[Show abstract][Hide abstract] ABSTRACT: Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid-liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG-salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid-liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.
[Show abstract][Hide abstract] ABSTRACT: The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential.
Applied biochemistry and biotechnology 06/2015; DOI:10.1007/s12010-015-1707-y · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Moschus und Patchouliöl spielen in der Herstellung von Parfüms, diversen Kosmetik- und Haushaltsprodukten eine wichtige Rolle: Es gibt kaum ein Parfüm das ohne Patchouli- oder Moschusduft auskommt. Während Patchouliöl bis heute überwiegend aus pflanzlichen Quellen gewonnen wird, sind die verwendeten Moschusduftstoffe schon längst nicht mehr natürlichen Ursprungs. Dank der modernen Synthesechemie existieren für Moschus zahlreiche synthetische Ersatzstoffe. Der Trend zu mehr nachhaltig produzierten Kosmetikprodukten, stellt die konventionellen Produktionsprozesse vor Herausforderungen. Die Biotechnologie steht daher schon in den Startlöchern, um Duft- und Aromastoffe in Zukunft wirtschaftlich, umweltfreundlich und qualitativ hochwertig, produzieren zu können.Synthetic musk odours and patchouli oil are widely used in the production of cosmetics and scented household goods. In many perfumes one or both substances are key fragrances to create a unique and luxury odour. While patchouli oil is an essential oil from the patchouli plant, natural musk is historically an animal secrete which is derived from a gland of the muskdeer. The progress in organic synthesis has led to numerous synthetic musk-like fragrance compounds. Both methods, the plant based production as well as the industrial synthesis of fragrances are connected to challenges such as ethic concerns and possible environmental risks. Therefore, sustainable chemical processes as well as biotechnological methods for the future production of important perfumery ingredients are developed.
Chemie in unserer Zeit 04/2015; DOI:10.1002/ciuz.201500676 · 0.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fabrication of individual labware is a sophisticated task that requires dedicated machines and skills. 3D-printing has the great potential to drastically simplify this procedure. In the near future scientists will be able to design labware digitally and 3D-print them afterwards directly in the laboratory. With the available rapid prototyping (RP) printer-systems it is possible to achieve this. The materials accessible are able to meet the needs of biotechnological laboratories which include biocompatibility and withstanding sterilization conditions. This will lead to a completely new approach of adapting the labware to the experiment or even tailor-made it to the organism it is being used for, not adapting the experiment to a certain standard labware. Thus it will encourage the creativity of scientists and will enrich the laboratory work of the future. We present different examples illustrating the potential and the possibilities of using 3D-printing for individualizing labware. This includes a well plate with different baffle geometries, a shake flask cap with build in luer connections and a filter holder for an in-house developed membrane reactor-system.This article is protected by copyright. All rights reserved
Engineering in Life Sciences 01/2015; 15(1). DOI:10.1002/elsc.201400093 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3D-printing techniques are continuously evolving, thus their application fields are likewise growing very fast. The applications shown here highlight the use of rapid prototyping (RP) in a dedicated biotechnology laboratory environment. The combination of improving prototypes using fused deposition modeling (FDM) printers and producing useable parts with selective laser sintering (SLS) printers enable a cost- and time-efficient use of these techniques. Biocompatible materials for 3D-printing are already available and the printed parts can directly be used in the laboratory. To demonstrate this, we tested 3D-printing materials for their in vitro biocompatibility. To exemplify the versatility of the 3D-printing process applied to a biotechnology laboratory, a normal well plate design was modified in silico to include different baffle geometries. This plate was subsequently 3D-printed and used for cultivation. In the near future this design and print possibility will revolutionize the industry. Advanced printers will become available for laboratories and could be used for creating individual labware or standard disposables on demand. These applications have the potential to change the way research is done and even how stock-keeping management is today, leading to more flexibility and promoting creativity of the scientists.This article is protected by copyright. All rights reserved
Engineering in Life Sciences 01/2015; 15(1). DOI:10.1002/elsc.201400094 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In numerous applications biomass or biochemically active substances, like pharmaceuticals, flavors or bio-ethanol, are produced in industrial-scale bioreactors. In order to ensure a constant and high quality and quantity of the particular product the biochemical environment within the reactor needs to be continuously controlled within narrow limits. Thus, sensitive sensor systems that allow continuous and preferably non-invasive monitoring of relevant parameters during the cultivation are required. In this work we present results of an analysis of exhaust gas of a bioprocess composed of growing phase and auto-inductive protein production phases of a recombinant Escherichia coli BL21 strain as model organism using a compact closed-loop ion mobility spectrometer (IMS) with gas chromatographic (GC) pre-separation. The used GC-IMS (in-house development) has a mobility resolution of about R = 90 (IMS drift time / peak width) and enables automatic sampling and analysis of the exhaust gas every 20 min. We compare the intensity of different IMS peaks with additional online and offline data like oxygen consumption, optical density or the fluorescence of a GFP-labeled protein which is produced by the organism after auto-induction. A great challenge in this context is to detect trace concentrations of possible precursors for a metabolic change or indicators for the efficiency of such a change in the presence of very high concentrations of water and compounds like acetone, ethanol and ammonia. Besides multiple peaks that show a significant and reproducible change during the cultivation we observe at least one peak that is assumed to be a precursor for the induction process.
International Journal for Ion Mobility Spectrometry 12/2014; 18(1-2). DOI:10.1007/s12127-014-0163-7
[Show abstract][Hide abstract] ABSTRACT: In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae . It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations.
[Show abstract][Hide abstract] ABSTRACT: A novel online sensor system for non-invasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°-scattered light and correlation to optical density by non-linear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms – Escherichia coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD600 ≤ 30. The determination of online OD showed relative errors of about 7.5% for E. coli K12 and 12% for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, non-varying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.This article is protected by copyright. All rights reserved
Engineering in Life Sciences 09/2014; 14(5). DOI:10.1002/elsc.201400026 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so-called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: i. simple dilution; ii. nanofiltration; and iii. electrodialysis. Lactose consumption, ethanol production and time-dependent yields were compared between the three methods. A dilution of DWP to 60 % (v/v) led to fermentation taking less than 80 h and yield above 7 % AbV (alcohol by volume). After nanofiltration, 7.5 % AbV was produced in about 80 h, and after electrodialysis, 11 % AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DLP could be enhanced by prior partial desalination.This article is protected by copyright. All rights reserved
Engineering in Life Sciences 09/2014; 14(5). DOI:10.1002/elsc.201300138 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present work, the quantification of different poly(ethylene glycol) (PEG) + potassium phosphate / sodium citrate aqueous two-phase system (ATPS) phase-forming components was investigated by using conductivity and refractive index measurements. For this purpose, refractive index and conductivity calibration curves were obtained for ATPS at different pH values in the presence of different bovine serum albumin (BSA) concentrations. While BSA had no effect on the conductivity, it had a considerable effect on the refractive index. Finally, a convenient dilution of the samples prior to the ATPS constituent determination is needed to ensure no significant influence from BSA.
[Show abstract][Hide abstract] ABSTRACT: Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. no.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4 % compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in E. coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co2+-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modelling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.
Protein Expression and Purification 05/2014; 97. DOI:10.1016/j.pep.2014.02.003 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Different poly(ethylene glycol) (PEG) + potassium phosphate or sodium citrate aqueous two-phase systems (ATPS) were investigated at 23 °C, containing different PEG types (molecular weights 2000 g·mol–1 to 8000 g·mol–1) and pH values (5 to 9). Furthermore, the effect of the added salt NaCl (0 wt % to 8 wt %) on the PEG + potassium phosphate/sodium citrate ATPS was studied at 23 °C. The experimental binodal data were successfully correlated with the empirical nonlinear equation proposed by Hu. The effects of increasing molecular weight of PEG, pH, NaCl, and salt type on the obtained binodal curves were determined, resulting in a binodal curve shift toward the origin. Thus, an expansion of the two-phase region occurred by increasing molecular weight of the PEG, pH, and NaCl and due to the Gibbs free energy of hydration of ions of phosphate. Furthermore, the phase equilibrium compositions, tie-line lengths, slopes of tie-lines, critical points, and effective excluded volumes were obtained for all studied systems. Finally, the experimental tie-line compositions were successfully correlated by using the Othmer–Tobias and Bancroft equations, and linear dependency was confirmed.
Journal of Chemical & Engineering Data 02/2014; 59(3):850–859. DOI:10.1021/je401002w · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction.
[Show abstract][Hide abstract] ABSTRACT: Flavonoide stellen eine große Gruppe pflanzlicher Sekundärmetabolite dar und haben eine Vielzahl unterschiedlicher biologischer Eigenschaften. Aufgrund dessen stehen sie im Interesse vieler Wissenschaftler, da sie zu vielen industriell interessanten Verbindungen führen können. Das anaerobe Darmbakterium Eubacterium ramulus kann über einen bislang unbekannten Abbauweg diese Flavonoide verstoffwechseln. Über eine Vollgenom‐Sequenzierung wurden in dieser Arbeit die entscheidenden Enzyme, eine Chalconisomerase (CHI) und eine Enoatreduktase (ERED), identifiziert, kloniert und funktionell sogar unter aeroben Bedingungen exprimiert. Mittels Biotransformation des Flavanons zum Dihydrochalcon konnte der Flavonoid‐Abbauweg bewiesen werden. Der beide Enzyme exprimierende E.‐coli‐Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden. Dies unterstreicht die Anwendbarkeit des in dieser Arbeit entwickelten biokatalytischen Systems. GermanDer biokatalytisch‐metabolische Weg für die Umsetzung von Flavonoiden aus Eubacterium ramulus wurde identifiziert. Eine Chalconisomerase und eine Enoatreduktase wurden erfolgreich kloniert und in E. coli exprimiert und unter aeroben Bedingungen eingesetzt, obwohl E. ramulus ein strikt anaerobes Bakterium ist. Der beide Enzyme exprimierende E.‐coli‐Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden.