Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from and, the previously unreported enzymes from the hyperthermophiles and, the thermophiles and, and the mesophile were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). and carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the kinase is efficiently converted to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) SSFF can be used for production of ethanol and biomass from wheat straw.Glucose present in the feed controlled the assimilation of xylose and acetic acid.The fungal growth rate was found not to be influenced by the feed composition.Rhizopus biomass yields of up 0.34 g/g and ethanol yields of 0.40 g/g were obtained. The economic viability of the 2 nd generation bioethanol production process cannot rely on a single product but on a biorefinery built around it. In this work, ethanol and fungal biomass (animal feed) were produced from acid-pretreated wheat straw slurry under an innovative simultaneous saccharification, fermentation, and filtration (SSFF) strategy. A membrane unit separated the solids from the liquid and the latter was converted to biomass or to both biomass and ethanol in the fermentation reactor containing Rhizopus sp. pellets. Biomass yields of up to 0.34 g/g based on the consumed monomeric sugars and acetic acid were achieved. A surplus of glucose in the feed resulted in ethanol production and reduced the biomass yield, whereas limiting glucose concentrations resulted in higher consumption of xylose and acetic acid. The specific growth rate, in the range of 0.013-0.015/h, did not appear to be influenced by the composition of the carbon source. Under anaerobic conditions, an ethanol yield of 0.40 g/g was obtained. The present strategy benefits from the easier separation of the biomass from the medium and the fungus ability to assimilate carbon residuals in comparison with when yeast is used. More specifically, it allows in-situ separation of insoluble solids leading to the production of pure fungal biomass as a value-added product. ARTICLE INFO ABSTRACT © 2016 BRTeam. All rights reserved.Journal homepage: www.biofueljournal.com FazeliNejad et al. / Biofuel Research Journal 9 (2016) [372][373][374][375][376][377][378] Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF).

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