Browsing by Subject "Proteine"

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Characterization and modulation of technofunctional properties of pea proteins
(2023) Moll, Pascal Bernd; Weiss, Jochen
Plant-derived ingredients for food formulation have gained increasing interest in recent years as animal products pose a higher burden on the environment. Among plant proteins, those from pea (Pisum sativum L.) are of particular interest because of their low allergenicity, low cost, high availability, and good reputation among consumers. However, the technofunctionality of pea proteins is often inferior to animal-derived proteins limiting a more widespread use in food products. These technofunctional properties include - among others - foaming, gelling, and binding of other ingredients and it depends on the food product, which functionality food scientists must utilize and optimize. Cost effective approaches to improve the technofunctionality of pea proteins are therefore desirable and would allow the industry to further implement the use of sustainable ingredients in foods. In line with these overall goals, the aim of the first section of this thesis was to characterize a commercial pea protein isolate and to modulate the physicochemical and technofunctional properties through homogenization for foaming application. The main goal of the second section was to mix pea proteins with pectin to obtain a suitable binder with desired properties for the application in meat alternatives. The mixing approach was based on previous research data that had shown that interacting protein-polysaccharide systems display a synergistic behaviour in terms of their functional properties. First section: Foams are two phase systems consisting of gas bubbles that are stabilized by surface-active ingredients such as proteins in the discontinuous, aqueous phase. The physico-chemical properties of proteins such as their solubility determines foaming performance. In Chapter I, a commercial pea protein isolate was fractionated into a water-soluble and a water-insoluble fraction for characterization. Although the two fractions were similar in protein composition, they showed distinct differences in physicochemical properties. For instance, the particle size of soluble pea proteins was around 40-50 µm at acidic pH (3-5), while no measurable particles were detected at neutral The insoluble pea proteins were large at pH 3 and 7 (> 80 µm) and ca. 40-50 µm close to their isoelectric point at pH 5. The results suggest that commercial pea protein isolates consisted of several fractions with differences in their physico-chemical properties. The yield of the water-insoluble fraction was higher and therefore used in Chapter II, where experimental results illustrated that dispersions of insoluble pea protein aggregates (5% w/w, pH 7) could be disrupted from 180 ± 40 µm (control) to 0.2 ± 0.0 µmm upon homogenization at pressures ≥ 125 MPa. This was attributed to a cleavage of intermolecular interactions such as disulphide bonds, hydrogen bonds, and hydrophobic interactions. The decrease in insoluble pea protein aggregate size was accompanied by an increase in solubility from 23 ± 1% to ≥ 80% that may be beneficial for its technofunctionality. Consequently, homogenization was applied to the same material at pH 3 and 5 with the aim of investigating its foaming performance in Chapter III. In general, unhomogenized dispersions of pea protein aggregates (5% w/w, pH 3 or 5) did not foam at both tested pH values due to large pea protein aggregates with low solubility and surface activity. At pH 3, the dissociation of pea protein aggregates into smaller, more soluble, and more surface-active proteins was responsible for a high foam capacity (FC = 360-520%) with medium foam stability as measured by drainage (FS = 19-30 min). Only a limited particle size reduction upon homogenization was observed at pH 5, which was close to the isoelectric point of the pea proteins. Nevertheless, the still large aggregates consisted of re-aggregated smaller protein particles that were able to form a smaller amount of rather stable foams with thick interfacial films (FC = 213-246%, FS = 32-42 min). Overall, homogenization of insoluble pea protein aggregates was shown to change its physicochemical properties thereby benefitting technofunctional properties such as foaming. Second section: Another technofunctionality of interest is binding of different structural elements in e.g., meat alternatives. For this, the binder must be i.) sticky to glue heterogeneous components together and ii.) able to readily solidify upon further processing thereby ensuring a coherent bulk matrix. In Chapter IV, the influence of pH (3.50, 4.75, 6.00) and biopolymer concentration (17.5-50.0% w/w) on the stickiness of a pea protein isolate – apple pectin mixture (mixing ratio r = 6:1) was investigated. It was found that biopolymer concentrations of 17.5-20.0% w/w led to low stickiness due to a lack of cohesive forces (WoA = 0.29-0.51 mJ). At high biopolymer concentrations of 40-50% w/w, the biopolymer mixtures were also not sticky because of adhesion being limited (WoA = 0.02-0.05 mJ). There was a good balance of adhesion and cohesion that facilitated a high stickiness (WoA = 0.48-0.65 mJ) at intermediate concentrations of 25-30% w/w, which was also indicated by a viscoelastic behavior (G’ ≈ G’’). At those concentrations, the mixtures at pH 6 were stickier due to increased swelling of the pea proteins. The importance of viscoelasticity for stickiness of biopolymer mixtures was confirmed in Chapter V, where pea protein isolate and apple pectin (25% w/w, pH 6) were mixed in different ratios r. Mixtures of pea protein and apple pectin and particularly the sample with r = 2:1 possessed high stickiness due to the development of a multiphase morphology that allowed for a good balance of adhesion and cohesion with distinct frequency dependency. Pea protein alone (r = 1:0, c = 25% w/w) had an elastic but soft texture with low stickiness due to limited viscous properties, whereas a sample solely consisting of apple pectin (r = 0:1, c = 25% w/w) was also not sticky because of its high cohesion and stiffness. The results of Chapter VI revealed that pea protein homogenization prior to mixing with apple pectin led to smaller protein particles in the blend that contributed to a higher cohesive strength. Interestingly, vacuum-dried pea proteins resulted in a higher network strength as this drying method prevented reaggregation of small protein particles to a higher extent as compared to freeze-drying. Overall, the mixture with homogenized and vacuum-dried pea proteins was nearly twice as sticky as the mixture with untreated pea proteins. In Chapter VII, sticky mixtures of different pea protein preparations (soluble, homogenized and unhomogenized pea proteins) and pectin (25% w/w, pH 6, r = 2:1) were tested for their ability to solidify upon different treatments, namely heating as well as the addition of transglutaminase, laccase, calcium, and combinations thereof. Calcium was found to facilitate crosslinking of pectin chains and thus induced solidification of the mixtures. For instance, the consistency coefficient K’ increased from 2800 ± 1000 Pasn for pea protein isolate – apple pectin mixtures to around 19000 Pasn when calcium was added. Heat treatment and transglutaminase did not lead to solidification indicating that pectin made up the continuous phase. Furthermore, laccase led to the highest degree of solidification when sugar beet pectin was used (K’ > 30000 Pasn) due to ferulic acid and pea protein tyrosine crosslinking. Consequently, the sticky mixture of pea protein and sugar beet pectin (25% w/w, pH 6, r = 2:1) with the addition of laccase for solidification was identified as the most suitable binder for a bacon type meat analogue, which was the object of the study carried out in Chapter VIII. This binder had the highest binding strength (W = 2.0-4.3 mJ) between textured protein, fat mimic, and both layers at 25 °C due to the introduction of covalent bonds by laccase within the binder and between the binder and the adherends. A control sample without laccase addition had lower binding properties (W = 0.7-1.0 mJ) and the binding strength of a methylcellulose hydrogel (6% w/w) serving as benchmark was only higher between two fat mimics at 70 °C (W = 1.8 ± 1.1 mJ) due to increased hydrophobic forces. Finally, the pea protein – sugar beet pectin binder (22.5% w/w, pH 6, r = 2:1) was tested in burger patty type meat analogues to glue textured vegetable protein and fat particles together (Chapter IX). The binder system did not influence the hardness of the burger patties suggesting that this property was governed by the structural elements and not the binder. However, the cohesiveness as determined by sensory analysis was found to be superior when the pea protein – sugar beet pectin binder was used (-0.7 ± 0.2) as compared to the methylcellulose benchmark (-2.9 ± 0.3). This was attributed to the sticky character of the biopolymer mixture that enabled improved binding of the different structural elements. Overall, this novel binder based on plant-derived ingredients was shown to be applicable in different meat alternatives. Last, Chapter X reviewed the functionality and binding mechanism of currently used binders in foods and showed that stickiness, hardening/solidification, and water holding capacity are of great importance. In many food products, the binder transitions from a sticky food glue to a solid matrix triggered by different process operations that depend on the characteristics of the applied binder. From the presented results, it can be concluded that pea proteins are useful functional ingredients in various application scenarios. The desired technofunctionality can be improved through different process operations such as fractionation, homogenization, or mixing with other plant-derived ingredients. For this, knowledge regarding structure-function relationship and other influential factors is needed. In some cases – such as in binders – process operations must be well orchestrated to induce structural transitions and therefore changes in functionality at the desired time during manufacturing. Overall, the results of this thesis contributed to a better understanding for a more widespread use of pea proteins to promote a more sustainable food system. The appended graphical abstract summarizes the key steps undertaken in this thesis to come to this conclusion.
Charakterisierung der akzessorischen Proteine vom felinen Coronavirus (FCoV) mit monoklonalen Antikörpern
(2014) Lemmermeyer, Tanja; Pfitzner, Artur J. P.
Little is known about the expression and function of the FCoV accessory proteins 3a, 3b, 3c, 7a and 7b. These proteins of FIPV strain 79-1146 were investigated in the present study. The obtained results are outlined: 1. The accessory FCoV proteins 3a, 3c and 7b were expressed in E. coli with a C-terminal his-tag. Furthermore the proteins 3a, 3b, 3c, the C-terminal half part of 3c, 7a, 7b as well as 7b without the amino acids 1 to 17 (7bdeltaSS) were expressed as fusion proteins with an N-terminal GST-tag and a C-terminal his-tag. The purification of all fusion proteins was performed by Ni2+ ion affinity chromatography under denaturing conditions. 2. To generate monoclonal antibodies the purified fusion proteins 3c and 7b were used for immunization of mice. ELISA screenings were established which enabled the identification of hybridoma cells that produce mabs against 3c and 7b. 3. The characterization of the anti-3c mabs led to the identification of regions in the C-terminus of the protein. The 3c protein could not be detected in an eukaryotic inducible expression system (Tet-on cell line BHKFIPV-3c) and also not in FCoV-infected cells. The anti-7b mabs bound within the region of amino acids 58 to 75 and reacted with a recombinant 7b fusion protein of a serotype I FCoV. 4. The expression of the 7b protein in infected cells was confirmed by western blot. An N-glycosylation site is located within the binding region. After incubation with tunicamycin the signal obtained with the anti-7b mabs was considerably stronger. Again after tunicamycin treatment the 7b protein was detected in the cytoplasm of infected cells by indirect immunofluorescence. The 7b protein colocalized partially with the ER. 5. The recombinant 7b protein was detected with all of the anti-FIPV 79-1146 sera and the ascites, but not with the anti-FECV 79-1683 sera. In contrast the recombinant fusion proteins 3a, 3b, 3c and 7a were not detected with the analyzed anti-FCoV sera.
Effekte der Proteinzufuhr während einer Gewichtsabnahme auf fettfreie Masse, Ruheenergieumsatz und physische Funktion bei übergewichtigen postmenopausalen Frauen - eine randomisierte, kontrollierte Studie
(2020) Englert, Isabell; Kohlenberg-Müller, Kathrin
Aim Weight loss in old age increases the risk of sarcopenia caused by the age-related reduction of fat-free mass (FFM). Due to the strong correlation between FFM and resting energy expenditure (REE), the maintenance of this must also be considered. In addition, the physical function (PF) must be maintained. The objective was to investigate the impact of protein intake on changes in fat-free mass (FFM), resting energy expenditure (REE), and physical function (PF) during weight loss. Methods 54 postmenopausal women (BMI 30.9 ± 3.4 kg/m²; 59 ± 7 years of age) were randomized into two groups with 0.8 g (K) or 1.5 g protein/kg body weight/d (P) energy-restricted diets (- 750 kcal of individual energy requirements) for 12 weeks, followed by six months weight maintenance with ad libitum food intake. The protein dose was evenly distributed to two liquid and one solid meal. The shakes of the P group were additionally enriched with pure whey protein. Four seminars were held to provide information on the course of the study and in particular on healthy nutrition. At the beginning and at the end of the study, the subjects kept a 7-day nutrition protocol. FFM (by bioelectrical impedance analysis), REE (by indirect calorimetry), PF (strength, endurance, and balance by short physical performance battery test (SPPB), 400 m walking speed and handgrip strength by hand dynamometer), blood parameters (lipid and carbohydrate profile, urea, vitamin D, calcium, magnesium, liver and kidney values from serum) and blood pressure were measured at baseline, after weight loss, and after follow up. The evaluation was primarily based on an intention to treat analysis with correlation and regression analysis, paired and unpaired t-tests, whereby the significance level was set ≤ at 0.05. The values given are continuous mean values ± standard deviation. Results 46 women completed the weight loss intervention and 29 were followed up after weight maintenance. Weight loss was -4.6 ± 3.6 kg (P) and -5.2 ± 3.4 kg (K) (both p < 0.001) and weight regain during follow up was 1.3 ± 2.8 kg (P, p = 0.028) and 0.4 ± 2.5 kg (K) (p = 0.392) with no differences between protein groups. Similar losses in FFM (-0.9 ± 1.1 kg (P) vs. -1.0 ± 1.3 kg (K)) and REE (-206 ± 136 kcal/d (P) vs. -239 ± 134 kcal/d (K), both p < 0.001) were observed in both groups. At follow-up, no changes in FFM were detected in both groups whereas in the NP group the REE increased again (138 ± 296, p = 0.02). The main determinants of the FFM loss were the energy deficit and the speed of weight loss. In the NP group, SPPB score improved with weight loss (0.6 ± 0.8, p < 0.001) and handgrip strength decreased (-1.7 ± 3.4 kg, p < 0.001) whereas no changes were observed in the HP group. The blood profile improved, especially regarding the carbohydrate profile, due to weight loss, and blood pressure. Conclusion A high protein weight loss diet without exercise had no impact on preserving FFM and REE but may help maintain muscle strength in postmenopausal women. As the handgrip strength can be a sensitive parameter for incipient sarcopenia even before the muscle mass decreases noticeably, it can be concluded that an increased protein intake during weight loss can counteract the risk of sarcopenia. Energy deficit and speed of weight loss should be considered as confounders in future studies. In addition, further strategies must be pursued to maintain FFM in weight loss in old age.
Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.
Plant protein gels as binders in meat product analogues
(2023) Herz, Eva Maria; Weiss, Jochen
In response to concerns about the environmental, ethical, and health impacts of meat consumption, plant-based meat analogues have become an important development in the food industry. To obtain prodcts with similar texture and nutritional properties, three major components of meat products (fibrous meat particles, adipose tissue, and myofibrillar meat proteins) need to be replicated. Furthermore, different binding mechanisms, such as heat, acid, and enzyme induction, and drying, are used to create coherent matrices for plant-based meat analogues. In Chapter 2, the study focuses on the use of soy protein gels as binders, with a particular emphasis on a combination of transglutaminase (TG) induced gels. The results indicate that TG-induced soy protein gels offer promising binding strength for meat analogues. Chapter 3 explores a combination of TG and slowly acidifying glucono-delta-lactone (GDL) as a binder, showing that this approach results in acidic gels with enhanced textural properties, making it suitable for acidic meat analogue products like fermented sausages. Chapter 4 applies previously studied soy protein gels as binders for sausage analogues. The research indicates that the choice of binder content influences the cohesiveness and hardness of the sausage analogues, with drying having a significant impact on hardness. In Chapter 5, hydrated gluten is used as a binder, leading to increased cohesiveness and springiness with rising binder content. It emphasizes the importance of adhesive properties between the binder and other particles in achieving desirable meat analogue texture. Overall, the thesis underscores that plant protein suspensions can serve as effective binders for meat analogue products, provided they exhibit both sufficient hardening through network formation and adhesive properties to ensure cohesiveness. It also discusses various formulation and process-based approaches to modulate the texture of meat analogue products.
Prediction of protein-protein complexes by combining size exclusion chromatography and mass spectrometric analysis
(2021) Gilbert, Max; Schulze, Waltraud
Two major objectives were pursued and met in this study. First, the goal was to add to the scientific toolbox a diligent method for uncovering PPi dynamics on a proteomic scale, with a focus on plant membranes. There are large-scale or high-throughput approaches, but they rely on genetically modified proteins or heterologous expression systems to describe PPi outside of their natural context. Similarly, those methods are incapable of describing the dynamics of protein interactions. In course of this study, a co-elution based approach was combined with modern mass spectrometric label free quantification in order to investigate PPi and interaction dynamics on a proteomic scale. A rigorous data processing pipeline was developed to not only address known fallacies of using co-elution based methods (such as for example random co elution), but also to access and utilize meta-information in form of protein abundance and protein network connectivity to draw conclusions not only on proteomic scale, but also for individual proteins. In total, 6.928 individual proteins extracted from Arabidopsis thaliana root membranes were detected under different nutritional conditions (full nutrition, nitrogen starvation and nitrogen resupply). The data processing pipeline described in this study was used to predict and discover connectivity information for at least 2.058 of these proteins. Each step in data processing was validated by comparison to database confirmed interactions to improve filtering criteria. Protein abundance was evaluated through a unique ranking system, allowing a seamless integration as network attributes for each condition. From the suggested interaction data, an interactome network of the various nutritional conditions was reconstructed. Using different network parameters from graph theory, protein significance and dynamic conditional changes were described. Second, this study applied the aforementioned approach to identify relevant proteins involved in nitrogen signaling in Arabidopsis thaliana root membranes. Through correlation analysis and network reconstruction, receptor kinase AT5G49770 was identified as a component of the nitrogen signaling network that collaborates with co-receptor QSK1, BAK1, the nitrogen transporter NRT2.1 and proton pump AHA2. In response to nitrogen deficiency, the network parameters of AT5G49770 reacted strongly and its involvement was demonstrated by a phenotypic similarity to knock-out lines of NRT2.1, NRT1.1 and AHA2 during a root growth assay of Arabidopsis seedlings. The interaction between QSK1 and BAK1 was further confirmed using FRET/FLIM microscopy and pulldown assays. These findings show that combining a co-elution based approach with a rigorous data processing pipeline and network analysis is suitable to study the protein interaction environment and signal response dynamics in plant root membranes. The modular experimental design allows for a simple adaptation to study different stimuli and the unbiased proteomic approach yields results for proteins regardless of the individual scientific focus. Meta-information such as protein abundance and network connectivity parameters can be used to prospect and identify important proteins involved in stress response dynamics. The author of this study is confident that the proteomic data produced can be utilized in further research and contributes to the understanding of nitrogen signaling in plant root membranes. Through integration of the data processing pipeline and adaptation to different scientific scenarios, valuable information beyond protein interaction is gained. Thus, this work makes an important contribution to the advancement of proteomic analysis and data interpretation methodology.
The function of E. coli YidC for the membrane insertion of the M13 procoat protein
(2018) Spann, Dirk; Kuhn, Andreas
The YidC/Oxa1/Alb3 family consists of insertase homologues that facilitate the insertion and folding of membrane proteins. YidC is located in the inner membrane of bacterial cells. Oxa1 is found in the inner membrane of mitochondria and Alb3 facilitates the insertion of membrane proteins in the thylakoid membranes of chloroplasts (Wang and Dalbey 2011, Hennon et al. 2015). An archaeal homologue was found in M. jannaschii showing that this insertase family is present in all domains of life (Dalbey and Kuhn 2015). The insertase family shares a structural feature that is conserved among all discovered members. This is a hydrophilic groove that is open towards the cytoplasm and the membrane core with a hydrophobic slide formed by transmembrane domain (TM) 3 and TM5. YidC functions on its own but also cooperates with the Sec translocon to facilitate the insertion of large membrane proteins. One protein that is membrane-inserted by YidC but is Sec-independent is the major coat protein of the M13 bacteriophage. The main objectives of this work are the analysis of the insertion mechanism of M13 procoat, the major capsid protein of the M13 bacteriophage, via the YidC-only pathway and the oligomeric state of the active YidC. The analysis of interactions between YidC and M13 procoat was performed via radioactive disulfide crosslinking mainly using copper phenanthroline as oxidizing agent. M13 procoat contacts YidC extensively in TM3 and TM5. The observed contacts suggest that the M13 procoat substrate “slides” along TM3 and TM5 of the insertase. Additional crosslinking experiments with the hydrophilic groove and the C1 loop of YidC were also performed to test their importance during the insertion process. A contact was found in the C1 loop that indicates a role in the insertion process, which is consistent with the proposed insertion model from Kumazaki et al. (2014a). Parallel to the radioactive disulfide crosslinking, a protocol using DTNB (Bis(3-carboxy-4-nitrophenyl) disulfide, Ellman’s reagent) as the oxidizing reagent and Western blot for detection was established. This method reliably promoted the formation of crosslinking products in vivo between YidC and M13 procoat over several hours and many, but not all, mapped at the same sites as in the radioactive approach. In addition, this protocol was used to purify small amounts of a YidC-substrate complex for biochemical analysis, which could also be applied to other substrates in the future. The oligomeric state of YidC was investigated by an artificial dimer of the insertase (dYidC) that was constructed by connecting two monomers together with a short linker. This dimer can complement YidC-depleted E. coli MK6S cells and facilitates the insertion of M13 procoat in vivo. For further analysis of the dYidC three functionally defective YidC mutants, T362A (Wickles et al. 2014), delta-C1 (Chen et al. 2014) and the 5S YidC mutant, were tested for their complementation and insertion capability. All three mutants were not able to complement under YidC depletion conditions. These mutants were then cloned in either one or both protomers of the dYidC. Complementation and insertion assays with these dYidC constructs show that in general one active protomer suffices to uphold cell viability and to facilitate the insertion of M13 procoat. Binding studies using cysteine mutants of the dYidC and M13 procoat for disulfide crosslinking with DTNB demonstrated that each protomer individually binds one substrate molecule. In summary, these experiments strongly support a monomer as the active state of the insertase for YidC-only substrates. Taken together, this study contributes to the understanding of the insertion of proteins into the inner bacterial cell membrane.
Untersuchung der Energie- und Nährstoffflüsse mikrobieller Gemeinschaften
(2017) Starke, Robert; Seifert, Jana
The activity of microorganisms was heavily investigated using the incorporation of stabile isotopes in the last decade. Here, all biomolecules but predominantly DNA, RNA, proteins and phospholipid derived fatty acids are used to trace the label in the biomass of active microbes. Thereby, the phylogenetic information decreases from DNA and RNA to proteins whereas the latter allow to describe the actual phenotype. In this work, protein stable isotope probing (protein-SIP) was applied to two different microbial systems: (a) the anaerobic mineralization of benzene and (b) the assimilation of plant-derived organic matter in soil. Labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community using 13C2-acetate: The well-described microbial community enriched from the Zeitz aquifer was fed with the postulated and fully 13C-labeled intermediate of syntrophic benzene fermentation, acetate, to unveil detailed secondary utilization processes. Additional acetate amended to the ongoing benzene mineralization showed no influence on sulfide produced by sulfate reduction. Instead, labeled acetate was incorporated by Campylobacterales, Syntrophobacterales, Archaeoglobales, Clostridiales and Desulfobacterales in descending order. The epsilonproteobacterial Campylobacterales featured the fastest and the highest 13C-incorporation to confirm previous metagenome-based studies and to assign a physiological role to this phylotype of the community for the first time. Metagenome based labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community: In this study, the population genome of the primary acetate utilizer was reconstructed from the metagenome of the benzene mineralizing community obtained by whole-genome shotgun sequencing. Genomic DNA originated from a starvation enrichment culture previously metabolizing m-xylen and enriched in the identical epsilonproteobacterial phylotype of this community. The presence of the sulfide quinone oxidoreductase (sqr) and the polysulfide reductase (psr) suggested a key role in sulfur cycling. Hence, the epsilonproteobacterial phylotype is able to oxidize otherwise toxic sulfid produced by sulfate reduction to polysulfide via SQR and its subsequent reduction to sulfide via PSR. Further, the detection of an acetate transporter (actP) and the acetyl-CoA synthetase (acsA) for acetate activation approved direct assimilation as shown in the previous study. Short-term assimilation of plant-derived organic matter in soil: In this protein-SIP study, the short-term assimilation of plant-derived organic matter in soil was demonstrated using 15N-labeled tobacco for the first time. In contrast to the postulated model in which fungi degrade plant-derived complex compounds and secrete low molecular weight compounds which are then degraded by bacteria, our study demonstrated the dominance of bacteria over fungi during the short-term assimilation of plant-derived organic matter. Bacteria outcompete fungi for the easy available plant-derived compounds until complex compounds such as cellulose and lignin are enriched and degraded by slow growing fungi. The use of multiOMIC techniques resulted in a multidimensional scheme to easily group and categorize different behaviours of microorganisms.
Untersuchungen zur Biogenese von Proteinen in der mitochondrialen Innenmembran
(2008) Randel, Olga; Rassow, Joachim
Mitochondria and prokaryotes show many similarities and it is a well established notion that they have common ancestors. It is therefore reasonable to expect significant similarities also in the biogenesis of their proteins. This study followed this idea in investigations on the biogenesis of protein complexes in the mitochondrial inner membrane. The yeast Saccharomyces cerevisiae served as a model organism. (1) γ-subunit of ATP synthases is highly conserved both in mitochondria and in prokaryotes. Previous studies demonstrated that deletions at the N- or C-terminus of the subunit entail only mild reductions in the enzymatic activity, and the reason of the conserved structure was enigmatic. The experiments of this study show that N- and C-terminus of the γ-subunit are essential for an efficient assembly in the ATP synthase. A deletion of 9 residues at the N-terminus or 10 residues at the C-terminus reduced the ratio of the subunit that assembled within 10 min. at 25°C by about 50%. Deletions of more than 9 N- or more than 20 C-terminal residues reduced the share of the assembled subunit by more than 90%. Yeast strains that synthesized a shortened γ- subunit did not grow on glycerole. N- and C-terminus are probably more relevant for assembly of the ATP synthase than for the transmission of energy. It is proposed that this is the case both for mitochondria and for prokaryotes. (2) The metabolite carrier proteins of the mitochondrial inner membrane were probably newly developed during the evolution of the eukaryotic cells. A common sequence motif of all carrier proteins is the carrier signature, P x (D/E) x x (K/R). The data of this study show that the carrier signature substantially facilitates the biogenesis of the dicarboxylate carrier (DIC). In particular, the translocation of this protein across the mitochondrial outer membrane is significantly accelerated. (3) The mitochondrial inner membrane protein Oxa1 is a member of a protein family that includes the bacterial protein YidC. Both Oxa1 and YidC act as mediators of protein insertion in their membranes, and both proteins participate in their own biogenesis. In this study, a series of experiments indicates that newly synthesized Oxa1 is not imported into the mitochondrial matrix, but accumulates in the inner membrane TIM23 translocase, for direct integration into the lipid bilayer. Oxa1 thereby shows a similar principle of membrane insertion as prokaryotic YidC. This was previously shown to first accumulate in the SecYEG translocase and then to directly integrate into the bacterial plasmamembrane. Oxa1 and YidC thus seem to resemble each other both in their structure and in their biogenesis. In summary, the experiments show that mitochondrial and prokaryotic proteins, after two billion years of separate evolution, have retained surprising similarities, even in molecular details of their function.
Variability of the protein and energy values of European dried distillers´ grains with solubles for ruminants
(2013) Westreicher Kristen, Edwin; Rodehutscord, Markus
The increasing demand of energy together with the implementation of the European Program for the use of energy from renewable sources are favourable scenarios to increment the ethanol production in the coming years in the EU. Ethanol production yields dried distillers´ grains with soluble (DDGS) as the main by-product, a valuable feedstuff for ruminants. A great number of publications mainly form USA and Canada has demonstrated the great variability of the feed value of corn-DDGS, the main by-product from ethanol production in these countries. In the EU, different and diverse technological conditions predominate and little was investigated to evaluate the feed value of DDGS. The variability of feeding value in conjunction with expected increase of DDGS production demands for further and more specific characterization of this by-product in the EU. Therefore, a project was conceived to characterize the chemical composition and evaluate the protein and energy value for ruminants of DDGS from different European countries. Thirteen samples of DDGS originating from wheat, corn, barley, and blends of different substrates were used. In the first study, the objective was to characterize variations in the composition and nutritive value of DDGS, and to estimate the undegradable crude protein (UDP) in DDGS. The rumen degradation of crude protein (CP) was determined using the nylon bag technique. Samples were incubated for 0, 1, 2, 4, 8, 16, 32, and 72 h, and in situ degradation kinetics were determined. UDP was estimated using a passage rate of 8 %/h. In vitro gas production was measured to estimate the metabolizable energy (ME), net energy for lactation (NEL) and in vitro digestibility of organic matter (IVDOM). Chemical profiles varied among samples (in g/kg dry matter (DM) ± standard deviation, the values were 310 ± 33 CP, 86 ± 37 ether extract, 89 ± 18 crude fibre, 408 ± 39 neutral detergent fibre, 151 ± 39 acid detergent fibre, and 62 ± 31 acid detergent lignin), as well as in protein fractions according to the Cornell Net Carbohydrate and Protein System (in g/kg CP, the values were 161 ± 82 for fraction A, 24 ± 11 for fraction B1, 404 ± 105 for fraction B2, 242 ± 61 for fraction B3, and 170 ± 87 for fraction C). ME, NEL (MJ/kg DM) and IVDOM (%), also varied among samples: 12.1 ± 0.59, 7.3 ± 0.39, and 72.5 ± 4.30, respectively. The in situ rapidly degradable CP fraction (a) varied from 10.2 to 30.6%, and the potentially degradable fraction (b) averaged to 66.8%. UDP varied from 8.6 to 62.6% of CP. This first study suggests significant variations in composition and nutritive value among different sources of DDGS. UDP could be predicted on the basis of analysed CP fractions, but the accuracy of UDP prediction improved upon the inclusion of neutral-detergent insoluble nitrogen, explaining 94% of the variation in the UDP values. To conclude, chemical protein fractions may be used to predict the UDP values of DDGS and the variability in the protein fractions of DDGS should be considered when formulating diets for dairy cows. To provide additional information on the nutritional value of DDGS, a second study was carried out to determine and compare the in situ ruminal degradation of CP and amino acids (AAs) of DDGS and to characterize the in vitro pepsin-pancreatin solubility of CP (PPS) from dietary DDGS (d-DDGS) and DDGS residue (DDGS-r) obtained after 16-h ruminal incubation. The rumen degradation of AAs and CP was determined using nylon bag incubations in the rumen of cows. Lysine and methionine content of d-DDGS varied from 1.36 to 4.00 and 1.34 to 1.99 g/16 g N, respectively. The milk protein score (MPS) of d-DDGS was low and ranged from 0.36 to 0.51, and lysine and isoleucine were estimated to be the most limiting AAs in d-DDGS and DDGS-r. DDGS-r contained slightly more essential AAs than did the d-DDGS. Rumen degradation of CP after 16 h varied from 44% to 94% between DDGS samples. Rumen degradation of lysine and methionine ranged from 39% to 90% and from 35% to 92%, respectively. Linear regressions showed that ruminal degradation of individual AAs can be predicted from CP degradation. The PPS of d-DDGS was higher than that of DDGS-r and it varied from 70% to 89% and from 47% to 81%, respectively. There was no significant correlation between the PPS of d-DDGS and PPS of DDGS-r (R2 = 0.31). The estimated intestinally absorbable dietary protein (IADP) averaged 21%. Moderate correlation was found between the crude fibre content and PPS of DDGS-r (R2 = 0.43). This study suggests an overestimation of the contribution of UDP of DDGS to digestible protein supply in the duodenum in currently used protein evaluation systems. More research is required and recommended to assess the intestinal digestibility of AAs from DDGS. Finally, in a third study, three sources of DDGS were evaluated in diets of mid-lactating dairy cows on milk production and milk composition and on digestibility in sheep. DDGS from wheat, corn and barley (DDGS1), wheat and corn (DDGS2) and wheat (DDGS3) were studied and compared with a rapeseed meal (RSM). RSM and DDGS were characterized through in situ CP degradability. Nutrient digestibility was determined in sheep. Twenty-four multiparous cows were used in a 4 × 4 Latin square design with 28-day periods. Treatments included total mixed rations containing as primary protein sources RSM (control), DDGS1 (D1), DDGS2 (D2) or DDGS3 (D3). RSM contained less rapidly degradable CP (fraction a), more potentially degradable CP (fraction b) and more UDP than the three DDGS. In vivo organic matter digestibility of RSM was similar to DDGS. Calculated NEL was lower for RSM (7.4 MJ/kg DM) than for DDGS, which averaged 7.7 MJ/kg DM. Cows? dry matter intake did not differ between diets (21.7 kg/d). Cows fed D1 yielded more milk than those fed D3 (31.7 vs. 30.4 kg/d); no differences were found between control and DDGS diets (31.3 vs. 31.1 kg/d). Energy-corrected milk was similar among diets (31.2 kg/d). Diets affected neither milk fat concentration (4.0%) nor milk fat yield (1.24 kg/d). Milk protein yield of control cows (1.12 kg/d) was significantly higher than D3 (1.06 kg/d) but not different from D1 and D2 (1.08 kg/d each). Feeding DDGS significantly increased milk lactose concentration (4.91%) compared to control (4.81%). DDGS can be a suitable feed compared to RSM and can be fed up to 4 kg dry matter per day in rations of dairy cows in mid-lactation. To conclude, DDGS is a suitable feedstuff for ruminants in terms of chemical composition, energy and protein value. However, the variability should be considered when included in diets of ruminants, especially in animals with high performance. For this purpose, prediction approaches initated in this study should be further developed into tools for routine application for rapid DDGS evaluation and estimation of feed values. These approaches might also be usefull for the evaluation of other feed protein sources and taked into consideration for practical feeding and diets formulation.