A simple and efficient approach for profiling the greenness of high performance liquid chromatography (HPLC) methods is presented. This environmental assessment tool (EAT) takes into consideration the environmental, health and safety issues for all solvents involved in the chromatographic method, and calculates a total score that can be used for comparison of the greenness of different methods. A software, HPLC-EAT, has been designed to facilitate the calculation and can be downloaded free of charge at http://www.biotek.lu.se/hplc-eat/. HPLC-EAT was successfully applied for a set of different HPLC methods from the literature, including both analytical and preparative chromatography. The performance of the tool was validated and it was further combined with another free software Eco-solvent tool to perform life cycle assessments of waste disposal options of distillation or incineration. HPLC-EAT can be routinely used in method development to calculate the greenness beside the conventional standards of accuracy, robustness and reproducibility.Although analytical chemists have considered environmental improvements important for a long time, the interest for green analytical chemistry has exploded during the last few years, as evident, for example, from the large number of publications and the special issue devoted to this subject by Trends in Analytical Chemistry (2010, volume 29, issue 7). 1-5 Green analytical chemistry goes hand in hand with the wellknown concept of green chemistry, 6,7 and can be summarized by

BackgroundMost of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development.ResultsWe report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin.ConclusionThe ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales’ procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.

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A biocatalysis based method for the solvent-free production of N-alkanoyl-N-methylglucamide (MEGA) surfactants was developed and used as a case study for the evaluation of different environmental assessment tools, such as the freeware package EATOS (Environmental Assessment Tool for Organic Synthesis). In order to also consider energy usage and process facilities, e.g. heating, stirring and vacuum, a complementary tool was needed; hence the EcoScale method and the use of an energy monitoring socket were also exploited. The solvent-free method followed by a simple hydrolysis step gave a final amide yield of 99% and a product essentially free of remaining substrate, N-methylglucamine (MEG). The latter is important since MEG can potentially be converted to carcinogenic nitrosamines. The absence of solvent in the reaction medium was also found to result in a significantly reduced potential environmental impact. The environmental tools used in this study were further scrutinized, and even if they represent some of the best freely available tools for evaluation of early stage process development, some points for further improvements are suggested.

The prevalence of extensively and pandrug-resistant Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes, nonetheless its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8 kb genome (50 ORFs), encoding an endolysin (LysMK34) with potent muralytic activity (1499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55 °C and a broad pH activity range (4-10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. Addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are re-sensitized in both Mueller-Hinton broth and 50 % human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic. Importance A. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last resort antibiotic and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure dependent intrinsic antibacterial activity, providing new insights in the mode-of-action of lysins with intrinsic activity against Gram-negative bacteria.

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Fungal endophytes are a major source of anti-infective agents and other medically relevant compounds. However, their classical blinded-chemical investigation is a challenging process due to their highly complex chemical makeup. Thus, utilizing cheminformatics tools such as metabolomics and computer-aided modelling is of great help deal with such complexity and select the most probable bioactive candidates. In the present study, we have explored the fungal endophytes associated with the well-known antimalarial medicinal plant Artemisia annua for their production of further antimalarial agents. Based on the preliminary antimalarial screening of these endophytes and using LC-HRMS-based metabolomics and multivariate analyses, we suggested different potentially active metabolites (compounds 1–8). Further in silico investigation using the neural-network-based prediction software PASS led to the selection of a group of quinone derivatives (compounds 1–5) as the most possible active hits. Subsequent in vitro validation revealed emodin (1) and physcion (2) to be potent antimalarial candidates with IC50 values of 0.9 and 1.9 µM, respectively. Our approach in the present investigation therefore can be applied as a preliminary evaluation step in the natural products drug discovery, which in turn can facilitate the isolation of selected metabolites notably the biologically active ones.