Abstract The significant root growth inhibition in zinc-treated soybean (Glycine max) seedling correlated with the increase of PODs and laccases activity. The increase of the activities of PODs (pI 8.3, pI 7.7, pI 4.8, pI 4.4 and pI 3.7) and laccases (pI 9.4, pI 8.3, pI 7.4, and pI 3.7) are accompanied by a rise of lignin contents in Zn-treated tissues. Our results suggested that laccases work during the early stage of Zn treatment. Laccases and peroxidases work cooperatively in lignin synthesis when the time of Zn treatment was prolonged.

Thesis (doctoral)--Helsinki University of Technology, 2008. / Includes bibliographical references. Also available on the World Wide Web.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES

Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.

No description available.

The present study aimed to investigate the methane yield from anaerobic digestion of a lignocellulosic substrate subjected to different pretreatments. The lignocellulosic forest residues materials were milled and then pretreated with the organic solvent NMMO (N-Methylmorpholine N-oxide) and/or the lignolytic enzymes laccase and versatile peroxidase at a dosage of 60 U g-1 total solids (TS) substrate. The amount of methane produced was studied in a biomethane potential assay with inocula from a thermophilic biogas reactor treating municipal waste. All samples were run in triplicates. Due to the large amount of samples, two biomethane potential assays were conducted: series 10 & 20 and series 30 & 40. The gas production results show that NMMO-treated forest residues yielded 130 NmL CH4 g-1 volatile solids (VS) substrate and the untreated forest residues yielded 95 NmL CH4 g-1 VS substrate for series 10 & 20. For series 30 & 40, both untreated and NMMO-treated forest residues yielded 140 NmL CH4 g-1 VS substrate. NMMO-treatment appears to be favourable and no advantages from the enzyme pretreatment could be seen in terms of gas yield. An analysis of the reaction fluid after the enzymatic treatment showed presence of phenols, an indication of successful lignin hydrolysis. / Studien avsåg att undersöka metanutbytet från anaerob nedbrytning med förbehandlad lignocellulosa som substrat. Lignocellulosamaterialet, i form av skogsavfall, maldes och förbehandlades därefter med det organiska lösningsmedlet NMMO (N-metylmorfolin-N-oxid) och/eller de lignolytiska enzymerna laccase och versatile peroxidas med dosen 60 U g-1 torrsubstanshalt (TS). Mängden producerad metan undersöktes i en biometanpotentialanalys med inocula från en termofil biogasreaktor, som behandlade hushållsavfall. Triplikat av varje prov användes för att öka den statistiska stabiliteten. På grund av det stora antalet prover genomfördes studien i två omgångar: Serie 10 & 20 samt serie 30 & 40. Resultaten visade att det NMMO-behandlade skogsavfallet gav 130 NmL CH4 g-1 organisk substans (VS) och det obehandlade skogsavfallet gav 95 NmL CH4 g-1 VS i serie 10 & 20. Både obehandlat och NMMO- behandlat skogsavfall gav 140 NmL CH4 g-1 VS i serie 30 & 40. Förbehandling med NMMO verkar vara fördelaktig medan enzymbehandling endast resulterade i en smärre ökning av gasproduktionen. En analys av vätskan efter enzymbehandlingen visade förekomst av fenoler, vilket visar på en lyckad ligninnedbrytning.

Les laccases sont des enzymes de type oxydase, réalisant de manière efficace la réduction du dioxygène en eau. Des études réalisées au laboratoire ont permis de montrer que l’irradiation sous atmosphère inerte d’un système de type, EDTA/[Ru(bpy)3]2+/laccase, conduisait à la photoréduction de l’enzyme via la formation d'une espèce [Ru(bpy)3]2+*. La substitution de l’EDTA par un alcène de type p-styrène sulfonate conduit également à la photoréduction de l’enzyme. L'ouverture à l'air du système permet une consommation d’oxygène concomitante à la détection par RMN de produits d’oxydation tels que l’époxyde, le diol et le p-benzaldéhyde sulfonate. L’influence de la concentration des différents partenaires, de la source d’irradiation et du pH sur l’efficacité de cette réaction a été évaluée. D’autres alcènes tels que le styrène, le cyclohéxène ou le cyclooctène sont également substrats. Le marquage isotopique en présence soit d'H218O soit d'18O2 ainsi que l’utilisation de générateurs d’espèces réactives de l’oxygène, ont permis de proposer un mécanisme majoritaire où l’espèce RuIII, photoproduite avec l'assistance de la laccase, pourrait arracher un électron du substrat qui à son tour réagirait avec le dioxygène présent dans le milieu pour conduire aux produits observés. D’autres complexes photoactivables à base de ruthénium ou de manganèse ont également été employés. Afin d’aborder le contrôle de la réactivité, le greffage covalent d’un photosensibilisateur à base de ruthénium sur une lysine unique à proximité du site d'oxydation des substrats de l'enzyme a été effectué. / Laccases are oxidases that efficiently perform the reduction of dioxygen into water. Studies in the laboratory have allowed to show that irradiation under inert atmosphere of a EDTA/[Ru(bpy)3]2+/laccase system, lead to the photoreduction of the enzyme via the irradiation of [Ru(bpy)3]2+*. The substitution of EDTA by the alkene p-styrene sulfonate results similarly in a photoreduction of the enzyme. Opening the system to air allows a dioxygen consumption with a simultaneous detection of oxidation products such as the epoxide, diol and p-benzaldehyde sulfonate detected by NMR. The influence of the concentration of the partners, the irradiation source and pH on the efficiency of the reaction was evaluated. Other alkenes such as styrene, cyclohexene and cyclooctene are also substrates. Isotopic labeling experiments in the presence of either H218O or 18O2, as well as the use of reactive oxygen species generators, allowed us to propose a main mechanism where the laccase assisted RuIII photogenerated specie would withdraw an electron from the substrate which in turn would react with dioxygen to yield the products observed. Other ruthenium and manganese photosensitizers were also used. To address the control of the reactivity, a covalent grafting of a ruthenium photosensitizer, on a unique lysine nearby the substrate oxidation site of the laccase was done.

Ce travail a pour objectif d’étudier l’effet de la diafiltration et de la polymérisation enzymatique sur l’hétérogénéité, la composition chimique et les propriétés physico-chimiques des lignosulfonates en solution. Le fractionnement par procédé membranaire a été mené par diafiltration. Les lignosulfonates ont été fractionnés en cinq fractions de différents poids moléculaire et polydispersité variant de 1400 g.mol-1 à 19500 g.mol-1 et de 1,4 à 3,5 respectivement. Les résultats indiquent que la diafiltration permet d’obtenir des fractions qui présentent des propriétés augmentées et/ou différentes du produit non fractionnés et une distribution moins hétérogène. Les fractions de poids moléculaire en poids de 2500 g.mol-1 et 4300 g.mol-1 ont la plus grande concentration en groupements hydroxyles et sulfoniques, ce qui affecte leurs propriétés, puisqu’elles présentent des activités de surface et antioxydantes supérieures aux lignosulfonates non fractionnés. La polymérisation enzymatique des lignosulfonates par la laccase a été étudiée en présence ou en absence de médiateur. La polymérisation des lignosulfonates a été constatée comme produit de leurs oxydations (SEC). Les principaux facteurs influant sur la polymérisation des lignosulfonates sont (i) une très grande concentration en lignosulfonates, (ii) l’utilisation des laccases fongiques (laccase de Trametes versicolor) avec un potentiel redox élevé et (iii) l'utilisation de l’acétosyringone ou l’acide violurique comme médiateur. L’effet du mode de conduite (discontinu, continu et semi continu) de la réaction de polymérisation des lignosulfonates a été rapporté. La comparaison des résultats des trois modes de mise en œuvre a indiqué que le mode en continu conduit à une augmentation importante du poids moléculaire (30600 Da) et à la diminution la plus importante de la polydispersité des polymères synthétisés (3,7). Ainsi, ce mode de conduite de la réaction est plus adapté pour obtenir des produits homogènes. / This work aims to study the effect of diafiltration and enzymatic polymerization on the heterogeneity, the chemical composition and physicochemical properties of lignosulfonates in solution. Membrane fractionation process was carried out by diafiltration. The lignosulfonates were fractionated into five fractions with different molecular weights and polydispersity ranging from 1400 g mol-1 to 19500 g mol-1 and from 1.4 to 3.5, respectively. The results indicate that diafiltration allows obtaining fractions which have enhanced and/or different properties from unfractionated product and a less heterogeneous distribution. Fractions with a weight average molecular weight between 2500 g mol-1 and 4300 g mol-1 have the largest concentration of hydroxyl and sulfonic groups which affect their properties, since they exhibit surface and antioxidant activities higher than unfractionated lignosulfonates. The enzymatic polymerization of lignosulfonates by laccase was studied in the presence or absence of mediator. The polymerization of lignosulfonates was observed as a product of their oxidation by SEC. The main factors influencing the polymerization of lignosulfonates are (i) a very high concentration of lignosulfonates (ii) the use of fungal laccases (laccase from Trametes versicolor) with a high redox potential (iii) the use of acetosyringone or violuric acid as mediator. The effect of the reactor mode (batch, continuous and semi continuous) of the polymerization of lignosulfonates has been reported. Comparison of the results of the three modes has shown that the continuous mode led to a significant increase in molecular weight (30600 Da) and the largest decrease of the polydispersity of the synthesized polymers (3.7). Thus, this mode of conducting the reaction is more suitable for homogeneous products.