Synthetische Glykobiotechnologie

The field of synthetic glycobiotechnology encompasses the synthesis and modification of free carbohydrates and carbohydrates linked to biomolecules. Our group develops bio-catalytic processes for the synthesis of carbohydrate building blocks, so-called sugar nucleotides, and cell-free multi-enzyme cascades to tailor carbohydrates linked to proteins. The technology can eventually help to advance our understanding of the roles of specific carbohydrates in nutrition and medicine and contribute to human health and well-being.

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

In vitro enzyme cascades possess great benefits, such as their synthetic capabilities for complex molecules, no need for intermediate isolation, and the shift of unfavorable equilibria towards the products. Their performance, however, can be impaired by, for example, destabilizing or inhibitory interactions between the cascade components or incongruous reaction conditions. The optimization of such systems is therefore often inevitable but not an easy task. Many parameters such as the design of the synthesis route, the choice of enzymes, reaction conditions, or process design can alter the performance of an in vitro enzymatic cascade. Many strategies to tackle this complex task exist, ranging from experimental to in silico approaches and combinations of both. This review collates examples of various optimization strategies and their success. The feasibility of optimization goals, the influence of certain parameters and the usage of algorithm-based optimizations are discussed.

Preparation of ZIF@ADH/NAD-MSN/LDH Core Shell Nanocomposites for the Enhancement of Coenzyme Catalyzed Double Enzyme Cascade

The field of enzyme cascades in limited microscale or nanoscale environments has undergone a quick growth and attracted increasing interests in the field of rapid development of systems chemistry. In this study, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and mesoporous silica nanoparticles (MSN) immobilized nicotinamide adenine dinucleotide (NAD+) were successfully immobilized on the zeolitic imidazolate frameworks (ZIFs). This immobilized product was named ZIF@ADH/NAD-MSN/LDH, and the effect of the multi-enzyme cascade was studied by measuring the catalytic synthesis of lactic acid. The loading efficiency of the enzyme in the in-situ co-immobilization method reached 92.65%. The synthesis rate of lactic acid was increased to 70.10%, which was about 2.82 times that of the free enzyme under the optimal conditions (40 °C, pH = 8). Additionally, ZIF@ADH/NAD-MSN/LDH had experimental stability (71.67% relative activity after four experiments) and storage stability (93.45% relative activity after three weeks of storage at 4 °C; 76.89% relative activity after incubation in acetonitrile-aqueous solution for 1 h; 27.42% relative activity after incubation in 15% N, N-Dimethylformamide (DMF) solution for 1 h). In summary, in this paper, the cyclic regeneration of coenzymes was achieved, and the reaction efficiency of the multi-enzyme biocatalytic cascade was improved due to the reduction of substrate diffusion.

A Multi-Enzyme Cascade Reaction for the Production of 2’3’‑cGAMP

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2’3’-cGAMP) formation was successfully developed. The 2’3’‑cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2’3’-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2’3’-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.