Marion B. Ansorge-Schumacher

Screening for Amidases: Isolation and Characterization of a Novel D-Amidase from Variovorax paradoxus

Using racemic tert-leucine amide as sole nitrogen source in minimal medium, 162 strains were isolated by enrichment techniques and shown to contain amidase activity. Among these isolates three D-amidase producers were found and identified as Variovorax paradoxus (two strains) and Klebsiella spec. The D-amidase from Variovorax paradoxus was purified to homogeneity by three chromatographic steps. With dl-Tle-amide as substrate Michaelis Menten kinetics were observed with a KM of 0.74 mM, a KI of 640 mM and a Vmax of 1.4 U/mg. The amidase has a broad pH-optimum between 7 and 9.5 and a temperature optimum at 47–49 °C. The amidase hydrolyzed amino acid amides as well as carboxamides and 2-hydroxy acid amides. The stereoselectivity of the reaction was variable, however. Hydrolyzing dl-Tle-amide the enantiomeric ratio E was >200 resulting in D-Tle with an ee of >99% and up to 47% conversion. Similar results were obtained with dl-Leu-amide and dl-Val-amide while dl-Phe-amide was hydrolyzed with an enantiomeric ratio E of only 5.

Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide

In two cycles of an error‐prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4‐fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild‐type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.

Enzymatic catalysis in gel-stabilized two-phase systems: improvement of the solvent phase

Gel-stabilized aqueous phases entrapping enzymes and surrounded by organic solvents have become promising tools for the biocatalytic conversion of hydrophobic compounds. In this work, we provide methods for an improvement of the solvent phase with special regard to the avoidance of gel agglomeration in batch as well as fluidized-bed reactors, and resulting effects on the catalyzed reaction. With alginate beads entrapping a lipase from Candida rugosa as investigation system, it was demonstrated that increasing the solvent polarity was only a limited measure to separate agglomerated beads, as water-unsaturated polar solvents extracted large amounts of water from the hydrogel. Water-saturated alcohols, however, were incorporated into side product esters by the entrapped enzyme. With non-polar solvents, like hexane, bead separation in batch reactors was achieved by the addition of certain surfactants to the organic phase. Best results were obtained with the cationic surfactant cetyl trimethyl ammonium chloride (CTAC), which in contrast to other surfactants only slightly affected the entrapped lipase and revealed no effects on the hydrogel structure. For the suspension of alginate beads in a fluidized-bed reactor, not only CTAC, but an additional increase in the solvent density was necessary, which affected the system’s productivity.

A new approach for the spatially resolved qualitative analysis of the protein distribution in hydrogel beads based on confocal laser scanning microscopy

To investigate the spatial distribution of white egg albumin (WEA) in alginate beads, a new method based on confocal laser scanning microscopy (CLSM) was developed. In contrast to the existing CLSM methods, misleading conclusions are prevented with the application of the new method which does not allow the attenuation of the exciting and emitted light by the opaque hydrogel matrices to be disregarded. By the application of this method, the distribution of WEA in alginate beads was shown to be dependent on the amount of protein loading. At low quantities of protein, a higher protein concentration occurs in the shell layer of the alginate bead while at higher loadings a more or less homogeneous distribution is observed.

Method for Quantitative Determination of Spatial Polymer Distribution in Alginate Beads Using Raman Spectroscopy

A new method based on Raman spectroscopy is presented for noninvasive, quantitative determination of the spatial polymer distribution in alginate beads of approximately 4 mm diameter. With the experimental setup, a two-dimensional image is created along a thin measuring line through the bead comprising one spatial and one spectral dimension. For quantitative analysis of the Raman spectra, the method of indirect hard modeling was applied to make use of the information contained in the entire recorded spectra. For quantification of the alginate signals from within the beads, a calibration curve acquired from sodium alginate solutions was used after it was shown that only negligible differences occur between signals from alginate solutions and alginate gels. The distribution of alginate over the bead gel matrix was acquired with high spatial (51 μm) and time (12 s) resolution. The inhomogeneous distribution obtained using the new measuring technique is qualitatively in excellent agreement with data from the literature. In contrast to known measuring techniques, correct quantitative information about the spatial polymer distribution within the matrix was derived. It gave an alginate mass fraction of approximately 0.045 g/g at the edges and 0.02 g/g in the center of the beads. Next to the determination of mere polymer concentrations, the excellent time resolution of the presented method will enable investigation of the dynamic process of gel formation and it will also serve as a basis for investigation of mass transfer of small diffusing molecules in alginate matrices.

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction – hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3–4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L−1 for CRL and 600 mmol L−1 for TLL in the reaction system.

Novel solvent-based method for preparation of alginate beads with improved roundness and predictable size.

Attempts to determine conditions or processes within alginate gel beads often suffer from inaccuracies due to an improper roundness of the analysed beads. Therefore, a novel solvent-based method for the preparation of alginate beads with improved shape was developed: An aqueous solution of 2% (w/v) alginate in water was injected into a solvent layering consisting of hexane, n-butanol, n-butanol with 1% (w/v) CaCl2 and finally 2% (w/v) CaCl2 in water. Beads of up to 3.5 mm in diameter obtained with this method had a roundness which was ∼5% better than comparable beads prepared by dropping an alginate solution into a CaCl2-hardening bath. This was determined by a software supported quantitative analysis of bead size and shape. Additionally, the novel solvent-based method allows for highly reproducible preparation of alginate beads with exactly predictable sizes. The biggest beads obtained with this method were 9 mm in diameter. Thus, with the solvent-based preparation of alginate beads it is now possible to easily obtain beads of exactly the type needed for a specific analytical purpose.

Use of polyvinyl alcohol cryogels for the compartmentation of biocatalyzed reactions in non-aqueous media

Separation into stable aqueous compartments is a promising concept to enable the synthetic application of biocatalysts in unfavourable non-aqueous media, and might provide the key for successful application of complex multistep synthesis. This study describes and evaluates the practical utility of polyvinyl alcohol (PVA) cryogels as a matrix for such compartmentation. The protocol for enzyme entrapment in the gels was highly efficient, giving an immobilization yield of >99% and a total loss of material less than 10%. The resulting gel compartments were between 2.2 and 4 mm in size and had high mechanical strength. Shrinking occurred in solvents with the ability for water uptake. The matrix enabled the synthetic use of benzaldehyde lyase and alcohol dehydrogenase in hexane, in which equilibrium concentrations were comparable to a conventional two-phase system. The results suggest a general suitability of PVA gels for the compartmentation of biocatalyzed reactions in non-aqueous media.