Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

Effects of drying on physical properties, phenolic compounds and antioxidant capacity of Robusta wet coffee pulp (Coffea canephora).

Wet coffee pulp (WCP), produced as waste from coffee production, is a rich source of bioactive compounds, especially caffeine and chlorogenic acid. However, it contains high moisture content, thus it is challenging for further utilization due to degradation and microbial deterioration. Dehydration is, therefore, necessary to minimize degradation and ease storage and transportation. As a waste, the common drying methods should be prioritized to be feasible for industrial application. This study aimed to determine the impact of different drying conditions of the three common drying methods including low temperature and pressure, vacuum and hot air drying on physical, phytochemical and antioxidant properties of WCP to identify the most suitable drying conditions. Browning index, moisture content, total phenolic content (TPC), flavonoids (TFC), proanthocyanidins, and chlorogenic acid as well as the antioxidant properties of the dried coffee pulp were significantly influenced by different tested conditions. Vacuum drying was found to be more suitable for drying the wet coffee pulp as compared to low temperature and pressure and hot air drying methods. Vacuum drying at 110 °C retained the highest TPC (14.4 mg gallic acid equivalents (GAE)/g dry weight (DW)), proanthocyanidins (6.8 mg catechin equivalents (CE)/g DW), TFC (13.2 CE/g DW), caffeine (2.9 mg/g DW) and antioxidant capacity. Chlorogenic acid (3.4 mg/g DW) was 13% lower, but energy consumption was 37% less than vacuum drying at 90 °C. Therefore, vacuum drying (3.75 mmHg) at 110 °C for 4h 05 min was suggested for dehydration of the wet coffee pulp for subsequent recovery and processing.