The EC135 was the first helicopter at ECD (Eurocopter Deutschland) to be qualified according to the Damage Tolerance requirements first known as ‘Special Condition’ of the German Luftfahrtbundesamt (LBA). Further investigations for other composite parts followed this approach.

Whole cell biocatalysis can effectively be used for the production of enantiomerically pure compounds, but efficiency is often low. Toxicity and poor solubility of substrates and products are the main obstacles. In this study, water immiscible ionic liquids are shown to have no damaging effects on the cell membranes of Escherichia coli and Saccharomyces cerevisiae. Thus, they can be used as biocompatible solvents for microbial biotransformations exemplified by an increase in yield of chiral alcohol synthesis. As key point to the success of these processes, the distribution ratio of the reactants between the ionic liquid and the aqueous phase was identified. The use of ionic liquids as substrate reservoir and in situ extracting agent for the asymmetric reduction of various ketones resulted in an increase of chemical yield from <50% to 80–90% in simple batch processes. (R)-1-(4-chlorophenyl)ethanol was produced at a higher initial reaction rate in the biphasic system (>50 μM s−1 L−1) compared to the aqueous system. This result demonstrates that good mass transfer rates can be obtained despite the relatively high viscosity of ionic liquids.

The reduction of methyl acetoacetate was carried out in continuously operated biotransformation processes catalyzed by recombinant Escherichia coli cells expressing an alcohol dehydrogenase from Lactobacillus brevis. Three different cell types were applied as biocatalysts in three different cofactor regeneration approaches. Both processes with enzyme-coupled cofactor regeneration catalyzed by formate dehydrogenase or glucose dehydrogenase are characterized by a rapid deactivation of the biocatalyst. By contrast the processes with substrate-coupled cofactor regeneration by alcohol dehydrogenase catalyzed oxidation of 2-propanol could be run over a period of 7 weeks with exceedingly high substrate and cosubstrate concentrations of up to 2.5 and 2.8molL−1, respectively. Even under these extreme conditions, the applied biocatalyst showed a good stability with only marginal leakage of intracellular cofactors.

A recombinant Saccharomyces cerevisiae strain over-expressing the fatty acid synthase of S. cerevisiae and the glucose dehydrogenase of Bacillus subtilis was applied for enantioselective reduction of ethyl 4-chloro acetoacetate (CAAE) to ethyl (S)-4-chloro-3-hydroxybutanoate (S-CHBE) as well as the reduction of ethyl benzoylacetate (EBA) to ethyl (S)-3-hydroxy-3-phenylpropionate (S-HPPE). The reaction conditions for these asymmetric reductions were optimised by combining simple screening procedures and a stochastic search strategy (Genetic Algorithm). Complete conversion of 200mM CAAE was achieved within 8h in a two liquid phase system (water/n-butyl acetate). The S-CHBE yield (94%) and the product/biocatalyst ratio (1.9mmol S-CHBE/g cell dry weight) were improved by 50 and 450%, respectively, compared to published data. The enantiomeric excess was 90%. Nearly complete conversion of 55mM EBA was achieved within 48h without addition of an organic solvent. The S-HPPE yield (74%) and the product/biocatalyst ratio (0.7mmol S-HPPE/g cell dry weight) were improved by 20 and 350%, respectively, compared to published data. The enantiomeric excess was >97% in the case of S-HPPE production.

A recombinant strain of Pichia pastoris coexpressing the carbonyl reductase of Candida magnoliae and the glucose dehydrogenase of Bacillus subtilis was used for the stereoselective reduction of ethyl 4-chloro acetoacetate 1 to (S)-4-chloro-3-hydroxybutanoate 2. The bioreduction was performed in a two-phase reaction system with n-butyl acetate as organic solvent. Complete conversion of 350mM 1 with a final yield of 91% and an enantiomeric excess of 95% was achieved by continuous feeding of cell suspension and ketoester 1.

Current strategies to improve the secretion of heterologous proteins in Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). A family of ER-specific protein s which share active-site homology wit protein disulfide isomerase (PDI) has been identified from other systems, many of which are inducible by agents which cause malfolding of proteins in the ER. Here we report identification of tigA from Aspergillus niger and erp38 from Neurospora crassa, two novel members of the PDI superfamily of proteins. TIGA and ERp38 show 66% identity at the amino acid level and are putative ER proteins. Both proteins show tandemly linked thiol-oxidoreductase domains followed by a functionally uncharacterised C-terminal domain. The most distal active site in TIGA is created by excision of a 66-bp intron. Although no Unfolded Protein Response elements can be seen in the tigA promoter, sequence homology has identified associated with protein trafficking (ERPTRE) in a gene encoding the related mammalian protein, ERp72, as well as a second motif conserved amongst the glucose-related protein family. Southern and dot blot analysis indicate that the tigA gene is present in single copy. Both the A. niger and N. crassa proteins show homology with a stress-inducible alfalfa, G1. Transcription of tigA is induced 2–3-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. Strains overexpressing a heterologous protein show no increased tigA mRNA levels.

Publisher Summary This chapter describes the preparation and use of interphase extracts from Xenopus eggs that promote assembly of nuclei around a chromatin substrate and, therefore, constitute the experimental basis to study nuclear architecture, function, and dynamics on a molecular level. Entry into mitosis of higher eukaryotic cells is accompanied by a series of reversible morphological rearrangements that are indispensible to achieve cell duplication. The most prominent among them includes chromosome condensation, spindle formation, and breakdown of the cell nucleus. All the cellular components recruited to perform mitosis-specific functions are recycled at the end of mitosis, resulting in the reformation of interphase cells. One of the most valuable systems to study the cell cycle in vitro is derived from unfertilized eggs from Xenopus laevis frogs. Depending on the way these extracts are used for the in vitro study of nuclear assembly nuclear disassembly or to perform several rounds of nuclear assembly and disassembly. The chapter also focuses on membrane assembly of the nuclear envelope, although it should be noted that the potential of Xenopus extracts has also been exploited in investigating structure, function, and dynamics of the nuclear pore complex and the nuclear lamina, the other components of the nuclear envelope.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.

The mitochondrial import receptors MOM19 and MOM72 form a complex with two other proteins of the mitochondrial outer membrane, MOM38 and MOM22. This receptor complex is involved in recognition, membrane insertion and translocation of precursor proteins with MOM38 constituting (at least part of) the general insertion site GIP.

We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavble ‘signal’ sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein wich is characterized by its extreme protease resistance.

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit beta. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor.

The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs-porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding").

The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs- porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding").

Recently it was reported that artificial targeting signals or signals specific for organelles other than mitochondria could direct proteins into mitochondria. Here we discuss findings which suggest that specific steps of mitochondrial protein import can be bypassed. Non-specific targeting signals appear to use this bypass pathway. Such import occurs at very low rates under physiological conditions and therefore does not affect the uniqueness of mitochondrial protein composition.