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.
Highlights
The 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
The secondary structure content estimated by deconvolution of the CD spectra of the Bacillus subtilis cellulase 5A (BsCel5A)-CBM3 was 26.4% α-helix and 20.7% β-strand and for the BsCel5A-CBM3 was 26.6% α-helix and 22.3% β-strand. These values are consistent with the sum of the secondary structure assignments from the crystallographic structure of the BsCel5A catalytic domain (PDB 3PZV, [27]) with either the CBM3 (PDB 2L8A [27], sum of 22.5% α-helix and 21.5% β-strand) or the Ruminiclostridium thermocellum CBM11 (RtCBM11) (PDB 1V0A [43], sum of 21.2% α-helix and 27.0% β-strand)
We have shown for the first time through direct measurements by Electron Paramagnetic Resonance (EPR) spectroscopy that the CBM3 of the BsCel5A-CBM3 does not bind to β-glucan substrate, suggesting that the CBM3 and catalytic domains may act independently
Summary
The 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. Exoglucanases or cellobiohydrolases (exo-1,4-β-glucosidases; CBH; EC 3.2.1.91) act on the reducing and non-reducing extremities in crystalline regions of the cellulose fibers to release glucose and cellobiose, and β-glucosidases hydrolyze short cell oligosaccharides and cellobiose to release glucose [3]. This synergistic hydrolysis of cellulose contributes to carbon recycling in the biosphere [3,4,5], and has been extensively studied from the viewpoint of biotechnological applications in the biofuels, animal feed, pulp bleaching, wastewater treatment, and paper bioconversion industries [6, 7]. The CBMs are currently grouped into 81 families (CAZy database, http://www. cazy.org) that display variable binding specificities against different plant cell wall polysaccharides such as crystalline or amorphous cellulose, β-glucans, xylans, laminarins, mannans, galactans, xyloglucans, arabinans, and others
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