Increased sensitivity and discrimination in screening through an immobilized-resin microbiological assay method

Problems with present bioactive microbial product screening techniques include low sensitivity and insufficient discrimination capabilities. These problems are addressed by our new immobilized-resin microbiological assay. This technique concentrates bioactive samples on macroporous polymeric resins that are immobilized in hydrogel beads. These beads are then subjected to elution in the wells of an agar diffusion microbiological assay medium. With a strong base anion exchanger, the sensitivity to ampicillin of the β-lactam-supersensitive Escherichia coli mutant ESS-22-31 was increased 10-fold. Similar increases in sensitivity were obtained in the detection of streptomycin using a weak acid cation exchanger with Bacillus subtilis and for cycloheximide by a neutral resin and Saccharomyces cerevisiae NRRL-Y-139. A judicious choice of resin type and eluent permitted a selective sensitivity increase based on the charge or hydrophobic nature of the desired product. This selectivity imparts a discrimination capability to the technique.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47948/1/10295_2005_Article_BF01569546.pd

Post-acute COVID-19 neuropsychiatric symptoms are not associated with ongoing nervous system injury

A proportion of patients infected with severe acute respiratory syndrome coronavirus 2 experience a range of neuropsychiatric symptoms months after infection, including cognitive deficits, depression and anxiety. The mechanisms underpinning such symptoms remain elusive. Recent research has demonstrated that nervous system injury can occur during COVID-19. Whether ongoing neural injury in the months after COVID-19 accounts for the ongoing or emergent neuropsychiatric symptoms is unclear. Within a large prospective cohort study of adult survivors who were hospitalized for severe acute respiratory syndrome coronavirus 2 infection, we analysed plasma markers of nervous system injury and astrocytic activation, measured 6 months post-infection: neurofilament light, glial fibrillary acidic protein and total tau protein. We assessed whether these markers were associated with the severity of the acute COVID-19 illness and with post-acute neuropsychiatric symptoms (as measured by the Patient Health Questionnaire for depression, the General Anxiety Disorder assessment for anxiety, the Montreal Cognitive Assessment for objective cognitive deficit and the cognitive items of the Patient Symptom Questionnaire for subjective cognitive deficit) at 6 months and 1 year post-hospital discharge from COVID-19. No robust associations were found between markers of nervous system injury and severity of acute COVID-19 (except for an association of small effect size between duration of admission and neurofilament light) nor with post-acute neuropsychiatric symptoms. These results suggest that ongoing neuropsychiatric symptoms are not due to ongoing neural injury

Enzymic conversions in organic and other low-water media

This comprehensive manual gives reliable answers to all questions on enzyme catalysis - from searching for suitable catalytic converter systems via choosing the optimal reaction conditions to implementing modern synthesis strategies

Biocatalysis with undissolved solid substrates and products

Closing a gap in the literature, this comprehensive book examines and discusses different non-aqueous systems from organic solvents to ionic liquids for synthetic applications, thus opening the door to new successful methods for biocatalytic reactions. It gathers into one handy source the information otherwise widely spread throughout the literature, combining useful background information with a number of synthetic examples, including industrial scale processes for pharmaceutical and fine chemicals. Extremely well structured, the text introduces the fundamentals of non-aqueous enzymology, before going on to new reaction media and synthetic applications using hydrolases and non-hydrolytic enzymes. This is the one-stop reference for everyone working in this hot field

Solid-to-solid biocatalysis: thermodynamic feasibility and energy efficiency

Enzymes can catalyse solid-to-solid condensation reactions in highly concentrated aqueous substrate suspensions. Reaction products precipitate from the reaction mixture and very high conversion yields can be obtained in low volume reactors. Solid-to-solid biocatalysis combines the advantages of using enzymes in aqueous media with the high conversion yields that are typically associated with non-aqueous biocatalysis. In this article, methods are presented for the calculation of the Gibbs free energy changes and heats of reaction of condensation reactions to form amides. The overall enthalpy change of the enzymatic reaction was compared to that of the conventional chemical methods and it was found that the enzymatic reaction produces a third of the heat with better atom efficiency

Operational stability of high initial activity protease catalysts in organic solvents

The first studies on the operational stability of cross-linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol-rinsed enzyme preparations (PREPs) of subtilisin and alpha-chymotrypsin)

Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid-base buffers

We have previously demonstrated that a solid-state buffer could be successfully used to control the ionization state of subtilisin Carlsberg cross-linked microcrystals (CLECs) suspended in supercritical ethane (sc-ethane) in the presence of acid-base active species such as salt hydrates and zeolite molecular sieves. Here we studied the effect of six zwitterionic proton/sodium (pH-pNa) solid-state acid-base buffers on the catalytic activity of subtilisin CLECs in sc- ethane at high and low water activity (a(W)). CLECs were strongly activated by increasing a(W). At high a(W), and despite the high hydrolysis rates, transesterification activities were still about one order of magnitude higher than those observed at lower a(W). This is in contradiction with what was previously reported in the absence of acid-base control and supports the hypothesis that the poor catalytic performance of subtilisin CLECs at high a(W) observed in those studies was due to the inhibitory effect of the hydrolytic by- product, rather than to the competition of water with propanol for the acyl-enzyme intermediate. Although the catalytic activity of subtilisin showed a general positive correlation with the aqueous pK(a) of the acid-base buffers tested here, our results also show that as expected, the acid-base behavior of the buffers in nonaqueous media is more complex than what can be predicted from aqueous-based parameters alone. This work further confirms the usefulness of solid-state acid-base buffers in supercritical biocatalysis but highlights the need for further research on the topic

Very high activity biocatalysts for low water systems : propanol rinsed enzyme preparations (PREPs)

Depending on the preparation method used enzymes under low water conditions can exhibit differences in catalytic activity that vary by several orders of magnitude. Since this can make the difference between whether a particular biotransformation is practically useful or not it is important to control enzyme history carefully. The basic problem is simple: how to transfer the enzyme from an aqueous environment to one where it is dehydrated whilst ensuring firstly it remains in a native conformation and secondly the active site residues are in the correct protonation state. Although the importance of retaining native structure is well recognised potential detrimental changes to protonation state are often not considered. Usually it is assumed that the enzyme will exhibit 'pH memory' of the previous aqueous solution. As discussed in detail in chapter ?? [sic] Partidge this is not always a valid assumption

Operational stability of subtilisin CLECs in organic solvents in repeated batch and in continuous operation

The operational stability of cross-linked crystals (CLECs) of subtilisin Carlsberg in organic media is compared between repeated batch and continuous flow operation. The study was designed to create similar conditions in the two modes, although these can never be identical in all respects. The biocatalyst inactivates rapidly during the first two batches of 2 h each, losing 50% or more of its activity. The inactivation becomes slower over the following longer batches of 24 h. This pattern of rapid and slower phases of inactivation is similar to that observed in continuous reactors. However, in both acetonitrile and tetrahydrofuran, inactivation is somewhat faster over the first 4h of continuous operation than in the equivalent batch mode