Determination of xylooligosaccharides produced from enzymatic hydrolysis of beechwood xylan using high-performance anion-exchange chromatography tandem mass spectrometry.

High-performance anion-exchange chromatography (HPAEC) coupled with triple quadrupole mass spectrometry (HPAEC-QqQ-MS) was applied to the determination of xylooligosaccharides (XOS) derived from enzymatically hydrolysed commercial xylan from beechwood and the analytical performance and advantages of the method explored. Separation, eluent suppression, electrospray ionisation, and detection options to enhance XOS sensitivity and selectivity were evaluated, delivering a new simple, fast, selective, and sensitive solution for the characterisation of these complex compounds. The method was fully validated in terms of its analytical performance for those XOS for which standards were available, i.e., degree of polymerisation from 1 to 6. The new method was applied to the analysis of xylan hydrolysates obtained by different enzymatic hydrolysis treatments using endo-xylanase from Thermomyces lanuginosus, characterising 25 different XOS and demonstrating the method's utility for future tailoring of enzymatic hydrolysis conditions to obtain desired XOS profiles in such hydrolysates. Linear XOS and 4-O-methyl glucuronic acid (MeGluA) branched XOS were detected by direct injection of the xylan hydrolysates after a simple 10-fold sample dilution and filtration. Identification of XOS detected by HPAEC-QqQ-MS was additionally confirmed using high-resolution orbitrap mass spectrometry (HR-orbitrap-MS). Further, an ultra-sensitive and -selective method was developed by using selected reaction monitoring acquisition mode (SRM), increasing signal-to noise ratio and decreasing the limits of detection, opening future applications to low concentrated sample analysis.

Towards preparative asymmetric synthesis of beta-hydroxy-alpha-amino acids: L-allo-threonine formation from glycine and acetaldehyde using recombinant GlyA.

The diastereospecific formation of L-allo-threonine, catalyzed by the serine hydroxymethyltransferase GlyA form Escherichia coli, was studied with regard to the application in continuous processes. Process design will rely on a suitable description of enzyme stability and kinetics under relevant process conditions. Therefore, the effects of addition of organic co-solvents--methanol and acetonitrile--to the reaction mixtures on activity, stability, and diastereoselectivity were investigated. A series of progress curves from batch reactions at 35 degrees C in 50mM sodium phosphate buffer pH 6.6 and 50mM sodium phosphate buffer pH 6.6 in 20% methanol was used to estimate the respective kinetic parameters for an appropriate kinetic model. The experimental data agreed well with a kinetic model for an ordered reaction mechanism of the type bi-uni including the formation of a ternary complex and a pseudo-equilibrium assumption. The model was then applied in order to simulate the performance of the enzyme in an enzyme membrane reactor (EMR) and gave an excellent agreement with the corresponding experimental data. A space time yield of 227g L(-1)d(-1) was achieved in a continuous running EMR without significant loss of enzyme activity over 120 h of operation.

Characterization of the AlkS/P(alkB)-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations.

The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P(alkB) expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single-cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed-back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned on at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations decreased from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose-limited fed-batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein.

Integrated operation of continuous chromatography and biotransformations for the generic high yield production of fine chemicals.

The rapid progress in biocatalysis in the identification and development of enzymes over the last decade has enormously enlarged the chemical reaction space that can be addressed not only in research applications, but also on industrial scale. This enables us to consider even those groups of reactions that are very promising from a synthetic point of view, but suffer from drawbacks on process level, such as an unfavourable position of the reaction equilibrium. Prominent examples stem from the aldolase-catalyzed enantioselective carbon-carbon bond forming reactions, reactions catalyzed by isomerising enzymes, and reactions that are kinetically controlled. On the other hand, continuous chromatography concepts such as the simulating moving bed technology have matured and are increasingly realized on industrial scale for the efficient separation of difficult compound mixtures - including enantiomers - with unprecedented efficiency. We propose that coupling of enzyme reactor and continuous chromatography is a very suitable and potentially generic process concept to address the thermodynamic limitations of a host of promising biotransformations. This way, it should be possible to establish novel in situ product recovery processes of unprecedented efficiency and selectivity that represent a feasible way to recruit novel biocatalysts to the industrial portfolio.

Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv. syringae B301 D-R.

Strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae secrete a family of structurally closely related peptide derivatives dubbed syringolins, of which syringolin A is the major variant. The function of syringolins in the interaction of P. syringae pv. syringae with their host plants presently is unknown. It is hypothesized that they may constitute virulence factors. However, syringolins are determinants recognized and reacted to by nonhost plant species, and syringolin A has been shown to induce hypersensitive death of cells colonized by powdery mildew in wheat and, thus, to reprogram a compatible interaction into an incompatible one. Syringolin A is an unusual derivative of a tripeptide that contains a 12-membered ring consisting of the amino acids 5-methyl-4-amino-2-hexenoic acid and 3,4-dehydrolysine, two nonproteinogenic amino acids. Here we report the cloning, sequencing, and analysis of genes involved in the biosynthesis of syringolin A. The genes encode proteins consisting of modules typical for nonribosomal peptide synthetases and type I polyketide synthetases, as well as proteins likely involved in the transcriptional regulation of syringolin A biosynthesis and in syringolin A export. The structure and arrangement of the modules lead to the formulation of a model explaining the synthesis of the tripeptide, including the formation of the two nonproteinogenic amino acids in the ring structure of syringolin A.