Methods for the preparation of carrier-free insoluble enzymes are reviewed. The technology of cross-linked enzyme aggregates has now been applied to a range of synthetically useful activities. Fusion proteins are also gaining momentum because they allow a relatively selective aggregation or even a specific self-assembly of the desired enzyme activity into insoluble particles in the absence of potentially denaturing chemicals required for precipitation and cross-linking. Recycling of insoluble protein particles for multiple rounds of batchwise reaction has been demonstrated in selected biotransformations. However, for application in a fully continuous biocatalytic process, low resistance to mechanical stress and high compressibility are issues for consideration on carrier-free enzyme particles.

AimsInsulin-dependent positive inotropic effects (PIE) are partially Ca 2þ independent. This mechanism is potentially glucose dependent. In contrast to most animal species, human myocardium expresses high levels of sodiumglucose-transporter-1 (SGLT-1) mRNA besides the common glucose-transporters-1 and -4 (GLUT1, GLUT4). Methods and resultsWe used ventricular myocardium from 61 end-stage failing human hearts (ischaemic cardiomyopathy, ICM and dilated cardiomyopathy, DCM) and 13 non-failing donor hearts. The effect of insulin on isometric twitch force was examined with or without blocking of PI3-kinase, GLUT4-translocation, or SGLT-1. Substrate-dependent (glucose vs. pyruvate vs. palmitoyl-carnitine) effects were tested in atrial myocardium. mRNA expression of glucose transporters was analysed. Insulin increased developed force by 122 + 7.4, 121.7 + 2.5, and 134.1 + 5.7% in non-failing, DCM, and ICM (P , 0.05 vs. DCM), respectively. Positive inotropic effect was partially blunted by inhibition of PI-3-kinase, GLUT4, or SGLT1. Combined inhibition of PI3-kinase and glucose-transport completely abolished PIE. Positive inotropic effect was significantly stronger in glucose-containing solution compared with pyruvate or palmitoyl-carnitine containing. mRNA expression showed only a tendency towards elevated GLUT4-expression in ICM. ConclusionsPositive inotropic effect of insulin is pronounced in ICM, but underlying mechanisms are unaltered. The Ca 2þ -independent PIE of insulin is mediated via glucose-transporters. Together with the Ca 2þ -dependent PI-3-kinase mediated pathway, it is responsible for the entire PIE. Substrate-dependency affirms a glucose-dependent part of the PIE.--

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HighlightsFreeze concentration of bovine serum albumin studied in a laboratory freeze container.Protein micro-segregation visualized by confocal laser scanning microscopy.Freezing protocol affected protein freeze concentration.Spatial heterogeneities created by freeze concentration.Micro-segregation was affected by freezing parameters.