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Doctor of Philosophy / Department of Chemistry / Ping Li / Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which have received much attention due to their potential applications in degradation of lignin and anthraquinone dyes. In this research, studies of two types of DyPs are carried out and reported in the following three sections. The first section includes the identification and characterization of class-A TcDyP from Thermomonospora curvata, a thermophilic actinomycete found in composted manure. The TcDyP was found to be highly active toward a wide range of substrates including phenolic lignin model compounds. Transient- and steady state- kinetics involving wild-type (wt) and mutant TcDyPs revealed that Asp220 and Arg327 are essential for compound I formation and reduction of compound II to resting state is the rate-limiting step. Additionally, replacement of His312 and Arg327 shifted the redox potential (E°′) to a more negative value. In the second section, the residues involved in the radical generation and substrate oxidation were explored. TcDyP contains 7 tryptophans and 3 tyrosines, which are the likely candidates of protein radicals and substrate oxidation sites. Crystal structure of TcDyP solved at 1.75Å revealed Trp263, Trp376 and Tyr332 as surface-exposed protein radical sites. Further studies using site-directed mutagenesis, steady-state and stopped-flow kinetics determined that the Trp263 is also one of the surface-exposed substrate oxidation sites. The Trp376 was characterized as the residue essential for covalent crosslinking of the enzyme units and an off-pathway electron sink. The highly conserved Tyr332 was found to be unimportant for substrate oxidation due to its extremely narrow surface exposure. The final section involves mechanistic study of a class-B DyP from Enterobacter lignolyticus (ElDyP), a bacterium capable of growing on lignin anaerobically. The crystal structure of ElDyP revealed the presence of two heme access channels measuring at ~3.0 and 8.0 Å in diameter and a water molecule as the sixth ligand to the heme center. Bisubstrate Ping-Pong mechanism was found operational in the catalytic cycle of ElDyP, in which conformational change of the enzyme resting state was proposed as the final step and the rate limiting step in the presence of ABTS. Microscopic events leading to Compound I formation was analyzed using D₂O₂. A kinetic isotope effect (KIE) of 2.4 at pD 3.5 suggested that Compound 0 is formed initially with protonation/deprotonation as the rate-limiting step. Compound I was directly reduced to the enzyme resting state via a 2-electron process, for which the rate increases as the pH decreases. Based on viscosity effect and solvent KIE (sKIE) with the reducing substrate, aquo release was found to be mechanistically important. Distal aspartate was proposed as the key residue that modulates the acidic pH optimum in Compound I reduction. These findings will pave the way for engineering DyPs for their applications in the degradation of lignin and synthetic dyes.

As the world moves forward to the development of biorefineries, the interest to replace chemicals and materials derived from petroleum is increasing exponentially. Lignin is a macromolecular by-product derived from the wood pulping industry, mainly used for heating purposes. The development of new processes to produce high value-added lignin products such as multifunctional aromatic chemicals and high-tech carbon materials are required to fulfill the needs for biorenewable feedstocks. Such processes are likely to include selective oxidation catalysis. The aim of this Thesis is to advance the state-of-the-art for the oxidation of lignin models and lignin extracts using homogeneous catalysts based on vanadium, an inexpensive and abundant transition metal, using air as the only oxidant. Lignin models containing the most important features of lignin (e.g., β-O-4 and β-1 linkages) were initially used to assess the catalytic potential. Previously reported (HQ)2V(O)(OiPr) and (dipic)V(O)(OiPr) catalysts (dipic = dipicolinate and HQ = 8-oxyquinoline) displayed different selectivity for C-H, C-O and C-C bond cleavage upon varying of the solvent, the lignin model or the catalyst. Moreover, these catalysts cleave the C-H bond of secondary alcohols through a two-electron oxidation process and the C-C bond cleavage of the oxidation product ketone in the presence of exogenous base. Several amine bis(phenolate) oxovanadium(V) catalysts were synthesized and fully characterized, and demonstrated very good activity for the oxidation of lignin models and the depolymerization of organosolv lignin. These new catalysts overcome the need for added base, display higher reaction rates of oxidation, and improve the selectivity for the disassembly of lignin models. The different selectivities involving C-H vs. C-O vs. C-C bond cleavage are discussed together with a novel redox-neutral C-C bond cleavage of lignin model 1,2-diphenyl-2-methoxyethanol. The oxovanadium(V) catalysts, along with a metal-free variant and other transition metal catalysts, were employed to assess their performance for the oxidation and depolymerization of organosolv lignin. Although most catalysts oxidized the lignin extracts, the oxovanadium(V) complexes demonstrated the highest degree of lignin depolymerization as shown by Gel Permeation Chromatography (GPC), quantitative-Heteronuclear Single Quantum Correlation (q-HSQC) and quantitative 31P NMR spectroscopy of derived phosphite esters. In a complementary study, oxovanadium(V) catalysts also established their utility for the valorization of cellulose-derived substrates (e.g., diols). Two trialkoxyamine oxovanadium(V) complexes bearing a triethoxyamine and tris[2-(3,5-di-tert-butyl-phenoxy)methyl]amine ligand respectively, selectively cleaved the C-C bond in 1,2-diols with excellent rates and using air as the only oxidant. In a stoichiometric investigation of this reaction, it was determined that the transformation proceeds through an unusual direct oxidative two-electron cleavage of diols, affording a non-oxo monometallic V(III) intermediate. DFT calculations support a single-site proton-coupled electron-transfer of the hydroxyl hydrogen to the V oxo orbital. In summary, this Thesis describes new developments in vanadium catalysis such as mechanistic implications and catalyst optimization for the valorization of lignocellulosic biomass utilizing air as an oxidant.

Lignin is the second most abundant biopolymer on Earth. It is an amorphous, cross-linked, aromatic polymer composed of phenylpropanoid units. There has been an ever growing interest in the biodegradation of this complex polymer for the last 30 years. White-rot fungi have been found to be an important lignin degraders in the natural environment. With the discovery of two groups of hemoprotein enzymes, lignin peroxidases and manganese(II)-dependent peroxidases, from the lignin degrading culture of a white-rot fungus, Phanerochaete chrysosporium, rapid progress has been made in understanding the mechanism of lignin biodegradation. Synthetic metaUoporphyrins, the iron(III) and manganese(III) complexes of meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrin (TDCSPPFeCl and TDCSPPMnCl) and meso-tetra(2,6-dichloro-3-sulfonatophenyl)-B-octachloroporphyrin (Cl₁₆TSPPFeCl and Cl₁₆TSPPMnCl), were used in this study to mimic the functions of the "lignin degrading" enzymes. Factors affecting the catalytic activities of these biomimetic catalysts were studied. TDCSPPFeCl could closely mimic lignin peroxidase in the degradation of a number of lignin model compounds, including veratryl alcohol, B-l, B-O-4, B-5, 5-5' biphenyl, phenylpropane, and phenylpropene model compounds. The reactions catalyzed by TDCSPPFeCl include benzyl alcohol oxidation, C[formula omitted],-C[formula omitted] side chain cleavage, demethoxylation, aromatic ring cleavage, benzylic methylene hydroxylation, and C[formula omitted]-C[formula omitted] double bond hydroxylation (glycol formation). Novel solvent incorporated compounds isolated from the oxidation of veratryl alcohol give insights about the site of attack of substrate cation radical by solvent molecules. The isolation of a solvent incorporated product from the oxidation of a phenylpropene model compound suggests a cation radical mechanism for the oxidation of this lignin substructure. The formation of a number of direct aromatic ring cleavage products during the oxidation of some model compounds supports the previously proposed mechanism of aromatic ring cleavage. TDCSPPFeCl was also able to catalyze the oxidation of environmental pollutants such as pyrene and 2,4,6-trichlorophenol. Veratryl alcohol and manganese(II)-complexes have been suggested to function as redox mediators for lignin biodegradation. Evidence has been provided to demonstrate their mediating power during electrochemical and biomimetic degradation of lignin model compounds. In addition to the mechanistic information obtained, the successful oxidation of the model compounds suggests that metalloporphyrins can be important catalysts for the pulp and paper industry and for pollution control. / Science, Faculty of / Chemistry, Department of / Graduate

Lack of a selective holocellulose isolation procedure and the problem of limited material have long frustrated the attempts of wood scientists to accurately measure and describe carbohydrate yields within coniferous growth zones. A new method has been devised for micro-cellulose determination, Alpha-(formula omitted) cellulose yield maybe quantitatively estimated as the corrected yield of nitrated wood meal. Three - O.1 g wood meal samples provide a statistically reliable determination. A major limitation of the new technique, however, is that it cannot be applied to all woods. Sixty positions within ten increments representing five Canadian coniferous woods of different genera were examined and intra-incremental patterns were constructed. Anova and Duncan's test showed latewood (formula omitted) -cellulose yield to be greater than that of earlywood by a highly significant degree. Alpha-cellulose content throughout mature growth zones was estimated reliably by linear correlation or, more accurately, by logarithmic transformation used in a recent mathematical model. The successful application of the latter is its first reported use describing the non-linear behaviour of a wood chemical property across a coniferous increment. These patterns showed relationship of the long-chain carbohydrate fraction to seasonal development within coniferous growth zones. In addition, six of the ten patterns demonstrated new chemical evidence pertaining to a physiologically significant phenomenon in earlywood. Therein, minimum (formula omitted) -cellulose yield occurs at considerable cellular depth following cambial reactivation in the growing season. First-formed earlywood appears to retain some similarity at the chemical level of organization to last-formed tissues of the preceding season. Later-formed earlywood (i. e. , from the present year) does not appear to retain such similarity. From earlier work of this laboratory, lignification patterns were described for the same materials, using ultraviolet measurements on acetyl bromide-acetic acid digestion products of two wood meal samples. Examination of (formula omitted) -cellulose and lignification patterns provided evidence for their mutually exclusive behaviour. For the ten increments studied, the (formula omitted) -cellulose estimate (x̅ = 45.9 + 2.0%) was the exact complement of lignification (x̅ = 27. 4±1. 9%) at all positions but one. The linear regression for data from all increments was highly significant ( r = - 0.785). In addition, micro (formula omitted) -cellulose and micro lignin values, when combined, showed a definite tendency to cluster about a central value (x̅ = 73. 4 ± 1. 2%) suggesting that certain species require a common, critical measure of high molecular weight material. Dispersion about combined lignin and (formula omitted) -cellulose estimates was significantly less than about either of their individual means. This suggests much closer physiological control over the combination of these chemical entities, indicating that tree physiology is oriented more towards the finished bio synthetic product than towards the individual components involved in such a system. As a means of measuring successful nitrocellulose preparation, intrinsic viscosity (formula omitted) was used to indicate presence or absence of extensive degradation. Because of the highly variable at each position tested, no consistent trends in chain length were found across growth zones. However, in four increments, significant differences in (formula omitted) throughout the earlywood provided further evidence of two earlywood types. / Forestry, Faculty of / Graduate

In order to investigate the relative merits of a selective oxidant for the degradation of natural polymers, humic acid extracts from three Alberta soil sites and three British Columbia soil sites, and two Kraft's prepared wood lignins were oxidized with 1.6 N NaOCl at room temperature (23°C) for 5 days. The oxidation products included CO₂, highly volatile acids and organic solvent soluble (OSS) products with the relative size of each fraction determined by carbon content. OSS products were characterized by Infrared (IR), Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) techniques and identified after methylation and separation by Gas Liquid Chromatography (GLC) methods involving co-injecting authentic compounds and matching elution time and temperature of some of the components with that of the authentic compounds. Proceeding from the known chemistry of the NaOCl reaction, the CO₂ and highly volatile acid products could only be derived from the aliphatic chain or saturated ring components of humic acids or lignin and represented 66 to 82% of the products assuming no destruction of aromatic structure. Benzene carboxylic acid products were derived from the aromatic component of the starting materials. Estimates of the degree of aromaticity of the starting materials, using GLC and potentiometric titration data, were substantially less than those calculated from proposed model humic acid and lignin structures in the soils literature. An unidentified oily component was found in the oxidation products of two of the humic acid preparations. The results of this study indicated humic acid and lignin are composed of a mixed aliphatic-aromatic compound system. The relatively more mature humic acid preparations were found to be of greater aromaticity than the less mature samples. It was found 2 that NaOCl was not totally selective in differentiating Sp² from Sp³ carbon hybrids; as a result the total discrimination between aliphatic and aromatic structures was not a safe assumption. It is postulated that aromatic ring opening may occur at sites of hydroxyl group substitution on the ring structures resulting in an apparent less aromaticity and the generation / Land and Food Systems, Faculty of / Graduate

C-lignin (caffeyl-lignin) is a novel linear lignin polymer found in the seed coats of several non-crop plants, notably Vanilla planifolia (Vanilla), Jatropha Curcas (Jatropha), and Cleome hassleriana (Cleome). C-lignin has several advantages over normal G/S-lignin, found in the majority of lignocellulosic biomass, for valorization in the context of bioprocessing: less cross-linking to cell wall polysaccharides (less recalcitrant biomass), ordered linkages between monomers (homogeneous polymer), and no branching points (linear polymer). These properties make C-lignin an attractive replacement for native lignin in lignocellulosic biomass crops. The seed coats of Cleome hassleriana (Cleome) synthesize G-lignin during early seed maturation, then switch to synthesis of C-lignin during late maturation. This switch to C-lignin in Cleome seed coats is accompanied by loss of caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) and caffeic acid 3-O-methyltransferase (COMT) activities, along with changes in transcript abundance of several lignin related genes. The focus of this research thesis is to understand the biochemical changes leading to C-lignin deposition in Cleome hassleriana seed coats, and to explore the ability of Arabidopsis thaliana seedlings to polymerize caffeyl alcohol to C-lignin. In this thesis, candidate transcripts were implicated in C-lignin biosynthesis by differential gene expression analysis of transcripts in seed coat tissues at 8-18 days after pollination (DAP) and in non-seed coat tissues. Three candidate genes were selected for recombinant expression and their in vitro kinetic properties were measured with potential substrates. Of the three candidates, a cinnamyl alcohol dehydrogenase (ChCAD5) was found to have high transcript levels during C-lignin formation and have a novel preference for converting caffealdehyde to caffeyl alcohol, the precursor of C-lignin. To determine if accumulation of caffeyl alcohol is sufficient for polymerization of C-lignin, Arabidopsis seedlings grown in a xylem induction system were supplied caffeyl alcohol. Polymerization of caffeyl alcohol was not found to occur in this Arabidopsis system, suggesting the need for a C-lignin specific polymerization mechanism.