Theoretical investigations on the treatment of bicyclic endoperoxides (1,2-dioxines) with ozone at the HF/6–31G*, MP2/6–31G* or 6–311G*, and DFT(B3LYP)/6–31G* levels of theory indicate that the estimated activation energies for formation of the possible endo-endo, endo-exo, exo-endo, or exo-exo transition states along with the formation of the primary ozonides and product ozonides are very sensitive to effects of electron correlation and basis set. This study suggests that MP2/6–311G* is the best level of theory for evaluating such systems. At the MP2/6–311G* level of theory it was found that the transition state for primary ozonide formation was lowest in energy when ozone approaches in an endo facial fashion with a further 3 kJ mol–1 stabilisation seen when the central oxygen within the primary ozonide protrudes outwards (exo) as opposed to inwards (endo). The product ozonides are predicted to be more stable than the combined starting materials by some 380–580 kJ mol–1 depending on the level of theory, clearly highlighting the substantive exothermic nature of this type of ozonolysis reaction.
Cellulosic biomass represents a huge reservoir of renewable carbon, but converting it into useful products is challenging. Attempts to transfer cellulose degradation capability to industrially useful micro-organisms have met with limited success, possibly due to poorly understood synergy between multiple cellulases. This is best studied by co-expression of many combinations of cellulases and associated proteins. Here, we describe the development of a test platform based on Citrobacter freundii, a cellobiose-assimilating organism closely related to Escherichia coli. Standard E. coli cloning vectors worked well in Cit. freundii. Expression of cellulases CenA and Cex of Cellulomonas fimi in Cit. freundii gave recombinant strains which were able to grow at the expense of cellulosic filter paper or microcrystalline cellulose (Avicel) in a mineral medium supplemented with a small amount of yeast extract. Periodic physical agitation of the cultures was highly beneficial for growth at the expense of filter paper. This provides a test platform for the expression of combinations of genes encoding biomass-degrading enzymes to develop effective genetic cassettes for degradation of different biomass streams.Biofuels have been shown to be the best sustainable and alternative source of fuel to replace fossil fuels. Of the different types of feedstocks used for producing biofuels, lignocellulosic biomass is the most abundant. Converting this biomass to useful products has met with little success. Different approaches are being used and microbial platforms are the most promising and sustainable method. This study shows that Citrobacter freundii is a better test platform than Escherichia coli for testing various combinations of cellulases for the development of microbial systems for biomass conversion.
Guaiacol and 4-methylguaiacol are routinely used as markers to determine extent of smoke impact on winegrapes and wines. However, smoke contains a complex group of compounds that may contribute to smoke taint in winegrapes and wine. In this study, a gas chromatography-mass spectrometry (GC-MS) based analytical method was developed and validated for the profiling of various smoke taint compounds in wines made from smoke affected fruit. A total of 22 analytes were separated and identified in the GC-MS chromatogram, all of which were selected to evaluate the samples and precision of the method. The GC-MS method showed good repeatability/reproducibility with intra- and inter-day relative standard deviation (RSD) of ±14%. The method was used to demonstrate that the smoked grapes and resultant wines, compared to unsmoked wines, contained significantly enhanced levels of guaiacol and 4-methylguaiacol along with other lignin derived phenols such as cresols and syringol. In smoke affected grapes and young wines, volatile phenols exist as glyco-conjugates (potential taint), which hydrolyse slowly leading to unacceptable levels of taint accumulation in wine during storage. The GC-MS method reported here, in conjunction with the optimised acid hydrolysis of phenol glycoconjugates, was successfully used to determine potential levels of smoke taint compounds in wines. Thus, the method can be used for screening smoke exposed grapes for potential taint levels prior to wine making. The results presented here highlight the need to include an array of smoke derived phenols to develop a complete picture of smoke taint and associated aroma in affected grapes and wines. Keywords: Acid hydrolysis, gas chromatography-mass spectrometry, glycosides of phenols, lignin, smoke taint, solid phase extraction, volatile phenols, wine.
The ozonolysis of bicyclic 1,2-dioxines was investigated using a variety of 1,4-disubstituted 1,2-dioxines along with a 1,3-dialkyl and steroidal example, with yields ranging from moderate to excellent. Two different pathways were observed upon reaction of the 1,4-disubstituted 1,2-dioxines with ozone; one pathway saw the "expected" results, that is, cleavage of the olefinic moiety with generation of 1,4-dicarbonyl 1,2-dioxines, while the other pathway revealed a previously unobserved rearrangement involving cleavage of the peroxide linkage along with loss of either CO or CO(2). Several unsymmetrical ozonolyses were also performed to further investigate the origins of this rearrangement, and initial mechanistic insights into the fragmentation pathways are discussed.
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