Sascha Beutel

Leibniz Universität Hannover, Hanover, Lower Saxony, Germany

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Publications (96)181.78 Total impact

  • Anna Glyk · Dörte Solle · Thomas Scheper · Sascha Beutel
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    ABSTRACT: Since the mechanism governing the partitioning behavior of biomolecules, such as proteins and enzymes, in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) is complex and not easily predictable, many laborious experiments have to be performed for an optimization of these systems, causing increased overall cost. However, the multivariate statistical design of experiments (DoE) methodology is representing a promising and efficient optimization technique which can overcome the limitations of traditional optimization methods. Therefore, DoE has emerged as a powerful and efficient optimization tool for PEG-salt ATPS, since it is faster, more efficient and cost-effective, allowing a simultaneous and rigorous evaluation of process/system parameters. In the present review, different DoE process steps are represented to highlight the feasibility of this approach to operate as a promising and efficient optimization tool, thus facilitating the evaluation of the partitioning behavior, recovery and purification of different proteins and enzymes in PEG-salt ATPS. In this context, several experimental designs, such as factorial and response surface designs, have been discussed and evaluated by statistical regression analysis and analysis of variance (ANOVA), as well as various applications of PEG-salt ATPS using DoE have been outlined which may further promote the optimization of these systems.
    No preview · Article · Dec 2015 · Chemometrics and Intelligent Laboratory Systems
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    ABSTRACT: The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay.
    No preview · Article · Oct 2015 · Applied biochemistry and biotechnology
  • Michail Nakos · Sascha Beutel · Thomas Scheper
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    ABSTRACT: Vitamin B12 is an essential nutrient in human dietary, as it has an important role in promoting carbohydrate and normal fat metabolism, as well as it is essential in the formation of red blood cells, and the normal functioning of the nervous system. Naturally occurring vitamin B12 is only found in animal products such as meat, milk, dairy products, fish, oysters and clams, but it is well-known for its absence in plant-based foods. Therefore, strict vegetarians (vegans) are at higher risk for developing cobalamin deficiency. Due to the need for investigation and accurate determination of vitamin B12 in plant matrices, a HPLC-UV method with a combined purification and concentration step by immunoaffinity column (IAC) was developed. The method demonstrated linear response with r2 > 0.994, with LOD and LOQ values 0.003 μg/ ml and 0.010 μg/ ml, respectively. The validation studies shows very good specificity and accuracy, as well as precision. Actinorhizal plants, such as Hippophae rhamnoides are symbiotic with actinobacteria Frankia Alni, they are potential hosts for vitamin B12. The method was applied in several plant samples, but significant amounts of vitamin B12 were detected only in Hippophaes rhamnoides (37 μg/ 100 g dry weight). These initial findings provide the basis for detection of vitamin B12 also in other plants, and can be a good measure of prevention for the vitamin B12 deficiency in vegetarians.
    No preview · Conference Paper · Sep 2015
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    ABSTRACT: Abstract Online pH control during microbial shake flask cultivation has not been established due to the lack of a practical combination of an online sensor system and an appropriate control unit. The objective of this investigation was to develop a minimum scale dosage apparatus, namely shake flask controller ("SFC"), which can control the pH during a complete cultivation and serves as technical example for the application of small liquid dispensing lab devices. A well evaluated optical, chemosensor based, noninvasive, multisensory platform prototype for online DO (dissolved oxygen)-, pH- and biomass measurement served as sensor. The SFC was designed as cap-integrated, semi-autarkical control unit. Minimum scale working parts like the commercial mp6 piezoelectric micropumps and miniature solenoid valves were combined with a selective laser sintering (SLS) printed backbone. In general it is intended to extend its application range on the control of enzymatic assays, polymerization processes, cell disruption methods or the precise dispense of special chemicals like inducers or inhibitors. It could be proved that pH control within a range of 0.1 pH units could be maintained at different cultivation conditions. A proportional-integral-derivative- (PID) controller and an adaptive proportional controller were successfully applied to calculate the balancing solution volume. SLS based 3D printing using polyamide combined with state-of-the-art micro pumps proved to be perfectly adaptable for minimum size, autoclavable lab devices.
    No preview · Article · Aug 2015 · Sensors and Actuators B Chemical

  • No preview · Article · Aug 2015 · Genetic engineering & biotechnology news: GEN
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    Anna Glyk · Thomas Scheper · Sascha Beutel
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    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.
    Preview · Article · Jul 2015 · Applied Microbiology and Biotechnology
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    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.
    Full-text · Article · Jun 2015 · Applied biochemistry and biotechnology
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    Thore Frister · Sascha Beutel
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    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.
    Full-text · Article · Apr 2015 · Chemie in unserer Zeit

  • No preview · Article · Apr 2015
  • S Hartwig · T Frister · S Alemdar · Z Li · T Scheper · S Beutel
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    ABSTRACT: An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in E. coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L(-1) were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni(2+)-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg(2+) containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC-MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis-Menten model, kinetic parameters of KM = 1.111 μM (± 0.113), vmax = 0.3245 μM min(-1)(± 0.0035), kcat = 2.95 min(-1), as well as a catalytic efficiency kcat/KM = 4.43x10(4) M(-1)s(-1) were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Feb 2015 · Biochemical and Biophysical Research Communications
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    ABSTRACT: In the present study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and BCA assay. Each phase-forming component above 1 and 5 wt.% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing PEG/salt concentration and PEG molecular weight. Therefore, a convenient dilution of both components (up to 1 and 5 wt.%) before protein quantification is recommended in both assays, respectively, in which the BCA assay is favored in comparison to the Bradford assay. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Feb 2015 · Analytical Biochemistry
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    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
    Full-text · Article · Jan 2015 · Engineering in Life Sciences
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    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
    Full-text · Article · Jan 2015 · Engineering in Life Sciences
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    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.
    No preview · Article · Dec 2014 · International Journal for Ion Mobility Spectrometry
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    ABSTRACT: Membrane clogging and biofilm formation are the most serious problems during water filtration. Silver nanoparticle (Agnano) coatings on filtration membranes can prevent bacterial adhesion and the initiation of biofilm formation. In this study, Agnano are immobilized via direct reduction on porous zirconia capillary membranes to generate a nanocomposite material combining the advantages of ceramics being chemically, thermally and mechanically stable with nanosilver, an efficient broadband bactericide for water decontamination. The filtration of bacterial suspensions of the fecal contaminant Escherichia coli reveals highly efficient bacterial retention capacities of the capillaries of 8 log reduction values, fulfilling the requirements on safe drinking water according to the U.S. Environmental Protection Agency. Maximum bacterial loading capacities of the capillary membranes are determined to be 3×10(9)bacterialcells/750mm(2) capillary surface until back flushing is recommendable. The immobilized Agnano remain accessible and exhibit strong bactericidal properties by killing retained bacteria up to maximum bacterial loads of 6×10(8)bacterialcells/750mm(2) capillary surface and the regenerated membranes regain filtration efficiencies of 95-100%. Silver release is moderate as only 0.8% of the initial silver loading is leached during a three-day filtration experiment leading to average silver contaminant levels of 100μg/L. Copyright © 2014 Elsevier B.V. All rights reserved.
    Full-text · Article · Dec 2014 · Materials Science and Engineering C
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    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 [1]. 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.
    Full-text · Article · Sep 2014 · Sensors
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    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
    No preview · Article · Sep 2014 · Engineering in Life Sciences
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    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
    No preview · Article · Sep 2014 · Engineering in Life Sciences

  • No preview · Article · Jul 2014 · New Biotechnology

  • No preview · Article · Jul 2014 · New Biotechnology