Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Background: The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber \u3e 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results: The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that \u3e 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions: This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

PLoS ONE. Volume 7, Issue 8, 29 August 2012, Article number e43902.The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots. © 2012 Kishko et al

Biphasic kinetic behavior of E. coli WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase.

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots

Substrate binding sites. A. NADH.

<p>View of the active site with NADH bound in the optimized position found by docking as described in the text. Green, molecular surface of holoWrbA calculated from the 2.05 Å crystal structure (PDB ID 3B6J) after removal of the FMN cofactor. Oxidized FMN is depicted as a skeletal model in atomic colors with cyan carbon, and docked NADH with white carbons for differentiation from FMN. Dashed lines represent the indicated distances in Å between nicotinamide C4 and each indicated electron acceptor site of FMN. <b>B. Mutual exclusivity of NADH and BQ.</b> Viewpoint of the binding cavity as in panel A but slightly zoomed out to better depict the steric environment of the full pocket. Translucent white indicates the molecular surface of NADH in the position identified by docking as in panel A; red indicates the molecular surface of BQ calculated from the 1.99 Å crystal structure of the BQ/WrbA complex (PDB ID 3B6K). The part of each substrate that is occluded by the other is represented by the overlap between the red and translucent white surfaces.</p

Substrate affinity.

<p><b>A.</b> NADH binding to 50 µM apoWrbA determined by UV spectroscopy. Difference absorbance at 265 nm (see text) is plotted <i>vs.</i> [NADH]. The solid line is intended only to guide the eye and does not represent a fit to the data. <b>B.</b> NAD binding to 200 µM apoWrbA detected by ³¹P NMR. Spectra at 100, 200, 500, 1000 and 2000 µM NAD from bottom to top, respectively, are overlaid. The bracket with four arrows indicates the doublet pair characteristic of free NAD.</p

Kinetic constants of WrbA^a.

a<p>Kinetic data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone-0043902-g003" target="_blank">Figure 3</a>.</p>b<p>Errors are one standard deviation derived from triplicate measurements and propagated to the ratio Vmax/Km.</p

Steady-state kinetics.

<p>Initial velocity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#s4" target="_blank">Methods</a>) is plotted <i>vs.</i> substrate concentration. <b>A.</b> NADH at constant [BQ] = 50 µM. <b>B.</b> BQ at constant [NADH] = 50 µM. <b>C.</b> DCPIP at constant [NADH] = 50 µM. Each plot depicts three temperature treatments of WrbA prior to assay (see text): squares, 5°C; triangles, 23°C; circles, 5°C after 23°C. Solid lines are intended only to guide the eye and do not represent fits to the data.</p

Sedimentation velocity.

<p>Each panel shows the sedimentation velocity profile using the whole boundary g(s*) approach of Stafford <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone.0043902-Stafford1" target="_blank">[28]</a> for apoWrbA (black), WrbA+50 µM FMN (red), and WrbA+50 µM FMN+0.5 mM NAD (blue). A, 3 µM total protein (monomer) at 5°C; B, 3 µM total protein (monomer) at 20°C; C, 20 µM total protein (monomer) at 5°C; D, 20 µM total protein (monomer) at 20°C.</p

Product Inhibition^a.

a<p>Kinetic data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone.0043902.s003" target="_blank">Figure S3</a>.</p

Ping-pong kinetics.

<p>Initial velocities were determined at 23°C to limit the reaction to a single kinetic phase as much as possible. <b>A.</b> Titration of NADH at [BQ] = 10 µM (open squares), 20 µM (triangles), 50 µM (circles), 100 µM (filled squares). <b>B.</b> Titration of BQ at [NADH] = 10 µM (open squares), 20 µM (triangles), 50 µM(circles), 100 µM(filled squares). Solid lines represent non-linear least-squares best fit of the Michaelis-Menten equation to the data points. The values of apparent Km and apparent Vmax returned from the fit are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone-0043902-t001" target="_blank">Table 1</a>.</p