Engineering of camel chymosin for improved cheese properties

More than 20 Mio tons of cheese are produced world-wide per year. By improving cheese yield and quality through process optimization, the amount of milk needed for manufacturing can be reduced significantly. Chymosin, an aspartic acid protease, is initiating milk coagulation in cheese manufacturing by cleaving off the glycomacropeptide (GMP) from the surface of casein micelles. Non-specific proteolysis of casein molecules by chymosin during this milk clotting process releases soluble peptides into the whey, resulting in protein losses from the cheese. The ratio between specific clotting activity (C) and non-specific proteolysis (P) of a coagulant can therefore be used as predictor for cheese yield. During ripening of the cheese, remaining coagulant continues proteolytic break-down of the caseins with significant impact on cheese properties. While the main proteolytic activity, the release of N-terminal peptides from alphaS1 casein (alphaS1-N), is associated with cheese softening and loss of firmness, cleavage of the C-terminal end of beta casein (beta-C) contributes to unwanted bitterness of the cheese [1]. The chymosin from Bos taurus (bovine chymosin) is traditionally used as milk coagulant in cheese manufacture. However, the homologous enzyme from Camelus dromedarius (camel chymosin) has been shown to be a superior alternative for various cheese types, since it reveals higher specific activity (C) and specificity (C/P) for the milk clotting reaction [2], as well as lower alphaS1 and beta casein proteolysis during ripening (Fig. 1). Please click Additional Files below to see the full abstract

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function

Multidimensional engineering of Chymosin for efficient cheese production by machine learning guided directed evolution

The global cheese market today exceeds $100B/year. Chymosin (a.k.a. rennin) is an aspartic endopeptidase produced by the stomach lining of new-born mammals. During cheese production chymosin is added to the milk where it cleaves the glycomacropeptide (GMP) from the surface of casein micelles to initiate milk coagulation. Current commercial recombinant chymosin enzymes derived from Bos taurus (cow) or Camelus dromedarius (camel) are limited in their proteolytic specificity leading to incomplete milk-to-cheese conversion. Increasing the chymosin specificity for GMP cleavage would significantly decrease the amount of milk needed for cheese production thereby reducing cost and decreasing environmental footprint of the dairy industry. Separate from milk coagulation, chymosin dependent release of N-terminal peptides from alphaS1 casein during cheese ripening leads to unwanted softening, accompanied with cheese loss during industrial processing such as slicing and shredding. Furthermore, chymosin dependent cleavage of the C-terminal end of beta casein contributes to unwanted bitterness of the cheese. Improvement of chymosin proteolytic specificity in both milk coagulation and cheese ripening is consequently of high commercial relevance. Please click Additional Files below to see the full abstract

A large aperture reflective wave-plate for high-intensity short-pulse laser experiments

We report on a reflective wave-plate system utilizing phase-shifting mirrors (PSM) for a continuous variation of elliptical polarization without changing the beam position and direction. The scalability of multilayer optics to large apertures and the suitability for high-intensity broad-bandwidth laser beams make reflective wave-plates an ideal tool for experiments on relativistic laser-plasma interaction. Our measurements confirm the preservation of the pulse duration and spectrum when a 30-fs Ti:Sapphire laser beam passes the system

Fate of ptaquiloside—A bracken fern toxin—In cattle

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites

Long-term outcome and patterns of failure in patients with advanced head and neck cancer

<p>Abstract</p> <p>Purpose</p> <p>To access the long-time outcome and patterns of failure in patients with advanced head and neck squamous cell carcinoma (HNSCC).</p> <p>Methods and materials</p> <p>Between 1992 and 2005 127 patients (median age 55 years, UICC stage III n = 6, stage IV n = 121) with primarily inoperable, advanced HNSCC were treated with definite platinum-based radiochemotherapy (median dose 66.4 Gy). Analysed end-points were overall survival (OS), disease-free survival (DFS), loco-regional progression-free survival (LPFS), development of distant metastases (DM), prognostic factors and causes of death.</p> <p>Results</p> <p>The mean follow-up time was 34 months (range, 3-156 months), the 3-, 5- and 10-year OS rates were 39%, 28% and 14%, respectively. The median OS was 23 months. Forty-seven patients achieved a complete remission and 78 patients a partial remission. The median LPFS was 17 months, the 3-, 5- and 10-year LPFS rates were 41%, 33% and 30%, respectively. The LPFS was dependent on the nodal stage (p = 0.029). The median DFS was 11 months (range, 2-156 months), the 3-, 5- and 10-year DFS rates were 30%, 24% and 22%, respectively. Prognostic factors in univariate analyses were alcohol abuse (n = 102, p = 0.015), complete remission (n = 47, p < 0.001), local recurrence (n = 71, p < 0.001), development of DM (n = 45, p < 0.001; median OS 16 months) and borderline significance in nodal stage N2 versus N3 (p = 0.06). Median OS was 26 months with lung metastases (n = 17). Nodal stage was a predictive factor for the development of DM (p = 0.025). Cause of death was most commonly tumor progression.</p> <p>Conclusions</p> <p>In stage IV HNSCC long-term survival is rare and DM is a significant predictor for mortality. If patients developed DM, lung metastases had the most favourable prognosis, so intensified palliative treatment might be justified in DM limited to the lungs.</p

Entwicklung eines Screening-Systems zur systematischen Untersuchung der Wechselwirkungseigenschaften fluoralkyl-substituierter Aminosäuren mit Polypeptiden

Titelblatt u. Inhalt 1 1\. Introduction 1 2\. Fluorine in Bioactive Molecules 4 3\. Aim of the Work 31 4\. Concepts of a Peptide-Based Screening System 32 5\. Development and Implementation of the Screening System 54 6\. Summary and Outlook 127 7\. Experimental Procedures and Methods 130 8\. Literature 189Fluorinated amino acids are powerful tools in the development of peptide-based drugs. Besides the use of organic fluorine as NMR label, fluoroalkyl groups can significantly improve structural and metabolic stability as well as the biological activity of peptides and peptidomimetics. Furthermore, such non- natural building blocks can be applied for the structural stabilization of proteins and for dictating their folding and oligomerization behavior. However, the applicability of fluorinated amino acids for the rational design of peptide-protein interaction sites is severely limited due to a lack of fundamental knowledge about the interaction properties of fluoroalkyl groups with native polypeptides, such as size, polarity, and the significance of fluorous interactions. In this thesis, a coiled coil-based model polypeptide with substitution sites for fluoroalkyl-substituted amino acids within specific hydrophobic and polar interfaces was designed to systematically investigate the interaction properties of fluoroalkyl groups in protein environments. Screening methods for the evaluation of size and polarity as well as for the characterization of fluorous interactions have been established. This screening system was sensitive enough to detect differences of a single fluorine atom between amino acid side chains. Systematically altered amino acids that vary in side chain length and fluorine content were incorporated into both interfaces of the model polypeptides via solid phase peptide synthesis and their influence on stability of the folding motif was evaluated. Based on thermostability measurements of the substituted coiled coils, opponent steric and electronic consequences of alkyl-fluorination on hydrophobic protein interactions were described. While an increase in side chain volume upon stepwise fluorine substitution stabilizes hydrophobic protein cores, a polarization of alkyl moieties in proximity to the fluorination site disturbs hydrophobic interactions. Coiled coil self- replication studies of revealed that fluorous interactions between substituted amino acids are strong enough to affect peptide and protein folding. Further investigations on Ca,a-dialkylated amino acids that are known to increase the metabolic stability of peptides, suggested that these building blocks can also improve the structural stability of coiled coil-based drugs. To select the best native binding partners for fluoroalklyl-substituted amino acids, the designed model polypeptide was adapted for library screening by phage display. A coiled coil-based stem loop peptide library was constructed and optimized regarding its size and diversity. Coiled coil formation and covalent linkage of the screening peptide to the library peptide were demonstrated on the surface of the soluble protein MBP. The display of the stem loop peptide on phage coat could also be shown. Since the specific binding of the screening peptide to the library peptide could not yet be accomplished on phage surface, library screenings were not performed in this work.Fluorierte Aminosäuren stellen wertvolle Werkzeuge für die Entwicklung Peptid- basierter Wirkstoffe dar. Neben ihrem Einsatz als NMR-Sonde können Fluoralkylgruppen eingebaut werden, um die Bioverfügbarkeit und die pharmakokinetischen Eigenschaften von Peptiden und Peptidmimetika zu verbessern. Desweiteren können diese nicht-natürlichen Bausteine für die strukturelle Stabilisierung von Proteinen und zur Steuerung deren Faltung und Oligomerisierung genutzt werden. Die Anwendbarkeit fluorierter Aminosäuren für das rationale Design von Peptid-Protein-Interaktionsdomänen ist bisher jedoch stark limitiert. Ursache dafür ist der Mangel an grundlegendem Wissen über die Wechselwirkungs-eigenschaften von Fluoralkylgruppen mit nativen Polypeptiden, wie z. Bsp. Größe, Polarität, Fluor-Fluor-Wechselwirkungen und deren Bedeutung für Faltung und Stabilität. In der vorliegenden Arbeit wurde ein coiled coil- basiertes Modell-Polypeptid mit Substitut-ionspositionen für fluoralkyl- modifizierte Aminosäuren in spezifischen hydrophoben und polaren Wechselwirkungsdomänen etabliert, um die Wechselwirkungseigenschaften der Fluoralkylgruppen in Proteinumgebung systematisch zu studieren. Zur Charakterisierung dieser Eigenschaften wurden zwei Untersuchungsmethoden optimiert und angewendet. Das entwickelte screening-System war empfindlich genug um den Unterschied eines einzelnen Fluoratoms zwischen Aminosäure- Seitenketten zu detektieren. Aminosäuren mit systematisch variierten Seitenketten wurden mittels Festphasenpeptidsynthese in die Wechselwirkungsdomänen der Modell-Polypeptide eingebaut. Basierend auf Thermo- stabilitätsuntersuchungen der substituierten Peptide konnte gezeigt werden, dass eine Vergrößerung des Seitenkettenvolumens durch zunehmende Alkylfluorierung hydrophobe Protein-domänen stabilisiert. Eine Polarisierung von Alkylgruppen nahe der Fluorierungs-stelle stört dagegen hydrophobe Wechselwirkungen. Selbstreplikations-Studien zeigten, dass Fluor-Fluor- Wechselwirkungen stark genug sind, um die Faltung von Peptiden und Proteinen zu beeinflussen. Desweiteren konnte mithilfe des screening-Systems gezeigt werden, dass Ca,a-dialkylierte Aminosäuren die Strukturstabilität von coiled coil-basierten Peptidwirkstoffen erhöhen können. Um die bevorzugten Wechselwirkungspartner fluoralkyl-substituierter Aminosäuren zu ermitteln, wurde das Modell-Polypeptid für ein Phage-Display-basiertes Bibliotheks- screening verwendet. Größe und Diversität einer entsprechenden Peptidbibliothek wurden optimiert und die coiled coil-Bildung von screening- und Bibliothekspeptid konnte an der Oberfläche des löslichen Proteins MBP nachgewiesen werden. Der Einbau des Bibliothekspeptids in die Phagenhülle konnte ebenfalls realisiert werden. Da die coiled coil-Bildung an der Phagenoberfläche noch nicht verwirklicht werden konnte, wurden im Rahmen dieser Arbeit keine Bibliotheks-screenings durchgeführt