Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase

The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptidesurfactin, a highly potent biosurfactant synthesized by a largemultimodular peptide synthetase. We report the genetic engineeringof the surfactin biosynthesis resulting in the production ofa novel lipohexapeptide with altered antimicrobial activities.A combination of in vitro and in vivo recombination approacheswas used to construct a modified peptide synthetase by eliminatinga large internal region of the enzyme containing a completeamino acid incorporating module. The remaining modules adjacentto the deletion were recombined at different highly conservedsequence motifs characteristic of amino acid incorporating modulesof peptide synthetases. The primary goal of this work was toidentify permissive fusion sites suitable for the engineeringof peptide synthetase genes by genetic recombination. Analysisof the rearranged enzymes after purification from B.subtilisand from the heterologous host Escherichia coli revealed thatthe selection of the recombination site is of crucial importancefor a successful engineering. Only the recombination at a specificHHIIxDGVS sequence motif resulted in an active peptide synthetase.The expected lipohexapeptide was produced in vivo and firstevidence of a reduced toxicity against erythrocytes and an enhancedlysis of Bacillus licheniformis cells was shown.     

The role of intumescents for construction products with improved fire safety

Using a brief review of the history of fire safety technology, the author outlines the development of intumescents and their effect under fire conditions, in particular discussing silicate-based materials. Finally a classification system based on the height of the foam and the foam pressure developed under fire exposure along with the operational stresses to which the intumescent may be subjected is put forward. It is suggested that this kind of classification needs to be taken into account in the development of guidelines for European Technical Approvals.     

Voltammetric and spectroelectrochemical studies on 4-aminophenol at gold electrodes in aqueous and organic media

Voltammetric and UV-Vis spectroelectrochemical studies on 4-aminophenol (4-AP) at gold electrodes have been carried out in conventional electrochemical and in novel spectroelectrochemical cells. As base electrolytes acetonitrile with 10⁻² M tetrabutylammonium hexafluorophosphate as supporting electrolyte and acetate buffer (pH 4.6) solutions have been used. 4-AP was electrochemically oxidized at gold electrodes at potentials >+0.1 V in aqueous solutions and at potentials >+0.5 V in acetonitrile solutions. The formation of an oxidation product has been characterized in situ by UV-Vis spectroscopy in a novel optical thin-layer spectroelectrochemical cell.     

Biocatalytical synthesis and monolayer studies of multiple hydroxylated wax esters

The continuous production of aleuritic acid decyl ester has been investigated with Lipozyme^TM as biocatalyst in a fixed-bed reactor. To avoid the limiting effects of mean substrate solubility, the direct synthesis without organic solvents was chosen, whereby a yield of 90% was obtained depending on the residence time. A comparison of different reaction paths to synthesize a centrally dihydroxylated decyl ester indicated a preference for transesterification of the methyl ester. Under external compression, the interfacial behavior of several enzymatically produced hydroxy wax esters was determined with a Langmuir film balance to find a structure/efficiency relationship. Brewster-angle microscopy allowed direct visualization of a wax ester monolayer at the air/water interface.     

Enzymatic production and physicochemical characterization of uncommon wax esters and monoglycerides

Wax esters from fatty alcohols and uncommon fatty acids were synthesized in yields up to 90% when commercially available microbial lipases fromRhizomucor miehei (Lipozyme^™) andCandida antarctica (SP 435) were used with limited water content in nonpolar solvents under mild conditions. The corresponding fatty acids were prepared by chemical conversion of naturally occurring resources (agricultural surpluses). Also, when phenylboronic acid was added as solubilizing agent in a nonpolar solvent, the direct enzymatic monoacylation of glycerol with uncommon fatty acids was successful. The measurement of π/A-isotherms by means of a Langmuir film balance indicated medium film pressures, medium or large molecular areas, and interesting phase behavior. The monolayer of a wax ester at the air/water interface could be directly visualized by Brewster angle microscopy.     

A system for the generation and application of probability fields for wear rate curves

In experiments with combustion engines it is desirable that conclusions on long-term wear should be obtained from short-term wear measurement data. Continuous wear measurements on critical diesel engine components made over a short period of time were treated with known mathematical statistics. Application of the results for a short time experiment indicated a percentage of engine components identical with those used in the single experiment whose rate would not exceed the wear rate found in the experiment. The suitability of the methods presented for work on engine development is demonstrated using several practical examples.