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Fully exploiting the potential of enzymes in cell-free biocatalysis requires stabilization of the catalytically active proteins and their integration into efficient reactor systems. Although in recent years initial steps towards the immobilization of such biomolecules in metal-organic frameworks (MOFs) have been taken, these demonstrations have been limited to batch experiments and to aqueous conditions. Here we demonstrate a MOF-based continuous flow enzyme reactor system, with high productivity and stability, which is also suitable for organic solvents. Under aqueous conditions, the stability of the enzyme was increased 30-fold, and the space-time yield exceeded that obtained with other enzyme immobilization strategies by an order of magnitude. Importantly, the infiltration of the proteins into the MOF did not require additional functionalization, thus allowing for time- and cost-efficient fabrication of the biocatalysts using label-free enzymes.

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECF&sigma, ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3&rsquo, 5&rsquo, )-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism.

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis.

Enzymatic synthesis of sugar fatty acid esters in organic solvents is a well-described procedure to synthesize glycolipids. This study aims at replacing these solvents with deep eutectic solvents (DES), a group of solvents that gained more and more interest during the last years, since they can be easily produced from non-toxic resources. Enzymatic glycolipid synthesis in deep eutectic solvents was investigated, employing Candida antarctica lipase B (Novozyme 435) in various deep eutectic solvents. A successful lipase-catalyzed synthesis of glucose fatty acid esters gave proof of this concept, while using the two deep eutectic solvents consisting of choline chloride and urea (CC : U) and choline chloride and glucose (CC : Glc). Additionally the DES consisting of choline chloride and glucose was observed to act as solvent and substrate for the synthesis at the same time. 1 Practical applications Glycolipids find applications in many everyday products like cosmetic and pharmaceutical formulations, food and classic cleaning products, utilizing their good detergent or emulsification properties. Glycolipids can, among other routes, be synthesized via lipase-catalyzed reactions, which are often carried out in organic solvents. By replacing these organic solvents with more ecologically friendly solvents like deep eutectic solvents, the reaction might be improved and the amount of waste produced could be reduced. The lipase-catalyzed synthesis of glucose-6-O-hexanoate employing glucose and vinyl hexanoate in different deep eutectic solvents was demonstrated within the manuscript.

Organic micropollutants (MPs), in particular xenobiotics and their transformation products, have been detected in the aquatic environment and the main sources of these MPs are wastewater treatment plants. Therefore, an additional cleaning step is necessary. The use of activated carbon (AC) is one approach to providing this additional cleaning. Industrial AC derived from different carbonaceous materials is predominantly produced in low-income countries by polluting processes. In contrast, AC derived from sewage sludge by hydrothermal carbonization (HTC) is a regional and sustainable alternative, based on waste material. Our experiments demonstrate that the HTC-AC from sewage sludge was able to remove most of the applied MPs. In fact more than 50% of sulfamethoxazole, diclofenac and bezafibrate were removed from artificial water samples. With the same approach carbamazepine was eliminated to nearly 70% and atrazine more than 80%. In addition a pre-treated (phosphorus-reduced) HTC-AC was able to eliminate 80% of carbamazepine and diclofenac. Atrazine, sulfamethoxazole and bezafibrate were removed to more than 90%. Experiments using real wastewater samples with high organic content (11.1 g m−3) succeeded in proving the adsorption capability of phosphorus-reduced HTC-AC.

Glucocorticoids, steroid hormones of the adrenal gland, are an integral part of the stress response and regulate glucose metabolism. Natural and synthetic glucocorticoids are widely used in anti-inflammatory therapy but can have severe side effects. In vivo tests are needed to identify novel glucocorticoids and to screen compounds for unwanted effects on glucocorticoid signaling. We created the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY assay to monitor glucocorticoid signaling in vivo. The GRIZLY assay detects stress-induced glucocorticoid production in single zebrafish larvae, measures disruption of glucocorticoid signaling by an organotin pollutant metabolite, and specifically identifies a compound stimulating endogenous glucocorticoid production in a chemical screen. Our assay has broad applications in stress research, environmental monitoring, and drug discovery.

P. aeruginosa living in biofilm populations sends out diffusive signalling molecules, called autoinducers, for example acylated homoserine lactone (AHL) or the P. aeruginosa quinolone signal (PQS). So far, two quorum-sensing systems, LasR and VsmR, have been identified in P. aeruginosa, both of which are required for all virulence determinants. The expression of specific genes involved in quorum-sensing regulatory mechanisms has been analysed with molecular biology methods. Real-time quantitative PCR is a highly sensitive and powerful technique for quantification of nucleic acids. Expression of the genes vsmR, lasI, and PA4296 was studied by use of reverse transcriptase and subsequent quantitative real-time PCR of the cDNAs. In parallel, expression of ribosomal 16S rRNA, used as a housekeeping gene that was constitutively expressed in all analyses, was also monitored. Biofilm was compared with planktonic bacteria, and in contrast to vsmR and Pa4296, the lasI gene was found to be down-regulated in biofilm. Extended experiments were run with synthetic signal molecules inducing regulated processes in bacterial populations. It was shown that the genes under investigation were up-regulated in mature biofilm in the presence of the signal molecule N-(3-oxododecanoyl)-L-homoserine lactone.

A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected.

Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. A PA4398 − mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398 − mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398 − mutant to the wild-type strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold ( P < 0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes, we were able to show that not only pvdQ but also pvdR , fptA , pchA , pchD , and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398 − mutant in addition to elevated c-di-GMP levels.

This book chapter focuses on non-R&D-intensive industry sectors and analyses their economic importance for Germany. Therefore we compare non-R&D-intensive industry sectors with R&D-intensive-industry-sectors and service sectors regarding R&D activities, domestic value added and import intensity, production, employment and skills. In order to not only include direct effects for these indicators we also analyze indirect effects via input-output (I/O) analysis by simulating the potential effect additional 1 billion euros demand impulse in the various sectors. On the one hand, our results show that the dynamics of non-R&D-intensive industries is less than that of the R&D-intensive industrial sectors. Moreover, R&D-intensive industries are found to contribute more to the employment of highly skilled professionals. On the other hand, our potential analyses show that non-R&D-intensive industries are of significant economic importance to Germany. This importance is evident based on a number of macroeconomic indicators: non-R&D-intensive industries are associated with strong indirect employment effects that also include qualified personnel. Overall, the analysis shows that the consideration of indirect macroeconomic effects is important to con-ducting an appropriate analysis of the role of non-R&D-intensive industries. Non-R&D-intensive companies have profound effects on upstream economic sectors through their spending on intermediate inputs (including business-related services and engineering). Policymakers should consider those linkages in determining an adequate selection of measures.

The wide variety of known and unknown endocrine disruptors demands highly efficient methods for detecting the relevant endocrine disrupting pollutants. Bioeffects-related analysis is expected to solve this problem, combining biomolecular recognition processes with chemical analysis, which therefore reduces costs of instrumental chemical analysis to relevant samples. The hyphenation of both steps provides information on potential bioeffects, structure and concentration of the substances of interest. In this work the hyphenation of an enzyme-linked receptor assay (ELRA) with liquid chromatography-tandem mass spectrometry (LC-MSMS) for the analysis of estrogens and xenoestrogens is proposed. Both test systems have been validated with standards and spiked real water samples. The ELRA achieved a detection limit of 0.1 μg/L for 17β-estradiol (without preconcentration). With the LC-MSMS method, steroids could be detected down to 1 ng/L (with preconcentration). Two concepts for a tighter hyphenation of biomolecular interaction and chemical analysis are discussed: the on-line coupling of receptor-affinity chromatography (RAC) to LC-MSMS and the direct measurement of receptor-ligand complexes by LC-MSMS.

From an innovation systems perspective the performance of the German medical devices industry, future challenges and barriers to innovation are assessed. Current performance indicators (e.g., RD intensity, export growth rates) paint a favourable picture. Nonetheless, a number of innovation barriers are identified: in particular, insufficient network integration of small and medium-sized enterprises (SMEs) and ineffective policy coordination. Finally, recommendations addressing identified future challenges and innovation barriers are developed.

Biotechnological methods have become an important tool in pharmaceutical drug research and development. Today approximately 15 % of drug revenues are derived from biopharmaceuticals. The most relevant indications are oncology, metabolic disorders and disorders of the musculoskeletal system. For the future it can be expected that the relevance of biopharmaceuticals will further increase. Currently, the share of substances in preclinical testing that rely on biotechnology is more than 25 % of all substances in preclinical testing. Products for the treatment of cancer, metabolic disorders and infectious diseases are most important. New therapeutic approaches such as RNA interference only play a minor role in current commercial drug research and development with 1.5 % of all biological preclinical substances. Investments in sustainable high technology such as biotechnology are of vital importance for a highly developed country like Germany because of its lack of raw materials. Biotechnology helps the pharmaceutical industry to develop new products, new processes, methods and services and to improve existing ones. Thus, international competitiveness can be strengthened, new jobs can be created and existing jobs preserved.

Zusammenfassung Die Methoden der Biotechnologie wurden in den letzten 10 Jahren zu einem wichtigen Instrumentarium in der pharmazeutischen Wirkstoffforschung und -entwicklung. Bereits heute machen Biopharmazeutika rund 15% des gesamten Umsatzes mit Arzneistoffen aus. Dabei sind die drei wichtigsten Indikationsgruppen die Onkologie, Stoffwechselerkrankungen und Erkrankungen des Bewegungsapparats. Fur die nachsten Jahre kann damit gerechnet werden, dass Biopharmazeutika weiter an Bedeutung gewinnen. Der Anteil von Substanzen in der praklinischen Forschung, die auf biotechnologischen Verfahren beruhen, liegt mittlerweile bei uber 25%. Neben Produkten fur die Onkologie und Stoffwechselerkrankungen spielen hier Antiinfektiva eine wichtige Rolle. Neuartige Therapieverfahren wie die RNA-Interferenz stecken dagegen mit 1,5% aller kommerziellen Forschungsprojekte noch in den Kinderschuhen. Investitionen in zukunftsfahige Spitzentechnologien wie die Biotechnologie sind fur ein hoch entwickeltes und rohstoffarmes Land wie Deutschland von zentraler Bedeutung. Die Biotechnologie hilft den deutschen Pharmaakteuren vor allem dabei, neue Produkte, Prozesse/ Verfahren und Dienstleistungen zu entwickeln und existierende zu verbessern. Dadurch kann die internationale Wettbewerbsfahigkeit gestarkt, neue Arbeitsplatze in Deutschland konnen geschaffen und bestehende gesichert werden.

Zusammenfassung Aus Innovationssystem-Perspektive werden die Leistungskraft der deutschen Medizintechnikindustrie, zukunftige Herausforderungen und Hemmnisse bewertet. Insgesamt zeichnen viele Leistungsindikatoren (z.B. F&E-Intensitat, Export-Wachstumsraten) ein gunstiges Bild. Es zeigen sich aber auch einige Innovationshemmnisse, etwa eine unzureichende Integration von kleinen und mittleren Unternehmen (KMU) in Netzwerke und verbesserungsbedurftige Koordinationsprozesse zwischen den relevanten politischen Institutionen. Auf dieser Grundlage werden Handlungsempfehlungen fur die Politik abgeleitet, die den kunftigen Herausforderungen und Hemmnissen Rechnung tragen.

Deutschland ist in der industriellen Biotechnologie im internationalen Wettbewerb sehr gut positioniert. Zwar gibt es Schwachen und Risiken (z. B. unzureichende Politikkoordination und Verfugbarkeit von Risikokapital, zu wenige Demonstrationsanlagen, zukunftige Engpasse beim qualifizierten Personal), jedoch ergeben sich auf Grund der Starken (u. a. starke technologische Wissensbasis, gut ausgebildete Arbeitskrafte, wettbewerbsfahige industrielle Basis in wichtigen Anwenderbranchen mit Zugang zu grosen Exportmarkten) zukunftig enorme Innovations-, Wachstums- und Beschaftigungspotenziale.

Einige wichtige Indikatoren deuten darauf hin, dass der Innovationsstandort Deutschland im Bereich der forschungs- und wissensintensiven Branchen seine derzeitige technologische Wettbewerbsposition im Vergleich zu wichtigen Konkurrenzländern nicht dauerhaft halten kann. Welche Schwächen sind entlang der gesamten Wertschöpfungskette erkennbar? Welche Maßnahmen zum Gegensteuern sollten ergriffen werden?

During the last few decades, increasing interest in biological surfactants led to an intensification of research for the cost-efficient production of biosurfactants compared with traditional petrochemical surface-active components. The quest for alternative production strains also is associated with new demands on biosurfactant analysis. The present paper gives an overview of existing analytical methods, based on the example of rhamnolipids. The methods reviewed range from simple colorimetric testing to sophisticated chromatographic separation coupled with detection systems like mass spectrometry, by means of which detailed structural information is obtained. High-performance liquid chromatography (HPLC) coupled with mass spectrometry currently presents the most precise method for rhamnolipid identification and quantification. Suitable approaches to accelerate rhamnolipid quantification for better control of biosurfactant production are HPLC analysis directly from culture broth by adding an internal standard or Fourier transform infrared attenuated total reflectance spectroscopy measurements of culture broth as a possible quasi-online quantification method in the future. The search for alternative rhamnolipid-producing strains makes a structure analysis and constant adaptation of the existing quantification methods necessary. Therefore, simple colorimetric tests based on whole rhamnolipid content can be useful for strain and medium screening. Furthermore, rhamnolipid purification from a fermentation broth will be considered depending on the following application.

The performance of the German pharmaceutical industry, future challenges and obstacles to quality improvement are assessed from a systems-of-innovation perspective, using appropriate innovation indicators. The current close-to-market performance indicators paint an unfavourable picture. Early RD indicators (e.g., publications, patents), however, reveal a positive trend. A lot of obstacles to quality improvements are identified with respect to knowledge base, knowledge/technology transfer, industrial RD processes, capital markets, market attractiveness and both regulatory and political framework conditions. On this basis, recommendations will finally be derived to improve quality in the pharmaceutical industry.

Aus Innovationssystem-Perspektive werden die Leistungskraft der deutschen Pharmaindustrie, zukünftige Herausforderungen und Hemmnisse für Qualitätsverbesserungen mit Hilfe von Innovationsindikatoren bewertet. Insgesamt zeichnen marktnahe Leistungsindikatoren ein ungünstiges Bild, wohingegen FuE-Frühindikatoren, wie z.B. Publikationen und Patente, einen positiven Trend erkennen lassen. Es zeigen sich vielfältige Hemmnisse für Qualitätsverbesserungen in den Bereichen Wissensbasis, Wissens-/Technologietransfer, industrielle FuE-Prozesse, Kapitalmärkte, Marktattraktivität und rechtliche und politische Rahmenbedingungen. Auf dieser Grundlage werden Handlungsempfehlungen für Qualitätsverbesserungen abgeleitet, die den zukünftigen Herausforderungen Rechnung tragen.

While the economic impact of biotechnology is disputed, empirical data and research about diffusion and economic impacts of biotechnology in application sectors is rather limited. In this paper, actual and prospective diffusion of biotechnology in different application industries is analysed on a disaggregated level by combining production data with a written expert survey. On the basis of these results, an input-output model was used to calculate the direct and indirect employment effects of biotechnology in Germany for the years 2004 and 2020. The results suggest a further diffusion of biotechnology in all application sectors. The subsequent employment occurs in both application industries and upstream sectors.