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Industrially, the use of high temperatures (40–60°C) in the l-malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50°C and highly stable even after 24 h of incubation at 40°C. The same gene from mesophilic Streptomyces coelicolor ( scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30°C and was rapidly denatured at 50°C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10°C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C. [ABSTRACT FROM AUTHOR]

Abstract Fumarase is one of the key enzymes in the TCA cycle and has been implicated in virulence and survival of some microorganisms under suboptimal environmental conditions. In this study, the fumC genes that encode fumarase C (FUMCs) from Neisseria meningitidis, N. gonorrhoeae and N. subflava were identified by homology-based analysis, cloned by polymerase chain reactions and fully sequenced. The inferred primary sequence of neisserial FUMCs showed a high degree of conservation with 97.898.7% amino acid identity. However, phylogenetic analysis revealed that these neisserial FUMCs are divergent from class II fumarases found in other microorganisms, rat and human. The putative fumC genes were subcloned into the expression vector, pGEX-6P-1 and efficiently expressed in Esherichia coli BL21. The purified recombinant fusion proteins obtained by affinity chromatography demonstrated high catalytic activities (120180 U/mg), thus authenticating the identities and functionalities of the cloned genes. Whether FUMC has any physiological relevance to the pathogenesisity of Neisseriae must await future gene disruption or mutagenesis studies. [ABSTRACT FROM AUTHOR]