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0964-0) contains supplementary material, which is available to authorized users.

Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum

“…This preference is confirmed by the lower Km observed for chimeric Dtur CelA compared with native Dtur CelA on barley β-glucan, displaying an enhanced affinity for this polymer exerted by the addition of Ct CBM11. These findings are in accordance with a very recent work 21 on the Bacillus subtilis endo-β-1,4-glucanase BsCel5A (a GH5 with high activity on β-1,3–1,4-linked glucans), where the exchange of the CBM3, present in the wild type BsCel5A, for Ct CBM11 (with high affinity for β-1,3–1,4 glucans) increased the catalytic efficiency of the enzyme on such substrates.…”

Mapping secondary substrate‐binding sites on the GH11 xylanase from Bacillus subtilis

“…These are significantly reduced in the presence of arabinoxylan, and are in agreement with previous MD studies of the reduction in the mobility of the PxXyn11B on binding xylopentaose [24]. Spin-label ESR spectroscopy has been previously applied to estimate the affinity of a carbohydrate-active enzyme-MTSSL conjugate for lignocellulose substrates by measuring the decrease in Ms values on titration of the substrate [52]. In the present study, the binding of arabinoxylan generally decreased the Ms values of the BsXynA-MTSSL conjugates indicating a reduced mobility of these positions on substrate binding and agrees with the results from the MD simulations.…”

“…Electron spin resonance (ESR) spectroscopy coupled with site-directed spin labeling (SDSL) with extrinsic paramagnetic probes has been established as a successful method to study the structural dynamics of proteins [45,[49][50][51] including the direct binding affinity of biomass-degrading enzymes to insoluble lignocellulosic substrates [52]. All four BsXynA single cysteine mutants were labeled with the spin probe MTSSL, and the mobility parameter (Ms) of the probe was assessed by analysis of the ESR spectra both in the absence and presence of arabinoxylan (Table 2 and Fig.…”

Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips

“…Thin-layer chromatography (TLC) analyses were performed on silica gel plates (10 × 10 cm) (Fluka, Darmstadt, Germany) at room temperature using a mobile phase composed of ethyl acetate, acetic acid, formic acid, and water in a ratio of 9:3:1:4 ( v / v ) [ 70 , 71 ]. Before the runs, the samples from 0, 30, 90, and 180 min (described in Section 3.10 ) were filtered in a 0.22-μm filter, precipitated overnight with 10% of trichloroacetic acid, and centrifuged at 12,000 × g for 10 min.…”