Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical-chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.

Efficient immobilisation protocols are the result of perfect matching of factors depending on the enzyme, the process and the support for immobilisation. Physical-chemical phenomena, such as partition, solvation and diffusion, strongly affect the efficiency of the biocatalyst in each specific reaction system. Therefore, tailored solutions must be developed for each specific process of interest. Indeed, direct investigation of what occurs at the molecular level in a reaction catalysed by an immobilised enzyme is a quite formidable task and observed differences in the performance of immobilised biocatalysts must be interpreted very carefully. In any study dealing with enzyme immobilisation the prerequisite is the rigorous planning and reporting of experiments, being aware of the complexity of these multi-phase systems.

Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.neuropsychiatric disorders ͉ neurotrophins S everal lines of evidence indicate that targeting of BDNF mRNA to dendrites plays a key role in mediating synaptic plasticity (1-4). However, the molecular mechanisms regulating this process and the differential subcellular localization of alternatively spliced BDNF transcripts, remain to be clarified.Multiple BDNF transcripts are generated by alternative splicing of one 5Ј exon with a shared 3Ј exon containing the entire BDNF coding region and either a short or long 3Ј UTR sequence (5, 6). In recent studies, we have demonstrated that BDNF transcripts differ in their subcellular localization (7). Exon 1 and 4 transcripts are localized in the cell soma, while exon 2 and 6 transcripts show a somato-dendritic localization. Thus, splice variants appear to encode spatial localization signals used to preferentially regulate BDNF expression in different subcellular domains (2, 3). A recent study has suggested that the long 3Ј UTR contains signals necessary for dendritic targeting of BDNF transcripts (4). However, it is unlikely that this mechanism can fully account for the differential dendritic targeting displayed by BDNF transcripts because more than one-third of exon 4 transcripts, which are retained in the soma, contain the long 3Ј UTR. Conversely, more than one-half of exon 6 transcripts, an isoform that displays targeting to dendrites, contain the short 3Ј UTR. To help define the mechanisms underlying differential localization of BDNF transcripts, we have tested the hypothesis that additional signals might be encoded by other BDNF mRNA regions.

There is an enormous potential for synthesizing novel bio-based functionalized polyesters under environmentally benign conditions by exploiting the catalytic efficiency and selectivity of enzymes. Despite the wide number of studies addressing in vitro enzymatic polycondensation, insufficient progress has been documented in the last two decades towards the preparative and industrial application of this methodology. The present study analyses bottlenecks hampering the practical applicability of enzymatic polycondensation that have been most often neglected in the past, with a specific focus on solvent-free processes. Data here presented elucidate how classical approaches for enzyme immobilization combined\ud with batch reactor configuration translate into insufficient mass transfer as well as limited recyclability of the biocatalyst. In order to overcome such bottlenecks, the present study proposes thin-film processes employing robust covalently immobilized lipases. The strategy was validated experimentally by carrying out the solvent-free polycondensation of esters of adipic and itaconic acids. The results open new perspectives for enlarging the applicability of biocatalysts in other viscous and solvent-free syntheses

Itaconic acid is ac hemically versatile unsaturated diacid that can be produced by fermentation and potentially it can replace petrol-based monomers such as maleic and fumarica cids in the production of curable polyesters or new biocompatible functionalized materials.U nfortunately,d ue to the presence of the unsaturated C=Cb ond, polycondensation of itaconica cid is hampered by cross reactivity and isomerization. Therefore,e nzymatic polycondensations wouldr espondt ot he need of mild and selective synthetic routes for the production of novelb io-based polymers.T he present work analyses the feasibility of enzymatic polycondensation of diethyli taconate and, for the first time,p rovidesc omprehensives olutions embracing botht he formulation of the biocata-lyst, the reaction conditions and the choice of the comonomers.C omputational docking was used to disclose the structural factorsresponsible for the low reactivity of dimethyl itaconate and to identify possible solutions.S urprisingly,e xperimentala nd computational analyses revealed that 1,4-butanediol is an unsuitable co-monomer for the polycondensation of dimethyli taconate whereast he cyclica nd rigid 1,4-cyclohexanedimethanol promotes the elongation of the oligomers.

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