Propionate sensor using coenzyme-A transferase and acyl-CoA oxidase

We developed an amperometric propionate sensor using comprised of two recombinant enzymes, propionate coenzyme A CoA transferase from Clostridium propionicum and short-chain acyl-CoA oxidase from Arabidopsis thaliana. Response current increased linearly with increase in propionate concentration from 10 microM to 100 microM. The detection limit was 10 microM propionate.     

Propionyl-coenzyme A synthetases of Ralstonia solanacearum and Salmonella choleraesuis display atypical kinetics

Propionyl-coenzyme A synthetases (PrpE) of Salmonella choleraesuis and Ralstonia solanacearum sharing 62% identity in amino acid sequence to each other were cloned, expressed in Escherichia coli and purified. Both enzymes catalyzed acetyl-, propionyl-, butyryl- and acrylyl-coenzyme A formation with the highest k(cat)/K(m) values for propionate. They displayed sigmoidal homotrophic autoactivation kinetics for propionate but not for the other acyl substrates tested. Besides, substrate inhibition kinetics was observed for co-substrates, i.e. ATP and CoA. Based on the kinetic data reported herein, the reaction mechanisms of the enzyme are discussed.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Volatile fatty acid-sensing system involving coenzyme-A transferase

On-site monitoring of volatile fatty acids (VFAs), such as propionate, is industrially and medically important. The present study developed a VFA biosensing system comprised of two recombinant enzymes, propionate coenzyme A (CoA) transferase (PCT) from Clostridium propionicum and acyl-CoA oxidase from Arabidopsis thaliana. This system produced hydrogen peroxide in the presence of acetyl-CoA, oxygen, and VFA substrates, which could be quantified by colorimetric methods using peroxidase and dye reagents (e.g., p-aminobenzoic acid plus 4-aminoantipyrine or Amplex Red). The use of PCT and acetyl-CoA, rather than acyl-CoA synthetases (ACS) and CoA-SH, obviated a background reaction of dye reagents with CoA-SH and enabled very sensitive detection of VFAs (down to 1 microM propionate, more than 100-fold more sensitive compared to previously developed ACS biosensors). We demonstrated its utility by measuring propionate concentrations in serum and fermentation samples. Results suggest that our biosensing system is applicable to the detection of propionate in medical and fermentation samples.