Highlights in Biocatalysis – Historical Landmarks and Current Trends

Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technology from the 19^th century onwards are highlighted. Considering the most relevant progress steps, the production of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manufacture (such as bread, cheese, beer), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivatives of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In organic synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.     

Establishment of a temperature-inducible cell line for human plasminogen activator (tissue-type) by transfection of monkey cells with expression constructs

An expression construct for human tissue-type plasminogen activator (t-pA) cDNA [containing a simian virus 40 (SV40) origin of replication] was introduced into CV1, COS-7 and COSts2 cells; in the latter cell line the amount of functionally active large T antigen of SV40 is regulated by the temperature. In a transient system, the expression in COSts2 cells at the permissive temperature for large T antigen was improved sixfold compared to COS-7 cells. By cotransfection with a plasmid conferring resistance to G418 into COSts2 cells, a cell line (COSts2Glob t-pA) could be isolated with barely detectable expression of t-pA at the semi-permissive and non-permissive temperature and inducible secretion of t-pA at the permissive temperature. The kinetics of induction, inducibility after continued propagation at the semi-permissive temperature and the influence of the temperature during previous propagation on inducibility were investigated. The biological activity of the secreted material was demonstrated by a functional assay. Inducibility of t-pA by temperature was accompanied by a dramatic increase of the copy number of episomal plasmids (up to 2000 copies per cell).     

Biochemical analysis and synergistic suppression of indoxacarb resistance in Plutella xylostella L.

Indoxacarb was treated to Plutella xylostella for 10 generations to develop a resistant strain and biochemical analysis of indoxacarb resistance in different tissues of P. xylostella was carried out. Biochemical analysis found maximum esterase activity in gut homogenates of indoxacarb resistant strains followed by whole body and cuticle homogenates. In gut homogenates of indoxacarb resistant strains, maximum increase in esterases was found as compared to the unselected strain. Acetylcholineesterase activity was higher in head homogenates of the resistant strain than in the unselected strain. Glutathione-S-transferase activity was highest in whole body homogenates. However, maximum increase was found in gut homogenates of indoxacarb resistant strains over the unselected. Induced resistance was suppressed using known synergists. Maximum synergism occurred using diethyl-maleate (DEM), followed by triphenyl phosphate (TPP).     

Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Shape and location of eukaryotic initiation factor eIF-2 on the 40S ribosomal subunit of rat liver. Immunoelectron-microscopic and hydrodynamic investigations

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.     

On differential responses of the melanophores of the Paradise fish, Macropodus opercularis

Chromatic adaptation of the Paradise fish, Macropodus opercularis L., to illuminated backgrounds ranging from "white" to "black" shows that the pigment is most readily dispersed in the deepest-lying melanophores and least readily in the uppermost layers.