In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self-assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring-shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co-localized on the PCNA ring by fusing them to the C-termini of the three PCNA subunits. However, an advantage of the specific non-covalent complex formation is inextricably associated with the disadvantage of its concentration-dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components.

We herein present a method for the analysis of 10 dithiocarbamate fungicides (DTCs) in beer, fruit juice, and malt samples, where the DTCs were converted into water-soluble sodium salts in the presence of NaHCO 3 prior to methylation using dimethyl sulfate. Extraction of the methylated compounds from matrices was performed using a "quick, easy, cheap, effective, rugged, and safe" (QuEChERS) method. Following a dispersive solid-phase extraction as a clean-up step, the methylated compounds were detected by liquid chromatography-tandem mass spectrometry. Performance evaluation was carried out on beer, fruit juice, and malt samples using representative compounds. Accuracies of the spiked compounds from the various matrices ranged from 92.2 to 112.6%, and the limits of quantification of propineb, mancozeb, and thiuram were <0.52, <0.55, and <6.97 µg/kg, respectively. The developed method was then applied in the determination of dithiocarbamate fungicide contents in commercial beer and fruit juice samples.

An LC-MS/MS method was developed for the simultaneous determination of 15 water-soluble vitamins that are widely used as additives in beverages and dietary supplements. This combined method involves the following simple pre-treatment procedures: dietary supplement samples were prepared by centrifugation and filtration after an extraction step, whereas beverage samples were diluted prior to injection. Chromatographic analysis in this method utilised a multi-mode ODS column, which provided reverse-phase, anion- and cation-exchange capacities, and therefore improved the retention of highly polar analytes such as water-soluble vitamins. Additionally, the multi-mode ODS column did not require adding ion pair reagents to the mobile phase. We optimised the chromatographic separation of 15 water-soluble vitamins by adjusting the mobile phase pH and the organic solvent. We also conducted an analysis of a NIST Standard Reference Material (SRM 3280 Multi-vitamin/Multi-element tablets) using this method to verify its accuracy. In addition, the method was applied to identify the vitamins in commercial beverages and dietary supplements. By comparing results with the label values and results obtained by official methods, it was concluded that the method could be used for quality control and to compose nutrition labels for vitamin-enriched products.