Studies into the chemoenzymatic modification of cellulose by the laccase/TEMPO system

Die vorliegende Arbeit befaßt sich mit der enzymatischen Modifizierung von Cellulosen durch das Laccase-Mediator-System (LMS). 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) ist ein solcher Mediator; der bisher allerdings nur in chemischen Systemen zusammen mit Hypochlorit/Hypobromit zur Oxidation von niedermolekularen Kohlenhydraten und Cellulosen eingesetzt wurde. Der Effekt des Laccase/TEMPO-Systems auf Cellulose wurde detailliert untersucht, insbesondere im Hinblick auf Molmasseveränderungen und die Einführung von funktionellen Gruppen, wofür Größenausschluß-Chromatografie mit selektiver Fluoreszenzmarkierung oxidierter Gruppen (Carbonyle, Carboxyle) zur Anwendung kam. Die LMS-Behandlung führte zu einer gleichmäßigen Oxidation des cellulosischen Materials auch im hochmolekularen Bereich, was ein untypisches Verhalten im Vergleich zur konventionellen, chemischen Celluloseoxidationen darstellt. Der enzymatische Weg wurde eingehend mit der herkömmlichen Oxidation mittels TEMPO/Hypochlorit verglichen. Drei verschiedene Enzym-Präparationen wurden hinsichtlich ihrer Eignung zum Einsatz in organischen Lösungsmitteln untersucht: lösliche (lyophilisierte) und immobilisierte Laccase sowie crosslinked enzyme crystals (CLECs). Letztere hatten eine deutlich erhöhte Aktivität im Vergleich mit der lyophilisierten oder immobilisierten Form. Ebenso wurde die Rolle von 4-Acetamido-TEMPO in der Celluloseoxidation untersucht, wobei der Schwerpunkt auf mögliche Nebenreaktionen sowie auf die chemische Struktur von TEMPO-Zersetzungsprodukten und Intermediaten gelegt wurde. Der Abbau war stark vom pH-Wert des Reaktionsmediums abhängig. Unter sauren Bedingungen (pH 5) wird 4-Oxo-TEMPO in einer oxidativen Deaminierung gebildet. Im alkalischen Medium läuft zuerst ein ähnlicher Prozess ab, auf den jedoch unmittelbar eine Favorskij-Umlagerung folgt, die zu einem Pyrrolidin-3-carbonsäure-Derivat (PROXYL) führt. Schließlich wurde eine neue Anwendung des Laccase-Mediator-Systems zur Regenerierung von NAD(P)+ aus NAD(P)H untersucht.The topic of this thesis is the enzymatic modification of cellulose by the laccase/TEMPO system. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), which has so far been applied mainly in chemical oxidation systems together with hypohalite to effect oxidation of low-molecular weight carbohydrates and cellulosics, is also an effective mediator in combination with laccase. The effect of the laccase/TEMPO system on cellulosics with regard to molecular weight changes and introduction of functional groups has been studied using gel permeation chromatography in combination with selective fluorescence labeling of oxidized groups (carbonyls and carboxyls). LMS treatment caused uniform oxidation of the cellulosic material, also in the high molecular-weight area, which is a non-typical behavior compared to conventional chemical oxidations of cellulose. The chemoenzymatic approach was compared to the well-known chemical approach employing TEMPO/hypohalite. The performance of three different laccase preparations was studied with regard to applicability in organic solvents for synthetic-organic carbohydrate and cellulose chemistry: soluble (lyophilized) laccase, immobilized enzyme and crosslinked enzyme crystals (CLECs). The latter showed a significantly higher activity compared to the immobilized and the lyophilized variants. We also examined the role of 4-acetamido-TEMPO as the mediator part of LMS in cellulose oxidation systems, with a special focus on possible side reactions, degradation pathways and the chemical structure and fate of the TEMPO-derived species. Degradation was strongly dependent on the pH value of the reaction mixture. Under acidic conditions (pH 5), 4-oxo-TEMPO is formed by an oxidative deamination process. In alkaline media, a similar process proceeds, but is immediately followed by a Favorskij rearrangement to form a pyrrolidine-3-carboxylic acid (PROXYL) derivative. Eventually, a novel LMS system for the regeneration of NAD(P)+ from NAD(P)H for use in dehydrogenase-catalyzed reactions was studied.submitted by Ilabahen PatelAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2010OeBB(VLID)193164

Increasing the coulombic efficiency of glucose biofuel cell anodes by combination of redox enzymes

A highly efficient anode for glucose biofuel cells has been developed by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) and cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). These two enzymes differ in how they oxidize glucose. AmPDH oxidizes glucose at the C(2) and C(3) carbon, whereas MtCDH at the C(1) carbon. Both enzymes oxidize efficiently a number of other mono- and disaccharides. They do not react directly with oxygen and produce no H(2)O(2). Electrodes were prepared by embedding (i) only AmPDH (in order to study this enzyme separately) and (ii) a mixture of AmPDH and MtCDH in an Os redox polymer hydrogel. Single-walled carbon nanotubes (SWCNTs) were added in order to enhance the current density. The electrodes were investigated with linear sweep and cyclic voltammetry in the presence of different substrates at physiological conditions. The electrochemical measurements revealed that the product of one enzyme can serve as a substrate for the other. In addition, a kinetic pathway analysis was performed by spectrophotometric measurements leading to the conclusion that up to six electrons can be gained from one glucose molecule through a combination of AmPDH and MtCDH. Hence, the combination of redox enzymes can lead to an enzymatic biofuel cell anode with an increased coulombic efficiency far beyond the usual yields of two electrons per substrate molecule

Wiring of pyranose dehydrogenase with osmium polymers of different redox potentials.

In this study, five different flexible osmium based redox polymers were investigated for their ability to efficiently "wire" the oxidoreductase pyranose dehydrogenase (PDH, EC 1.1.99.29) from Agaricus meleagris, on graphite electrodes for possible applications in biofuel cells. A series of newly synthesised osmium based redox polymers covering the potential range between -270 and +160mV vs. Ag|AgCl (0.1M KCl) was used. The performance of the redox polymers for enzyme wiring was investigated using glucose as substrate. The optimal operational conditions such as pH and potential were investigated

Treatment of wood fbres with laccases: improved hardboard properties through phenolic oligomerization

International audienceLaccase-treated wood fibres were tested for small-scale wet-process manufacture of hardboards. Two laccases of distinctredox potentials, one from Pycnoporus cinnabarinus and one from Myceliophthora thermophila, were compared in terms oftheir effect on the physical–chemical properties of the treated fibres and hardboards. Wood fibres were produced from Norway spruce (Picea abies) by thermomechanical pulping. The thermomechanical pulp was treated with each laccase in parallel, in the presence or absence of the synthetic laccase mediator 1-hydroxybenzotriazole (HBT). High-performance size-exclusion chromatography revealed that the ethanolic extractable phenolic compounds in the fibres underwent oligomerization upon enzymatic treatment, and that the extent of oligomerization was dependent on the enzyme source and concentration and on the presence or absence of mediator. Lower lignin oligomerization levels led to higher (up to two-fold) fibre internal bonding, whereas higher lignin oligomerization levels led to higher fibre hydrophobisation. X-ray photoelectron spectroscopy revealed a significant change in surface lignin content. These results demonstrate pre-treatment of spruce fibres with laccase–mediator systems prior to hot processing can improve the mechanical resistance of hardboards while using lower amounts of enzyme

Treatment of wood fibres with laccases: improved hardboard properties through phenolic oligomerization

International audienceLaccase-treated wood fibres were tested for small-scale wet-process manufacture of hardboards. Two laccases of distinct redox potentials, one from Pycnoporus cinnabarinus and one from Myceliophthora thermophila, were compared in terms of their effect on the physical-chemical properties of the treated fibres and hardboards. Wood fibres were produced from Norway spruce (Picea abies) by thermomechanical pulping. The thermomechanical pulp was treated with each laccase in parallel, in the presence or absence of the synthetic laccase mediator 1-hydroxybenzotriazole (HBT). High-performance size-exclusion chromatography revealed that the ethanolic extractable phenolic compounds in the fibres underwent oligomerization upon enzymatic treatment, and that the extent of oligomerization was dependent on the enzyme source and concentration and on the presence or absence of mediator. Lower lignin oligomerization levels led to higher (up to twofold) fibre internal bonding, whereas higher lignin oligomerization levels led to higher fibre hydrophobisation. X-ray photoelectron spectroscopy revealed a significant change in surface lignin content. These results demonstrate pre-treatment of spruce fibres with laccase-mediator systems prior to hot processing can improve the mechanical resistance of hardboards while using lower amounts of enzyme

MOESM1 of Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6

Additional file 1. Production, purification and characterization of lytic polysaccharide monooxygenases secreted by Pestalotiopsis sp. NCi6

Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos