Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Purification and properties of a beta-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli.

1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile "Caldocellum saccharolyticum" strain Tp8T6.3.3.1. It was found to have an M(r) of 42,000 and an isoelectric point of approx. 5.0. 2. The enzyme showed optimum activity at pH 5.0-7.7 and had an activation energy of 44 kJ mol-1. It was stable at room temperature at pH 4.5-11.5 in the presence of 0.5 mg ml-1 bovine serum albumin. The half-life of the enzyme at 75 degrees C was 20 min at pH 6.0 in the presence of 0.5 mg ml-1 bovine serum albumin. 3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The Km for oat spelts xylan was 0.021% (w/v) at pH 6.0. 4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl beta-D-cellobioside and p-nitrophenyl beta-D-cellobioside, but could not hydrolyse xylobiose. 5. It showed transferase activity on p-nitrophenyl beta-D-xylopyranoside. Xylose did not inhibit the enzyme.

Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli.

An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum'). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.