Implementation of Perforated Concentric Ring Walls Considerably Improves Gas-Liquid Mass Transfer of Shaken Bioreactors.

Since their first use in the 1930s, shake flasks have been a widely used bioreactor type for screening and process development due to a number of advantages. However, the limited gas-liquid mass transfer capacities-resulting from practical operation limits regarding shaking frequency and filling volumes-are a major drawback. The common way to increase the gas-liquid mass transfer in shake flasks with the implementation of baffles is generally not recommended as it comes along with several severe disadvantages. Thus, a new design principle for shaken bioreactors that aims for improving the gas-liquid mass transfer without losing the positive characteristics of unbaffled shake flasks is introduced. The flasks consist of cylindrical glass vessels with implemented perforated concentric ring walls. The ring walls improve the gas-liquid mass transfer via the formation of additional liquid films on both of its sides, whereas the perforations allow for mixing between the compartments. Sulfite oxidation experiments revealed over 200% higher maximum oxygen transfer capacities (OTRmax) compared to conventional shake flasks. In batch cultivations of Escherichia coli BL21 in mineral media, unlimited growth until glucose depletion and oxygen transfer rates (OTR) of up to 138 mmol/L/h instead of an oxygen limitation at 57 mmol/L/h as in normal shake flasks under comparable conditions could be achieved. Even overflow metabolism could be prevented due to sufficient oxygen supply without the use of unconventional shaking conditions or oxygen enrichment. Therefore, we believe that the new perforated ring flask principle has a high potential to considerably improve biotechnological screening and process development steps.

Neuromuscular electrostimulation techniques: historical aspects and current possibilities in treatment of pain and muscle waisting.

Application of electricity for pain treatment dates back to thousands of years BC. The Ancient Egyptians and later the Greeks and Romans recognized that electrical fishes are capable of generating electric shocks for relief of pain. In the 18th and 19th centuries these natural producers of electricity were replaced by man-made electrical devices. This happened in following phases. The first was the application of static electrical currents (called Franklinism), which was produced by a friction generator. Christian Kratzenstein was the first to apply it medically, followed shortly by Benjamin Franklin. The second phase was Galvanism. This method applied a direct electrical current to the skin by chemical means, applied a direct and pulsed electrical current to the skin. In the third phase the electrical current was induced intermittently and in alternate directions (called Faradism). The fourth stage was the use of high frequency currents (called d'Arsonvalisation). The 19th century was the "golden age" of electrotherapy. It was used for countless dental, neurological, psychiatric and gynecological disturbances. However, at beginning of the 20th century electrotherapy fell from grace. It was dismissed as lacking a scientific basis and being used also by quacks and charlatans for unserious aims. Furthermore, the development of effective analgesic drugs decreased the interest in electricity. In the second half of the 20th century electrotherapy underwent a revival. Based on animal experiments and clinical investigations, its neurophysiological mechanisms were elucidated in more details. The pain relieving action of electricity was explained in particular by two main mechanisms: first, segmental inhibition of pain signals to the brain in the dorsal horn of the spinal cord and second, activation of the descending inhibitory pathway with enhanced release of endogenous opioids and other neurochemical compounds (serotonin, noradrenaline, gamma aminobutyric acid (GABA), acetylcholine and adenosine). The modern electrotherapy of neuromusculo- skeletal pain is based in particular on the following types: transcutaneous electrical nerve stimulation (TENS), percutaneous electrical nerve stimulation (PENS or electro-acupuncture) and spinal cord stimulation (SCS). In mild to moderate pain, TENS and PENS are effective methods, whereas SCS is very useful for therapy of refractory neuropathic or ischemic pain. In 2005, high tone external muscle stimulation (HTEMS) was introduced. In diabetic peripheral neuropathy, its analgesic action was more pronounced than TENS application. HTEMS appeared also to have value in the therapy of symptomatic peripheral neuropathy in end-stage renal disease (ESRD). Besides its pain-relieving effect, electrical stimulation is of major importance for prevention or treatment of muscle dysfunction and sarcopenia. In controlled clinical studies electrical myostimulation (EMS) has been shown to be effective against the sarcopenia of patients with chronic congestive heart disease, diabetes, chronic obstructive pulmonary disease and ESRD.

Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures.

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30 degrees C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30 degrees C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of approximately 1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30 degrees C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.

Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-D-glucosamine--new strategies for the optimisation of neuraminic acid synthesis.

In this work, a model describing the complete enzyme catalysed synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc) is presented. It includes the combined reaction steps of epimerisation from GlcNAc to N-acetyl-D-mannosamine (ManNAc) and the aldol condensation of ManNAc with sodium pyruvate yielding Neu5Ac. The model is expedient to predict the reaction course for various initial and feed concentrations and therefore to calculate reaction times and yields. The equilibrium constants calculated from the kinetic constants via the Haldane relationship correspond with experimental values very well (0.26 calculated and 0.24 experimental value for the epimerisation, 27.4 l mol(-1) calculated and 28.7 l mol(-1) experimental for the aldol condensation). The actual relevance of the model is shown by a scale-up. Using the model, an optimisation of reaction conditions in consideration of different targets is possible. Exemplarily, it is presented how the optimal ratio of the two enzymes in the reaction can be determined and how the composition of the reaction solution in a fed-batch reactor can be designed to meet downstream processing needs.