Hendrik Hellmuth

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor‐containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin‐arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor‐containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.

Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer

BackgroundSince volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis.ResultsTo develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel’s industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer.ConclusionsIn this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.

Understanding Interactions of Surfactants and Enzymes: Impact of Individual Surfactants on Stability and Wash Performance of Protease Enzyme in Detergents

Abstract Enzymes and surfactants are both essential ingredients that determine the performance of modern laundry detergents. We have conducted an investigation of the interaction of surfactants and enzymes under laundry detergent application conditions in order to understand the influence of individual ingredients and to optimize detergent performance. We can show that for a given protease enzyme, individual surfactants in a constant detergent matrix have a significant impact on relevant stability and performance parameter. While certain anionic surfactants like e.g. linear alkylbenzene sulfonate show strong protease inactivation, nonionic surfactants did only show slight inactivation over time. On the other hand, proteolytic performance of protease on test stains was most driven by fatty alcohol ether sulfate. Knowledge about the impact of individual surfactants on proteases will enable the best choice of ingredients for mixed surfactant systems with optimized enzyme performance and stability.