Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC 261-388 ) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule.From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC 261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC 261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.
Cellulases and xylanases are enzymes of industrial significance, particularly in the pulp, paper, textile, and animal feed industries. Moreover, their utilization in the food industry, among them, bakery, brewery, winery and fruit and vegetable juice production, cannot be underestimated. One of the potential sources of enzymes is the filamentous fungi, and hence bio-prospecting of this specific group of microorganisms with the highest levels of cellulase and xylanase secretions is being continuously undertaken. The specific aim of this study was to isolate and characterize cellulase-and xylanaseproducing filamentous fungi from termite mounds. Termite mounds have long been established as very good sources of filamentous fungi with the ability to secrete high levels of lignocellulolytic enzymes, and hence an ideal target for the bio-prospecting of cellulases and xylanases. In this study, various groups of filamentous fungi were isolated through enrichment and repeated sub-culturing. This was followed by screening using the Congo red plate-based assay. Cellulase and xylanase activities during the solid-state fermentation of wheat bran were detected and analyzed through spectrophotometry via the 3,5-dinitrosalicylic acid detection system for reducing sugars. The obtained fungal isolates were then finally characterized through zymography, reaction kinetics and morphological studies. Overall, a total of eight different groups of fungi, capable of decomposing cellulose and hemicellulose, were isolated, and their tentative identities established as Fusarium, Didymostible, Penicillium, Phytophthora, Oedocephalum, Aspergillus, Monosporascus and Acremonium. Taken together, findings of this study conceivably showed that termite mounds are a good source of filamentous fungi that in turn are also a good source of cellulases and xylanases that arguably, can be recommended for use in industrial and commercial settings.
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