The enzymology of itaconic acid (methylenesuccinic acid) production by Aspergillus terreus has been investigated. The organism has been shown to possess many of the enzymes of the citric acid cycle when cultured under conditions which did support, and conditions which did not support, itaconate production. No significant differences in the levels of individual citric acid cycle enzyme activities between the two modes of culture were detected. A new, more sensitive, assay for itaconate has been devised. An unfractionated extract of Pseudomonas aeruginosa, capable of converting itaconate to pyruvate, provides the basis of the enzyme-linked assay in which the ultimate product, pyruvate, is detected spectrophotometrically as its phenylhydrazone derivative. The assay has been shown to be applicable to the monitoring of itaconate production during the fermentation of Aspergillus terreus. Furthermore, the enzyme-linked assay has been adapted to operate in a continuous mode and hence to permit measurement of the rate of formation of itaconate by cell-free extracts of Aspergillus terreus. cis-Aconitate decarboxylase (CAD) activity was detected in mycelia harvested in the acid-producing phase of growth, but was not found in mycelia harvested prior to the production of acid, nor when the organism was grown under conditions which did not support acid production. The enzyme present in the crude extract precipitated between 40% and 60% ammonium sulphate saturation. Studies on this partially purified extract indicated a Km value for cis-aconitate of 0.15muM and a pH optimum of 5.5. Investigation of Aspergillus terreus grown in batch culture revealed that the depletion of phosphate in the culture medium coincided with a rise in intracellular CAD activity and subsequent itaconate production. In addition, substantial CAD activity was detected in cell-free extracts of Aspergillus terreus even after the production of itaconate had ended, thus suggesting that other factors, which are possibly amenable to external control, may be responsible for the cessation of acidogenesis.
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