Kinetic Profiling of Homogeneous and Heterogeneous Biocatalysts in Continuous Flow by Online Mass Spectrometry.

Enzyme kinetics is normally assessed by performing individual kinetic measurements using batch-type reactors (test tubes, microtiter plates), in which enzymes are mixed with different substrates. Some drawbacks of conventional methods are the large amounts of experimental materials, long analysis times, and limitations of spectrophotometry. Therefore, we have developed a method for facile determination of enzyme kinetics using online flow-based mass spectrometry. A concentration ramp of substrate or product was created by dynamically adjusting flow rates of pumps delivering stock solution of substrate and diluent. Precise kinetic measurements were performed by reaction product quantification and initial rate calculation. In the presence of ascending substrate concentrations, the rate of a target enzyme (penicillinase)-catalyzed hydrolysis was varied. By measuring the reaction product continuously, Michaelis constants (KM) could be calculated. The enzyme kinetic measurements for hydrolysis of penicillins were conducted based on this simple, rapid, and low sample consumption online flow device. In the homogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 254.9 ± 14.5, 29.2 ± 0.3, 2.6 ± 0.1, and 5.4 ± 0.1 µM, respectively. In the heterogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 408.9 ± 75.1, 114.4 ± 8.0, 21.8 ± 0.7, and 83.3 ± 4.8 µM, respectively. Apart from enzyme assay, the showcased method for the generation of temporal concentration ramps can be utilized to perform rapid quantity calibrations for mass spectrometric analyses.

Rapid Acid/Base Switching in Flow Injection Analysis and Isocratic Elution Liquid Chromatography with Mass Spectrometric Detection for Improved Sensitivity.

Ion signals in electrospray ionization (ESI) mass spectrometry (MS) are affected by addition of acid or base. Acids or bases are typically added to samples to enhance detection of analytes in positive- or negative-ion mode, respectively. To carry out simultaneous monitoring of analytes with different ionogenic moieties by ESI-MS, a rapid acid/base switching system was developed. The system was further coupled with flow injection analysis (FIA) and liquid chromatography (LC) MS. The two variants enable detection of separated analytes immediately after alternating addition of acid and base. The methods were tested using a set of phospholipids (PLs) as analytes. The rapid acid/base switching enhanced signals of some of the PL analytes in both ion modes of MS. Both FIA-MS and LC-MS with acid/base switching show signal enhancements (∼1.3-23.2 times) of some analyte signals when compared with analysis conducted without acid/base switching. The proposed methods are suitable for simultaneous analysis of cationic and anionic analytes. The FIA-MS and LC-MS methods with acid/base switching were also applied in analysis of lipid extract from real samples (sausage and porcine liver). However, the FIA-MS results were affected by ionization competition and isobaric interference due to the complexity of the sample matrix and diversity of PL species. In contrast, the LC-MS mode provides adequate selectivity to observe signal enhancement for specific analyte ions. Overall, alternating addition of acid and base immediately before the ESI source can improve analytical performance without the need to carry out separate analyses targeting different types of analytes.

Eucalyptus red grandis pretreatment with protic ionic liquids: effect of severity and influence of sub/super-critical CO2 atmosphere on pretreatment performance.

Deconstruction of lignocellulosic biomass with low-cost ionic liquids (ILs) has proven to be a promising technology that could be implemented in a biorefinery to obtain renewable materials, fuels and chemicals. This study investigates the pretreatment efficacy of the ionoSolv pretreatment of Eucalyptus red grandis using the low-cost ionic liquid triethylammonium hydrogen sulfate ([N2220][HSO4]) in the presence of 20 wt% water at 10% solids loading. The temperatures investigated were 120 °C and 150 °C. Also, the influence of performing the pretreatment under sub-critical and supercritical CO2 was investigated. The IL used is very effective in deconstructing eucalyptus, producing cellulose-rich pulps resulting in enzymatic saccharification yields of 86% for some pretreatment conditions. It has been found that under a CO2 atmosphere, the ionoSolv process is pressure independent. The good performance of this IL in the pretreatment of eucalyptus is promising for the development of a large-scale ionoSolv pretreatment processes.

Green and Sustainable Solvents in Chemical Processes.

Sustainable solvents are a topic of growing interest in both the research community and the chemical industry due to a growing awareness of the impact of solvents on pollution, energy usage, and contributions to air quality and climate change. Solvent losses represent a major portion of organic pollution, and solvent removal represents a large proportion of process energy consumption. To counter these issues, a range of greener or more sustainable solvents have been proposed and developed over the past three decades. Much of the focus has been on the environmental credentials of the solvent itself, although how a substance is deployed is as important to sustainability as what it is made from. In this Review, we consider several aspects of the most prominent sustainable organic solvents in use today, ionic liquids, deep eutectic solvents, supercritical fluids, switchable solvents, liquid polymers, and renewable solvents. We examine not only the performance of each class of solvent within the context of the reactions or extractions for which it is employed, but also give consideration to the wider context of the process and system within which the solvent is deployed. A wide range of technical, economic, and environmental factors are considered, giving a more complete picture of the current status of sustainable solvent research and development.

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.