Nina Mußmann

Asymmetric synthesis of D-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol.

Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents.

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.

Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium

BackgroundCulture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097.ResultsBacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth.ConclusionsThe introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.