FY 2008 Project Quarterly Report BUSC 08. Enzymatic Polymerization. Research Team: Gisela Buschle Diller (PI), Shuying Long, Zhiwei Xie

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1 FY 2008 Project Quarterly Report BUSC 08 Enzymatic Polymerization Research Team: Gisela Buschle Diller (PI), Shuying Long, Zhiwei Xie Background In the light of rising cost of petro chemicals and concerns regarding the environment, alternative polymer synthesis routes are gaining increased importance that incorporate greener and more energy efficient approaches and the use of less toxic chemicals. ne possible venue is the application of highly specific enzymes in place of conventional catalysts that support a chemical reaction under mild and environmentally friendly conditions. These biocatalysts are highly selective and have extraordinary catalytic power under optimum process conditions. Enzymes are proteins that assist in the conversion of a chemical reaction by enhancing the reaction rate. Based upon the type of reaction they catalyze, they have been classified into six major categories: oxidoreductases, transferases, hydrolases, ligases, lyases, and isomerases. xidoreductases have shown great promise for oxidative polymerizations of small aromatic compounds as well as for other applications. orseradish peroxidase, for example, has been repeatedly used for bioremediation in the presence of hydrogen peroxide [Wu et al., 1999]. orseradish (RP) and soybean peroxidase (SBP) allowed the successful production of polyphenol resins that very well could replace conventionally polymerized novolac and resol resins without the involvement of formaldehyde [Ikeda et al., 1996]. The same research group also used laccase, an oxidoreductase that has shown to be involved in the biodegradation of lignin as well as in lignin s biosynthesis. By way of one electron transfer reactions polyphenols could be synthesized and a product polymer obtained with very similar structure for both RP and SBP. Enzymatic reactions generally only work well with water as the solvent. owever, the reaction with any of the oxidoreductases with low molecular weight phenols could be achieved in aqueous organic solution with a mixed acetone/acetate buffer. rvetto et al. (2001) achieved 40% conversion of phenols and near 70% conversion of aniline in the presence of RP and hydrogen peroxide. The research group suggested to apply RP to remove these toxic substances from waste effluent with the simultaneous benefit of generating polymeric products as a side effect. Polyphenol and polyaniline could then be used for further applications. It could be argued that without the formation of a valuable polymeric product that could be harvested, pure RP is currently too costly for wastewater treatment. A crude white rot fungus might be used instead with reasonable results for effluent treatment. Soybean peroxidase has also been explored, which is contained in the shells of soybeans and as such a waste product of the food industry itself, for a similar approach to industrial effluent clean up [Ghiuoreliotis and icell, 1999]. The research group however notes that small enzymatically formed products that are water soluble remain in the treated water and might present a problem. Laccases obtained from various microorganisms have been investigated for their capability of polymerizing guaiacol, 1 naphthol, hydroquinone, vanillic acid and other small substrates [Coll et al.,

2 1993]. The initial reaction proceeds via a one electron transfer (or hydrogen transfer) at the active site of the enzyme (see Figure 1), followed by further enzymatic or chemical reactions of the products: 2 laccase 2 non-enzymatic 1/2 2 2 Scheme 1: ne electron transfer in laccase oxidations. Radicals formed from substrates in the process might be able to couple to oligomers. The redox potential of a specific reactive center in an oxidoreductase strongly depends on its origin. It has been found that substrate oxidation occurs at the type I copper center which is also responsible for the blue color of these multi copper proteins due to the absorbance band of the cysteine bond at 600 nm. Types 2 and 3 form a trinuclear cluster and are thought to be responsible for the reduction of molecular oxygen and the release of water (Figure 2). Reactive radicals such as phenoxy are formed during the reduction of the substrates (substituted phenols, anilines, etc., Figure 3). They subsequently react to form oligomeric and polymeric products with covalent C C, C, and C bonds. Some of these products further proceed via non enzymatic cross linking reactions to polymeric networks or moderate molecular weight. Met S Type 1 Amino Acid Type 3 Type 3 Type 2 Figure 1: Active site of blue copper oxidoreductases (laccase B. subtilis).

3 Tyrosinases and laccases were found to catalyze phenols without the need for any cofactors. A binuclear containing active site (type 3 bound to 6 histidine residues) was shown to be responsible for o hydroxylation of monophenols to o diphenols and for oxidation of o diphenols to o ketones (Claus and Decker, 2006). 4 A, laccase 4 A 2 2 laccase(red) peroxide intermediate 2 Figure 2: Simplified reaction mechanism according to Wesenberg et al. (2003) Figure 3: Possible mechanisms for oxidative polymerization of small aromatic phenols (Shleev et al., 2006).

4 The fundamental research background for this current project is based on the reaction of laccases with small fragments of colorants as substrates to create colorant compounds (Stephen, 2007). Waterinsoluble colorants were incorporated into films made from natural compounds, such as carboxymethylcellulose and chitosan, or synthetic compounds, such as poly(vinyl alcohol); water soluble ones were used like dyes for natural fibers (cotton, linen, wool) for light, but washfast shades. It was found that the colorants formed faster and with more intense hue if lignin was present during the polymerization reaction. This observation was not surprising, because in earlier research pulp fibers were enzymatically modified to be hydrophobically coated by reaction of small ligninic substrate compounds with a laccase (Kenealy et al.,2003). The redox potentials of the screened laccases generally were sufficiently high for oxidation of o and p phenols, amino and polyphenols, and polyamides without the need for an additional mediator. The formed polymeric materials can serve as binding adhesive in many different applications in the forest products industry, thus lowering the demand for petrochemical resins. As part of that project a large number of ligninic constituents had been screened for their suitability to function as substrates. It was found that resorcinol, guaiacol and vanillic acid and others yielded such hydrophobic surfaces on pulp fibers which were orange, brown and dark purple in shade, respectively, under enzymatic catalysis. owever, only resorcinol based products were water soluble and could be isolated as a versatile dye. Experimental approach Most of the polymerization reactions documented in the literature have been performed with oxidoreductases especially extracted and purified from specific microorganisms in the respective research laboratories. These enzymes are prohibitively expensive for a larger scale process. Previous research in our laboratories has been carried out with an experimental laccase (ovozymes) with very promising results. owever, if the enzymatic oxidation of phenolic aromatics is to be used more broadly at reasonable cost of the enzyme, it is necessary to achieve equally good results with commercially available oxidoreductases. Thus, in the first phase of the current approach, various types of commercial enzymes will be screened for their suitability. Laccase from various microbial sources are available, as are horseradish peroxidases. The first series of experiments is performed with laccases from Trametes versicolor which are commercially obtainable at a somewhat reasonable price. Resorcinol had worked exceptionally well as a substrate for both colorant formation and as coating precursor. In this project it will serve as the control substrate and as an indicator whether the selected laccases possess a sufficiently high redox potential for the planned biocatalyzed coupling reactions. The goal of the first phase will be to polymerize simple compounds that are natural lignin precursors. It will be investigated whether auxiliary compounds, such as tannin or soluble lignin could be beneficial for the enzymatic process. References Claus,. and M. Decker, (2006), System. Appl. Microbiol. 29, Coll, P.M., Ferandez Abalos, J.M., Villanueva, J.R., Santamaria, R., Perez, P., (1993), Applied Environ. Microbiol. 59, 2607.

5 rvetto,.r., D. Figlas, A. Brandolin, S. B. Saidman,. Rueda, M. L. Ferreira, (2006), Biochem. Eng. J. 29, 191. Ghioureliotis, M., J. A. icell, (1999), Enzyme Microbiol. Technol. 25, 185. Ikeda, R., J. Sugihara,. Uyama, and S. Kobayashi, (1996), Macromolecules 29, W. Kenealy, J. Klungness, M. Tshabalala, E. orn, M. Akhtar, R. Gleisner,. Zulaica Villagomez, G. Buschle Diller, Modification of Lignocellulosic Materials by Laccase, TAPPI Fall Techn. Conf., Engineering, Pulping & PCE&I, Chicago, IL, ct , 2003 Shleev, S. P. Persson, G. Shumakovich, Y. Mazhugo, A. Yaropolov, T. Ruzgas, L. Gorton, (2006), Enzyme Microbiol. Technol. 39, 841. Stephen, R., Enzymatic Formation of Colorants, MS Thesis, Auburn University, Wesenberg, D. I. Kyriakides, S.. Agathos (2003), Biotechn. Adv. 22, 161. Wu, Y., Tailor, K.E., Biswas,., Bewra, J.K., (1999), J. Chem. Technol. Biotechnol. 74, 519.

PHENOLIC POLYMERIZATION USING Fe III -TAML

PHENOLIC POLYMERIZATION USING Fe III -TAML K. Entwistle*, D. Tshudy*, and T. Collins** *Gordon College, Department of Chemistry, Wenham, MA 01984 **Institute for Green Science, Carnegie Mellon University,