BACKGROUND:
Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed trasesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.
RESULTS:
We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50[degree sign]C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.
CONCLUSIONS:
Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
Title: Crystal Structure of Proteus mirabilis Lipase, a Novel Lipase from the Proteus/Psychrophilic Subfamily of Lipase Family I.1 Korman TP, Bowie JU Ref: PLoS ONE, 7:e52890, 2012 : PubMed
Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45 degrees C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca(2+) coordination may explain how these lipases can fold without specific chaperones.
Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity-dependent remodelling of excitatory synapses. Here, we report that Zn(2+) ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member-specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA-mediated knockdown, we could demonstrate that only the overexpression of zinc-sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn(2+) along with the knockdown of zinc-insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD-95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn(2+) is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.
