Prediction of the level of astringency in persimmon using visible and near-infrared spectroscopy

[EN] Early control of fruit quality requires reliable and rapid determination techniques. Therefore, the food industry has a growing interest in non-destructive methods such as spectroscopy. The aim of this study was to evaluate the feasibility of visible and near-infrared (NIR) spectroscopy, in combination with multivariate analysis techniques, to predict the level and changes of astringency in intact and in the flesh of half cut persimmon fruits. The fruits were harvested and exposed to different treatments with 95 % CO2 at 20 ºC for 0, 6, 12, 18 and 24 h to obtain samples with different levels of astringency. A set of 98 fruits was used to develop the predictive models based on their spectral data and another external set of 42 fruit samples was used to validate the models. The models were created using the partial least squares regression (PLSR), support vector machine (SVM) and least squares support vector machine (LS-SVM). In general, the models with the best performance were those which included standard normal variate (SNV) in the pre-processing. The best model was the PLSR developed with SNV along with the first derivative (1-Der) pre-processing, created using the data obtained at six measurement points of the intact fruits and all wavelengths (R2=0.904 and RPD=3.26). Later, a successive projection algorithm (SPA) was applied to select the most effective wavelengths (EWs). Using the six points of measurement of the intact fruit and SNV together with the direct orthogonal signal correction (DOSC) pre-processing in the NIR spectra, 41 EWs were selected, achieving an R2 of 0.915 and an RPD of 3.46 for the PLSR model. These results suggest that this technology has potential for use as a feasible and cost-effective method for the non-destructive determination of astringency in persimmon fruits.This work has been partially funded by the Institute Nacional de Investigacion y Tecnologia Agraria y Alimentaria de Espana (INIA) through research projects RTA2012-00062-004-01/03, RTA2013-00043-C02, and RTA2015-00078-00-00 with the support of European FEDER funds, and by the Conselleria d' Educacio, Investigacio, Cultura i Esport, Generalitat Valenciana, through the project AICO/2015/122. V. Cortes thanks the Spanish MEC for the FPU grant (FPU13/04202).Cortés López, V.; Rodríguez Ortega, A.; Blasco Ivars, J.; Rey Solaz, B.; Besada, C.; Cubero García, S.; Salvador, A.... (2017). Prediction of the level of astringency in persimmon using visible and near-infrared spectroscopy. JOURNAL OF FOOD ENGINEERING. 204:27-37. doi:10.1016/j.jfoodeng.2017.02.017S273720

A review of optical nondestructive visual and near-infrared methods for food quality and safety

This paper is a review of optical methods for online nondestructive food quality monitoring. The key spectral areas are the visual and near-infrared wavelengths. We have collected the information of over 260 papers published mainly during the last 20 years. Many of them use an analysis method called chemometrics which is shortly described in the paper. The main goal of this paper is to provide a general view of work done according to different FAO food classes. Hopefully using optical VIS/NIR spectroscopy gives an idea of how to better meet market and consumer needs for high-quality food stuff.©2013 the Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.fi=vertaisarvioitu|en=peerReviewed

In silico molecular modelling and design of heme-containing peroxidases for industrial applications

It is widely known that the development of modern chemistry and the consequent world industrialization have improved our quality of life to unimaginable levels. However, these advances have come with a high cost, causing environmental, health and societal concerns. As a consequence, during the past two decades a growing need has appeared to update the traditional chemistry industry processes towards greener and efficient alternatives. Along these lines, the use of enzymes has shown to be a suitable alternative to conventional industrial chemical processes. Enzymes are life-essential proteins that catalyze biochemical reaction and show several advantages over conventional chemical catalysts: they work under milder conditions, which decrease the energy requirements and consequently the capital costs of reactions; They show a high degree of selectivity and catalytic efficiency; and in addition, they are inherently non-hazardous, reusable and biodegradable catalysts, making them ideal environmentally friendly reagents. However, the main bottleneck for taking more benefit of enzymes in an industrial context is the lack of biocatalysts with the required selectivity, availability, and compatibility with industrial rigorous process conditions, and because of this, the development of enhanced enzymes by means of enzyme engineering is a main research field nowadays. Along these lines, in silico methodologies have progressively turned into highly valuable tools for the study and design of enzymatic systems, due to their unique potential to offer atomic and electronic-level insights into biocatalysts’ activity. Moreover, the continuous software and hardware improvements, and the cost-effectiveness and rapidness generally associated with these methods, make them very appealing for their application to the real problems that face the industry. Motivated by the advances on computational techniques and by the ease of obtaining valuable experimental data, which has been provided by our collaborators, the main goal of this thesis has been to understand the mechanisms of reaction of the heme-containing peroxidases under study (Auricularia auricula-judae DyP and Agrocybe aegerita UPO). Moreover, the acquired knowledge has been used to evaluate experimentally obtained enzyme variants and to guide the design of new ones towards desired properties. In this way, distinct computational techniques at different levels of accuracy (e.g. PELE, QM/MM or MD calculations) have been used to unravel the atomic and electronic mechanistic details under peroxidases mechanisms (e.g. long range electron transfer pathways, peroxidation and peroxygenation mechanisms) and to rationalize the molecular determinants that guide yield and selectivity in both natural occurring and experimentally designed peroxidases. Furthermore, the better understanding of the molecular principles under enzyme activity, along with the use of in silico semi-rational redesign methods, has enabled us to tailor UPO enzyme towards the enhanced production of high-value chemicals.A causa del desenvolupament de la química moderna i de la consegüent industrialització del món, la nostra qualitat de vida ha millorat a uns nivells que creiem inimaginables. Malauradament, tots aquests avenços han vingut acompanyats de repercussions mediambientals, socials i de salut. Per això, en les dues últimes dècades s'ha percebut una creixent necessitat de reemplaçar els processos químics tradicionals per alternatives més ecològiques i eficients. En aquest sentit, els enzims han demostrat ser una alternativa molt plausible als processos químics convencionals que s'usen avui en dia en la indústria. Els enzims són proteïnes essencials per a la vida que catalitzen reaccions bioquímiques, i l'ús dels quals aporta múltiples avantatges en comparació a les tècniques convencionals: permeten treballar en condicions suaus, fet que disminueix els requisits energètics i conseqüentment els costos de les reaccions a nivells industrials; en general són molt selectius i eficients; i a més a més, són inherentment reutilitzables, segurs i biodegradables, fet que els converteix en reactius respectuosos amb el medi ambient. Tot i això, les seves aplicacions a nivells industrials encara són limitades degut a la baixa tolerància a substrats, la poca disponibilitat d'enzims i a l'escassa resistència a les severes condicions industrials. Per aquesta raó, avui en dia el desenvolupament d'enzims millorats és un camp d'investigació important. Particularment, les tècniques in silico de modelització molecular s'estan convertint cada vegada més en eines clau per a l'estudi i el disseny de biocatalitzadors degut al seu potencial per a obtenir informació, tant a escala electrònica com molecular, sobre els mecanismes d'acció enzimàtics. A més a més, millores en el software i el hardware, i la rapidesa i bona relació cost-qualitat que mostren aquests mètodes, els fan molt atractius per a resoldre els problemes reals de la indústria. Motivada pels avenços en les tècniques computacionals i per la facilitat d'obtenir dades experimentals, que han estat proporcionades pels nostres col·laboradors, l'objectiu principal d'aquesta tesi ha sigut entendre els mecanismes de reacció de les peroxidases (en particular la DyP del fong Auricularia aurícula-judae i la UPO del fong Agrocybe aegerita). D'altra banda, els coneixements adquirits durant aquest procés de racionalització s'han utilitzat per avaluar variants millorats d'enzims obtinguts experimentalment i per guiar el disseny de nous biocatalitzadors cap a les propietats desitjades. Així doncs, s'han utilitzat diferents tècniques computacionals, a diferents nivells de precisió (p. ex. PELE, QM/MM o MD), per tal de comprendre els mecanismes electrònics i moleculars responsables de diferents mecanismes en les peroxidases (p. ex., mecanismes de transferència electrònica de llarg abast, peroxidació o peroxigenació), i racionalitzar els determinants moleculars que guien el rendiment i la selectivitat tant en les peroxidases naturals com en aquelles millorades experimentalment. A banda d'això, la millor comprensió dels principis moleculars responsables de l'activitat enzimàtica, juntament amb l'ús de mètodes computacionals per al disseny d'enzims, ens ha permès adaptar l'enzim UPO cap a la producció de productes químics valuosos

In silico molecular modelling and design of heme-containing peroxidases for industrial applications

[eng] It is widely known that the development of modern chemistry and the consequent world industrialization have improved our quality of life to unimaginable levels. However, these advances have come with a high cost, causing environmental, health and societal concerns. As a consequence, during the past two decades a growing need has appeared to update the traditional chemistry industry processes towards greener and efficient alternatives. Along these lines, the use of enzymes has shown to be a suitable alternative to conventional industrial chemical processes. Enzymes are life-essential proteins that catalyze biochemical reaction and show several advantages over conventional chemical catalysts: they work under milder conditions, which decrease the energy requirements and consequently the capital costs of reactions; They show a high degree of selectivity and catalytic efficiency; and in addition, they are inherently non-hazardous, reusable and biodegradable catalysts, making them ideal environmentally friendly reagents. However, the main bottleneck for taking more benefit of enzymes in an industrial context is the lack of biocatalysts with the required selectivity, availability, and compatibility with industrial rigorous process conditions, and because of this, the development of enhanced enzymes by means of enzyme engineering is a main research field nowadays. Along these lines, in silico methodologies have progressively turned into highly valuable tools for the study and design of enzymatic systems, due to their unique potential to offer atomic and electronic-level insights into biocatalysts’ activity. Moreover, the continuous software and hardware improvements, and the cost-effectiveness and rapidness generally associated with these methods, make them very appealing for their application to the real problems that face the industry. Motivated by the advances on computational techniques and by the ease of obtaining valuable experimental data, which has been provided by our collaborators, the main goal of this thesis has been to understand the mechanisms of reaction of the heme-containing peroxidases under study (Auricularia auricula-judae DyP and Agrocybe aegerita UPO). Moreover, the acquired knowledge has been used to evaluate experimentally obtained enzyme variants and to guide the design of new ones towards desired properties. In this way, distinct computational techniques at different levels of accuracy (e.g. PELE, QM/MM or MD calculations) have been used to unravel the atomic and electronic mechanistic details under peroxidases mechanisms (e.g. long range electron transfer pathways, peroxidation and peroxygenation mechanisms) and to rationalize the molecular determinants that guide yield and selectivity in both natural occurring and experimentally designed peroxidases. Furthermore, the better understanding of the molecular principles under enzyme activity, along with the use of in silico semi-rational redesign methods, has enabled us to tailor UPO enzyme towards the enhanced production of high-value chemicals.[cat] A causa del desenvolupament de la química moderna i de la consegüent industrialització del món, la nostra qualitat de vida ha millorat a uns nivells que creiem inimaginables. Malauradament, tots aquests avenços han vingut acompanyats de repercussions mediambientals, socials i de salut. Per això, en les dues últimes dècades s'ha percebut una creixent necessitat de reemplaçar els processos químics tradicionals per alternatives més ecològiques i eficients. En aquest sentit, els enzims han demostrat ser una alternativa molt plausible als processos químics convencionals que s'usen avui en dia en la indústria. Els enzims són proteïnes essencials per a la vida que catalitzen reaccions bioquímiques, i l'ús dels quals aporta múltiples avantatges en comparació a les tècniques convencionals: permeten treballar en condicions suaus, fet que disminueix els requisits energètics i conseqüentment els costos de les reaccions a nivells industrials; en general són molt selectius i eficients; i a més a més, són inherentment reutilitzables, segurs i biodegradables, fet que els converteix en reactius respectuosos amb el medi ambient. Tot i això, les seves aplicacions a nivells industrials encara són limitades degut a la baixa tolerància a substrats, la poca disponibilitat d'enzims i a l'escassa resistència a les severes condicions industrials. Per aquesta raó, avui en dia el desenvolupament d'enzims millorats és un camp d'investigació important. Particularment, les tècniques in silico de modelització molecular s'estan convertint cada vegada més en eines clau per a l'estudi i el disseny de biocatalitzadors degut al seu potencial per a obtenir informació, tant a escala electrònica com molecular, sobre els mecanismes d'acció enzimàtics. A més a més, millores en el software i el hardware, i la rapidesa i bona relació cost-qualitat que mostren aquests mètodes, els fan molt atractius per a resoldre els problemes reals de la indústria. Motivada pels avenços en les tècniques computacionals i per la facilitat d'obtenir dades experimentals, que han estat proporcionades pels nostres col·laboradors, l'objectiu principal d'aquesta tesi ha sigut entendre els mecanismes de reacció de les peroxidases (en particular la DyP del fong Auricularia aurícula-judae i la UPO del fong Agrocybe aegerita). D'altra banda, els coneixements adquirits durant aquest procés de racionalització s'han utilitzat per avaluar variants millorats d'enzims obtinguts experimentalment i per guiar el disseny de nous biocatalitzadors cap a les propietats desitjades. Així doncs, s'han utilitzat diferents tècniques computacionals, a diferents nivells de precisió (p. ex. PELE, QM/MM o MD), per tal de comprendre els mecanismes electrònics i moleculars responsables de diferents mecanismes en les peroxidases (p. ex., mecanismes de transferència electrònica de llarg abast, peroxidació o peroxigenació), i racionalitzar els determinants moleculars que guien el rendiment i la selectivitat tant en les peroxidases naturals com en aquelles millorades experimentalment. A banda d'això, la millor comprensió dels principis moleculars responsables de l'activitat enzimàtica, juntament amb l'ús de mètodes computacionals per al disseny d'enzims, ens ha permès adaptar l'enzim UPO cap a la producció de productes químics valuosos

Innovations in non-destructive techniques for fruit quality control applied to manipulation and inspection lines

Tesis por compendioLa industria alimentaria, concretamente el sector poscosecha, necesita innovar en sus procesos productivos, optimizando los mismos para rentabilizar sus actividades, garantizando productos de calidad capaces de satisfacer las necesidades de los consumidores. La presente tesis doctoral se centra en evaluar el potencial de la espectroscopia VIS-NIR para la caracterización e inspección de la calidad de la fruta tanto fuera de línea como a tiempo real en procesos automatizados. En un primer lugar, la viabilidad de la técnica se estudió a nivel de laboratorio en estado estático (off-line), con el fin de conocer y optimizar las condiciones de medición. Posteriormente, se evaluó la calidad interna y externa de diferentes tipos de frutas como son caqui, nectarina y mango. En una segunda etapa, se llevó a cabo una automatización de los procesos de inspección mediante el desarrollo de nuevos prototipos in-line. Para este propósito, y con el objetivo de completar y corroborar los resultados obtenidos de manera estática, se estudió la integración de dos sondas VIS-NIR en una garra robótica capaz de manipular mangos. Finalmente, se estudió la integración de una sonda VIS-NIR a una cinta transportadora. Los resultados obtenidos a nivel estático han demostrado que la espectroscopia VIS-NIR es un método no destructivo muy prometedor para predecir la astringencia en caqui. Así mismo, ha demostrado ser una adecuada herramienta para clasificar al 100% entre variedades de nectarinas como "Big Top" y "Diamond Ray" con una apariencia externa e interna muy similar, pero con diferentes propiedades organolépticas. De manera similar, fue posible clasificar al 100% variedades como "Big Top" y "Magique" de apariencia externa y composición similar pero distinto color de pulpa., y además se desarrolló un índice de calidad interna (IQI) para evaluar la calidad de las nectarinas. Por lo que respecta a los trabajos off-line realizados con mangos de la variedad "Osteen", fue posible predecir su calidad interna mediante los índices de madurez (RPI) y de calidad (IQI) con un gran rendimiento. A su vez, los ensayos experimentales efectuados con estos mismos mangos bajo la manipulación no destructiva de una garra robótica, demostraron que los mejores modelos eran capaces de predecir tanto la firmeza mecánica, el contenido en sólidos solubles, la luminosidad de la pulpa, así como el índice RPI de las muestras en base a la información obtenida por los acelerómetros instalados en los dedos de la garra robótica. En cuanto a los ensayos realizados de manera in-line, el primer prototipo desarrollado se basó en la integración de dos sondas VIS-NIR en una garra robótica dispuesta con dos acelerómetros. El sistema desarrollado permitió alcanzar una buena estimación de la calidad del mango a través del índice RPI fusionando la información tanto de los espectros VIS-NIR como del impacto no destructivo de los acelerómetros. De este modo quedó demostrado que era posible obtener una predicción similar trabajando de forma in-line como trabajando de manera off-line para la predicción del mismo índice de calidad en mangos. El segundo prototipo in-line desarrollado se basa en la integración de una sonda VIS-NIR en una cinta transportadora para la identificación de distintas variedades y orígenes de manzanas. El prototipo desarrollado permitió registrar resultados de clasificación tan buenos como los efectuados de manera off-line con, por ejemplo, nectarina. De este modo, se puede concluir que la espectroscopia VIS-NIR permite monitorear la calidad y clasificar fruta poscosecha tanto en modo off-line como in-line. Los nuevos prototipos desarrollados aportan claras ventajas respecto a los procesos tradicionales realizados a mano, como son la reducción del tiempo de inspección, la disminución de la cantidad de residuos generados y la posibilidad de inspeccionar toda la producción, obteniendo así un análisis más estandarizThe food industry, concretely the post-harvest sector, needs to innovate in their production processes, optimizing them to make their activities profitable, guaranteeing quality products capable of satisfying the needs of consumers. The present doctoral thesis focuses on evaluating the potential of visible and near infrared spectroscopy (VIS-NIR) for the characterization and inspection of fruit quality both off-line and in real time in automated processes. Firstly, the viability of the technique was studied at the laboratory level in a static mode (off-line), in order to know and optimise the measurement conditions. Subsequently, the internal and external quality of different types of fruits such as persimmon, nectarine and mango were evaluated. Secondly, an automation of the inspection processes was carried out through the development of new in-line prototypes. For this purpose, and with the aim of completing and corroborating the results obtained in a static mode, the integration of two VIS-NIR probes in a robotic gripper capable of manipulating mangoes was studied. Finally, the integration of a VIS-NIR probe to a conveyor belt was studied as an in-line monitoring tool on the inspection process of different apple varieties. The results obtained in static mode have shown that VIS-NIR spectroscopy is a very promising non-destructive method to predict the astringency in persimmon. Likewise, it has demonstrated to be an adequate tool to classify 100% between nectarine varieties such as 'Big Top' and 'Diamond Ray' with very similar external and internal appearance, but with different organoleptic properties. Similarly, it was possible to classify 100% varieties such as 'Big Top' and 'Magique' with external appearance and similar composition but different pulp colour. An internal quality index (IQI) was developed to evaluate the quality of nectarines, which can be predicted through VIS-NIR spectroscopy. Regarding the off-line work carried out with mangoes of 'Osteen' variety, it was possible to predict its internal quality through the indexes of maturity (RPI) and quality (IQI) with a high performance. Moreover, the experimental tests carried out with these same mangoes under the non-destructive manipulation of a robotic gripper, showed that the best models were able to predict both the mechanical firmness, the soluble solids content, the brightness of the pulp, as well as the RPI index of the samples based on the information obtained by the accelerometers installed on the fingers of the robotic gripper. Regarding the tests carried out in an in-line mode, the first developed prototype was based on the integration of two VIS-NIR probes in a robotic gripper fitted with two accelerometers. The developed system allowed reaching a good estimation of mango quality through the RPI index. In this way, it was demonstrated that it was possible to obtain a similar prediction working in-line as off-line mode for the prediction of the same quality index in mangoes. The second developed in-line prototype is based on the integration of a VIS-NIR probe in a conveyor belt for the identification of different varieties and origins of apples, achieving a success rate of 98% with the system. The developed prototype allowed to register classification results as good as those carried out off-line with, for example, nectarine. In this way, it can be concluded that VIS-NIR spectroscopy allows monitoring the quality and classifying post-harvest fruit in both off-line and in-line mode, being a tool that allows improving and guaranteeing the correct quality and food safety. The new developed prototypes provide clear advantages over the traditional processes performed by hand, such as the reduction of inspection time, the reduction of the amount of waste generated by destructive quality analysis and the possibility of inspecting full production, obtaining a more standardised analysis of the quality of the products.La indústria alimentària, concretament el sector postcollita, necessita innovar en els seus processos productius, optimitzant els mateixos per a rendibilitzar les seues activitats, garantint productes de qualitat capaços de satisfer les necessitats dels consumidors. La present tesi doctoral es centra en avaluar el potencial de l'espectroscòpia visible i infraroig pròxim (VIS-NIR) per a la caracterització i la inspecció de la qualitat de la fruita tant fora de línia com a temps real en processos automatitzats. En un primer lloc, la viabilitat de la tècnica es va estudiar a nivell de laboratori en estat estàtic (off-line), a fi de conéixer i optimitzar les condicions de mesurament. Posteriorment, es va avaluar la qualitat interna i externa de diferents tipus de fruites com són caqui, nectarina i mango. En una segona etapa, es va dur a terme una automatització dels processos d'inspecció per mitjà del desenvolupament de nous prototips in-line. Per aquest propòsit, i amb l'objectiu de completar i corroborar els resultats obtinguts de manera estàtica, es va estudiar la integració de dos sondes VIS-NIR en una garra robòtica capaç de manipular. Finalment, es va estudiar la integració d'una sonda VIS-NIR a una cinta transportadora. Els resultats obtinguts a nivell estàtic han demostrat que l'espectroscòpia VIS-NIR és un mètode no destructiu molt prometedor per a predir l'astringència en caqui. Així mateix, ha demostrat ser una adequada ferramenta per a classificar al 100% entre varietats de nectarines com "Big Top" i "Diamond Ray" amb una aparença externa i interna molt semblant, però amb diferents propietats organolèptiques. De manera semblant, va ser possible classificar al 100% varietats com "Big Top" i "Magique" d'aparença externa i composició semblant però distint color de polpa. Es va desenvolupar un índex de qualitat interna (IQI) per avaluar la qualitat de les nectarines. Pel que fa als treballs off-line realitzats amb mangos de la varietat "Osteen" va ser possible predir la seua qualitat interna mitjançant els índexs de maduresa (RPI) i de qualitat (IQI) amb un gran rendiment. Al mateix temps, els assajos experimentals efectuats amb estos mateixos mangos baix la manipulació no destructiva d'una garra robòtica, van demostrar que els millors models eren capaços de predir tant la fermesa mecánica, el contingut en sòlids solubles, la lluminositat de la polpa, així com l'índex RPI de les mostres basant-se en l'informació obtinguda pels acceleròmetres instal¿lats en els dits de la garra robòtica. En quant als assajos realitzats de manera in-line, el primer prototip desenvolupat es va basar en la integració de dos sondes VIS-NIR en una garra robòtica disposada amb dos acceleròmetres. El sistema desenvolupat va permetre aconseguir una bona estimació de la qualitat del mango a través de l'índex RPI fusionant l'informació tant dels espectres VIS-NIR com de l'impacte no destructiu dels acceleròmetres. D'esta manera va quedar demostrat que era possible obtindre una predicció semblant treballant de forma in-line com off-line per a la predicció del mateix índex de qualitat en mangos. El segon prototip in-line desenvolupat es va basar en la integració d'una sonda VIS-NIR en una cinta transportadora per a l'identificació de distintes varietats i orígens de pomes. El prototip desenvolupat va permetre registrar resultats de classificació tan bons com els efectuats de manera off-line. D'aquesta manera, es pot concloure que l'espectroscòpia VIS-NIR permet monitorar la qualitat i classificar fruita postcollita tant en mode off-line com in-line. Els nous prototips desenvolupats aporten clars avantatges respecte als processos tradicionals realitzats a mà, com són la reducció del temps d'inspecció, la disminució de la quantitat de residus generats pels anàlisis destructives de qualitat i la possibilitat d'inspeccionar tota la producció, obtenint així un anàlisi més estandarditzCortés López, V. (2018). Innovations in non-destructive techniques for fruit quality control applied to manipulation and inspection lines [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/110969TESISCompendi

Molekularbiologische Charakterisierung von Häm-Thiolat- und DyP-type-Peroxidasen ausgewählter Basidiomyceten

Peroxidasen des Typs ClassII sowie die Chlorperoxidase waren für mehrere Jahrzehnte die einzigen bekannten sekretorischen Hämperoxidasen aus Pilzen. Im Jahr 2004 wurde eine neue, ungewöhnliche Hämperoxidase aus dem Speisepilz Agrocybe aegerita isoliert und biochemisch charakterisiert. Diese Peroxidase, heute offiziell Unspezifische Peroxygenase (UPO, EC 1.11.2.1) genannt, vereint in sich Eigenschaften sowohl intrazellulärer Cytochrom-P450-Enzyme als auch klassischer extrazellulärer, pilzlicher Peroxidasen des Typs ClassII. In dieser Arbeit wurden für den UPO-Modellorganismus A. aegerita und für andere UPO-produzierende Pilze erstmals Nukleotidsequenzen der Enzyme ermittelt und phylogenetisch analysiert. Die Genome zweier Stämme von A. aegerita wurden sequenziert. Die Ergebnisse legen nahe, dass UPOs gemeinsam mit der Chlorperoxidase Vertreter einer phylogenetisch alten Protein-Superfamilie (Häm-Thiolat-Peroxidasen) darstellen, die in allen echten Pilzen (Eumycota) zu finden ist, aber in echten Hefen und einigen hefeartig wachsenden Basidiomyceten sekundär reduziert wurde. Die natürliche Funktion dieser extrazellulären Enzyme ist bislang unbekannt. Im Labor können UPOs ausschließlich unter Verwendung leguminosenhaltiger Kulturmedien induziert werden. Die induzierende Komponente aus Soja wurde in dieser Arbeit als proteinogen identifiziert. Eine zweite neue, extrazelluläre Hämperoxidase-Superfamilie ausserhalb der ClassII-Peroxidasen wurde im Totholz bewohnenden Pilz Bjerkandera adusta entdeckt. Die als DyP-type-Peroxidasen benannten Enzyme (DyP, EC 1.11.1.19) wurden später auch in anderen Pilzen gefunden, von denen in dieser Arbeit ebenso Nukleotidsequenzen erhalten wurden. Die phylogenetische Analyse ergab, dass pilzliche Sequenzen der DyP-type-Peroxidase-Superfamilie sehr ungleichmäßig sowie ausschließlich in den Genomen höherer Pilze (Dikarya) verteilt sind und vermutlich einen prokaryotischen Ursprung haben. Transkripte von UPO- und DyP-Genen wurden in nahezu allen untersuchten Laubstreuproben aus unterschiedlich stark genutzten Waldgebieten der Deutschen Biodiversitätsexploratorien nachgewiesen. Dies legt eine biologische Funktion dieser neuen Hämperoxidase-Superfamilien in entsprechenden Habitaten nahe.:Abkürzungsverzeichnis 1 1. Einleitung 5 2. Theoretische Grundlagen 7 2.1. Hämperoxidasen 7 2.1.1. Häm – Pigment des Lebens 7 2.1.2. Mechanismus der Hämperoxidase-vermittelten Katalyse 10 2.1.3. Systematik 16 2.2. Häm-Thiolat-Peroxidasen 36 2.3. DyP-type-Peroxidasen 38 3. Anliegen der Arbeit 41 4. Material und Methoden 43 4.1. Organismen 43 4.2. Chemikalien 53 4.3. Methoden 55 4.3.1. Kultivierung 55 4.3.2. Nukleinsäure-Isolation und cDNA-Synthese 56 4.3.3. PCR 57 4.3.4. Klonierung und DNA-Sequenzierung 62 4.3.5. Protein-Sequenzierung 62 4.3.6. Sequenzierungsstrategien 63 4.3.7. Genomsequenzierungen 73 4.3.8. Transkriptomsequenzierung 76 4.3.9. Phylogenetische Analysen 77 4.3.10. Vorhersage von Gen- und Protein-Eigenschaften 79 4.3.11. Photometrische Enzymaktivitätsmessung 80 4.3.12. Proteinkonzentrationsbestimmung 83 4.3.13. SDS-PAGE 83 4.3.14. Hämperoxidasen in der Streuschicht von Laubwäldern 83 4.3.15. UPO-Induktionsversuche 85 4.3.16. Partielle Charakterisierung sekretierter Peptidasen im Sojamedium 89 5. Ergebnisse 91 5.1. Häm-Thiolat-Peroxidasen 91 5.1.1. Sequenzen 91 5.1.2. Phylogenetische Analyse 110 5.1.3. UPO-Geninventar und UPO-Aktivität im Agrocybe-aegerita-Multispezieskomplex 113 5.1.4. Physiologie der UPO-Bildung in A. aegerita TM-A1 116 5.2. DyP-type-Peroxidasen 126 5.2.1. Sequenzen charakterisierter Enzyme 126 5.2.2. Sequenzen hypothetischer Enzyme 130 5.2.3. Phylogenetische Analyse 135 5.3. Genom- und Transkriptomsequenzierung von A. aegerita TM-A1 und AaN 137 5.4. Hämperoxidasen in Umweltproben 142 6. Diskussion 143 6.1. Sekretorische Peroxidasen aus Pilzen 143 6.2. Häm-Thiolat-Peroxidasen 144 6.2.1. UPO-Geninventar und -expression im Agrocybe-aegerita-Multispezieskomplex 147 6.2.2. Induktion der Unspezifischen Peroxygenase in A. aegerita TM-A1 150 6.2.3. Biologische Funktion 152 6.2.4. Phylogenetik 154 6.2.5. Cytochrom-P450-Enzyme 159 6.2.6. Ein Vorschlag zur Nomenklatur 165 6.3. DyP-type-Peroxidasen 166 6.3.1. Differentiell exprimierte Isoenzyme 166 6.3.2. Hypothetische Sequenzen 168 6.3.3. Biologische Funktion 168 6.3.4. Prokaryotischer Ursprung pilzlicher DyP-type-Peroxidasen 169 6.3.5. DyPs sind Teil der strukturellen CDE-Superfamilie 170 6.3.6. Heterologe Expression 171 7. Ausblick 173 8. Thesen 177 Literaturverzeichnis 179 A. Peptidsequenzen 225 B. Genspezifische Oligonukleotide 231 C. Verbreitung von HTP-Sequenzen 241 D. Verbreitung von DyP-Sequenzen 255 E. Charakterisierte ClassII-Peroxidasen 261 F. ClassII-Aktivitäten in Reinkulturen 267 G. Physiologie von A. aegerita TM-A1 in Sojamedien 271 H. Einfluss der Soja-Inhaltsstoffe auf die UPO-Produktion in A. aegerita TM-A1 275 I. Inhaltsstoffe von Sojamedien 283 J. Transkriptomanalyse A. aegerita TM-A1 285 K. Bakterielle DyP-type-Peroxidasen 289 L. Publikationen 293 Danksagung 371 Rechtliche Erklärungen 373For several decades ClassII peroxidases and chloroperoxidase have been the only known secretory heme peroxidases from fungi. In 2004, a novel heme peroxidase was isolated and characterized from the edible mushroom Agrocybe aegerita. This enzym, nowadays called unspecific peroxygenase (UPO, EC 1.11.2.1), combines characteristics of intracellular cytochrom P450 enzymes as well as extracellular classII fungal peroxidases. In the course of this PhD work, for the first time nuleotide sequences of UPO model organism A. aegerita as well as other UPO producing fungi were obtained and phylogenetically analyzed. The whole-genome sequences of two strains of A.aegerita were obtained. The results suggest that UPOs and the chloroperoxidase together represent a phylogenetically old protein superfamily (of heme-thiolate peroxidases) that is found in all true fungi (Eumycota), but is secondarily reduced in true yeasts and a few yeast-like growing basidiomycetes. The natural function of these enzymes remains unclear. In the laboratory, fungi secrete UPOs solely in legume-containing culture media. The eliciting component of the legume soybean has been determined as proteinogenic. A second novel heme peroxidase superfamily outside the classII peroxidases was discovered in deadwood inhabiting fungus Bjerkandera adusta. These so-called DyP-type peroxidases (DyP, EC 1.11.1.19) were later found expressed in several other fungi for which nucleotide sequences were obtained in this work, too. Phylogenetical analysis revealed that fungal DyP sequences are exclusively but very unequally distributed in genomes of higher fungi (Dikarya). Fungal DyP sequences are seemingly decended from prokaryotes. Transcripts encoding UPOs and DyPs were found in almost every sample of leave litter (derived from forests with different intensity of human usage) investigated. This suggests biological functions of these new heme peroxidase superfamilies in respective habitats.:Abkürzungsverzeichnis 1 1. Einleitung 5 2. Theoretische Grundlagen 7 2.1. Hämperoxidasen 7 2.1.1. Häm – Pigment des Lebens 7 2.1.2. Mechanismus der Hämperoxidase-vermittelten Katalyse 10 2.1.3. Systematik 16 2.2. Häm-Thiolat-Peroxidasen 36 2.3. DyP-type-Peroxidasen 38 3. Anliegen der Arbeit 41 4. Material und Methoden 43 4.1. Organismen 43 4.2. Chemikalien 53 4.3. Methoden 55 4.3.1. Kultivierung 55 4.3.2. Nukleinsäure-Isolation und cDNA-Synthese 56 4.3.3. PCR 57 4.3.4. Klonierung und DNA-Sequenzierung 62 4.3.5. Protein-Sequenzierung 62 4.3.6. Sequenzierungsstrategien 63 4.3.7. Genomsequenzierungen 73 4.3.8. Transkriptomsequenzierung 76 4.3.9. Phylogenetische Analysen 77 4.3.10. Vorhersage von Gen- und Protein-Eigenschaften 79 4.3.11. Photometrische Enzymaktivitätsmessung 80 4.3.12. Proteinkonzentrationsbestimmung 83 4.3.13. SDS-PAGE 83 4.3.14. Hämperoxidasen in der Streuschicht von Laubwäldern 83 4.3.15. UPO-Induktionsversuche 85 4.3.16. Partielle Charakterisierung sekretierter Peptidasen im Sojamedium 89 5. Ergebnisse 91 5.1. Häm-Thiolat-Peroxidasen 91 5.1.1. Sequenzen 91 5.1.2. Phylogenetische Analyse 110 5.1.3. UPO-Geninventar und UPO-Aktivität im Agrocybe-aegerita-Multispezieskomplex 113 5.1.4. Physiologie der UPO-Bildung in A. aegerita TM-A1 116 5.2. DyP-type-Peroxidasen 126 5.2.1. Sequenzen charakterisierter Enzyme 126 5.2.2. Sequenzen hypothetischer Enzyme 130 5.2.3. Phylogenetische Analyse 135 5.3. Genom- und Transkriptomsequenzierung von A. aegerita TM-A1 und AaN 137 5.4. Hämperoxidasen in Umweltproben 142 6. Diskussion 143 6.1. Sekretorische Peroxidasen aus Pilzen 143 6.2. Häm-Thiolat-Peroxidasen 144 6.2.1. UPO-Geninventar und -expression im Agrocybe-aegerita-Multispezieskomplex 147 6.2.2. Induktion der Unspezifischen Peroxygenase in A. aegerita TM-A1 150 6.2.3. Biologische Funktion 152 6.2.4. Phylogenetik 154 6.2.5. Cytochrom-P450-Enzyme 159 6.2.6. Ein Vorschlag zur Nomenklatur 165 6.3. DyP-type-Peroxidasen 166 6.3.1. Differentiell exprimierte Isoenzyme 166 6.3.2. Hypothetische Sequenzen 168 6.3.3. Biologische Funktion 168 6.3.4. Prokaryotischer Ursprung pilzlicher DyP-type-Peroxidasen 169 6.3.5. DyPs sind Teil der strukturellen CDE-Superfamilie 170 6.3.6. Heterologe Expression 171 7. Ausblick 173 8. Thesen 177 Literaturverzeichnis 179 A. Peptidsequenzen 225 B. Genspezifische Oligonukleotide 231 C. Verbreitung von HTP-Sequenzen 241 D. Verbreitung von DyP-Sequenzen 255 E. Charakterisierte ClassII-Peroxidasen 261 F. ClassII-Aktivitäten in Reinkulturen 267 G. Physiologie von A. aegerita TM-A1 in Sojamedien 271 H. Einfluss der Soja-Inhaltsstoffe auf die UPO-Produktion in A. aegerita TM-A1 275 I. Inhaltsstoffe von Sojamedien 283 J. Transkriptomanalyse A. aegerita TM-A1 285 K. Bakterielle DyP-type-Peroxidasen 289 L. Publikationen 293 Danksagung 371 Rechtliche Erklärungen 37

Visible and Hyperspectral Imaging Systems for the Detection and Discrimination of Mechanical and Microbiological Damage of Mushrooms

Horticultural products such as mushrooms are exposed to environmental conditions during their postharvest life, which may affect product quality. Loss of whiteness during storage is particularly important in the mushroom industry. Rough handling and distribution, fruiting body senescence and bacterial infections are among the main causes of mushroom discolouration. The aim of this work was to study the use of visible and hyperspectral imaging (HSI) systems for the detection and discrimination of mechanical and microbiological damage of mushrooms. This piece of research involved a) monitoring the browning of mushroom with visible computer imaging systems, b) investigating the effect of mechanical damage on the kinetics of enzymes responsible for mushroom browning, c) exploring the potential use of Vis-NIR HSI to predict PPO activity in mushroom caps and d) studying the potential application of Vis-NIR HSI for microbial and viral detection on mushroom caps and for their discrimination from mechanical damage. Results presented in this thesis show that the efficacy of commercial webcams was limited in the detection of mechanical damage on mushroom caps. Damage increased the activity of PPOs on mushroom pileipellis, but the effect of the extent of damage was not significant at the levels of study. Vis-NIR HSI showed some potential as a tool to estimate the activity of PPO enzymes on mushroom caps. The combination of HSI with chemometric tools allowed for the differentiation of mechanically and microbiologically damaged mushroom classes. Results from this study could be used for developing non-destructive monitoring systems for mechanical and microbiological damage detection and discrimination. The potential application of such systems as on-line process analytical tools would facilitate rapid assessment of mushroom quality.

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

Understanding Peroxidase immobilisation on Bioinspired Silicas and application of the biocatalyst for dye removal

Dyestuff industry is responsible for up to 20% of the industrial water pollution, due to dye loss in effluents. Compared to research on treatment of azo dyes (largest category), research of anthraquinone dyes (second largest category) is neglected. Environmental considerations about industrial chemical processes for water treatment have led to a shift towards green chemistry and biocatalysis. Although peroxidases are vastly applied in bioremediation, they cannot be industrially implemented due to low stability, lack of reusability and difficulty in scale-up. Immobilisation offers reusability and can improve the catalytic functions and operational stability of biocatalysts. Novel approaches, include bioinspired supports, synthesised fast and economically, avoiding the environmentally un-friendly methods used in “conventional” immobilisation. This project focused on understanding the immobilisation of Horseradish Peroxidase (HRP) on bespoke Bioinspired Silicas (BIS), by examining factors affecting the synthesis and performance of the biocatalysts. We immobilised HRP on BIS via in-situ encapsulation and adsorption, and compared the outcomes to that of HRP adsorbed on commercial silicas. We also examined the effect of the controlled presence of amine functionalisation on BIS, of the point of HRP addition during synthesis of the biocatalyst and of increasing HRP concentration, to the immobilisation efficiency and performance of biocatalysts. BIS showed high potential as immobilisation supports, offering high loading (about 20% HRP on BIS-HRP composite) of active enzyme and their ability to protect HRP under exposure to non-optimal conditions. Biocatalysts were characterised for their morphology and porosity before assessing their performance a standard peroxidase assay based on 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate acid oxidation (ABTS assay) and an application assay based on enzymatic degradation of a model anthraquinone dye, Reactive Blue 19 (RB19 assay). Further examination of the best performing BIS-HRP samples, revealed a competitive action of BIS to enzymatic activity, where the support acts as an excellent adsorbent, hindering the diffusion of substrate and product(s) through the pore network. Although free HRP outperforms immobilised HRP (especially via encapsulation), immobilisation results in a highly reusable biocatalyst, for up to 20 times with 60% performance retention towards dye removal, with enhanced storage stability, retaining almost 100% activity over 50 days of storage, compared to 3 days of storage reached with free HRP. Through this work, we showed the importance of individual factors crucial for enzyme immobilisation, regarding both biocatalyst synthesis and expected performance, as well as the importance of the combination of enzyme, substrate and immobilisation support on biocatalyst performance. This work can be a great base for further optimisation of BIS as enzyme immobilisation support, and its exploration in other applications in the area of water treatment