American Journal of Biochemistry and Biotechnology 9 (4): 365-394, 2013 ISSN: 1553-3468 ©2013 Science Publication doi:10.3844/ajbbsp.2013.365.394 Published Online 9 (4) 2013 (http://www.thescipub.com/ajbb.toc) REACTION KINETICS OF VERSATILE PEROXIDASE FOR THE DEGRADATION OF LIGNIN COMPOUNDS 1Busse, N., 1D. Wagner, 2M. Kraume and 1,3,4 P. Czermak 1Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen, Germany 2Department of Chemical and Process Engineering, Technische Universität Berlin, Berlin, Germany 3Department of Chemical Engineering, Kansas State University, Manhattan KS, USA 4Faculty of Biology and Chemistry, Justus-Liebig-University of Giessen, Giessen, Germany Received 2013-06-24, Revised 2013-08-27; Accepted 2013-08-28 ABSTRACT The H 2O2-dependent degradation of adlerol by a crude versatile peroxidase from Bjerkandera adusta , a new ligninolytic enzyme, was investigated. Adlerol (1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3- propanediol)) is a non-phenolic β-O-4 dimer whose structural architecture represents the most abundant unit (50-65%) of the valuable renewable biopolymer lignin. Lignin removel plays a key role in utilizing lignocellulosic biomass in biorefineries. Steady-state analyses in the µL scale showed saturation kinetics for both, H 2O2 and adlerol with quite sensitive response to H 2O2. This was characterized through slow transient states (lag phases) prior steady-state and were enhanced by increasing H 2O2 concentration. The major reason for such phenomena was found to be an accumulation of compound III (E III ) via reaction of II 0 compound II (E ) with H 2O2; instead with adlerol to the enzyme’s ground state E in order to restart another catalytic cycle. As result, the enzyme deviated from its normal catalytic cycle. A corresponding threshold was determined at ≥ 50 µM H 2O2 and an adlerol to H 2O2 ratio of 15:1 for the given conditions. Furthermore, EIII did not represent a catalytical dead-end intermediate as it is generally described. By an additional decrease of the adlerol to H 2O2 ratio of ca. 3 at the latest, considerable irreversible enzyme deactivations III occurred promoted through reaction of E with H 2O2. At a mL scale deactivation kinetics by H 2O2 were further examined in dependence on adlerol presence. The course followed a time-dependent irreversible deactivation (two step mechanism) and was diminished in the presence of adlerol. The deactivation could be sufficiently described by an equation similar to the Michaelis-Menten type, competitive inhibited by adlerol. S1 S2 app app Finally, first estimates of the kinetic parameters v max , K m (S 1: H 2O2), K m (S 2: adlerol), k i and K i were made. Moreover, the peroxidase reaction mechanism was reviewed and recommendations are given preventing permature enzyme losses. Keywords: Versatile Peroxidase (VP), Lignin Model Compound (LMC), Steady-State Kinetics, Slow Transient States, Deactivation 1. INTRODUCTION (Sánchez, 2009)) of vascular plants (Wong, 2009), i.e. wood or straw. Lignocellulose is the most abundant Lignin is the only naturally synthesized aromatic renewable organic raw material on earth, and based on its biopolymer (Dashtban et al ., 2010). Together with the main constituents of high value, with an annually polysaccharides cellulose and hemicellulose, lignin forms production of many billion tons (Villas-Bôas et al ., 2002). a complex lignin-carbohydrate network, well-known as Lignocellulosic material is of great interest as lignocellulose, the major compound (around 50% feedstock for bio-based industrial products and Corresponding Author: Czermak, P., Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstrasse 14, 35390 Giessen, Germany Science Publications 365 AJBB Busse, N. et al. / American Journal of Biochemistry and Biotechnology 9 (4): 365-394, 2013 biorefineries. Especially wood is focused on biofuel still attracted double attention, “as model enzymes and production of second generation due to several reasons: as a source of industrial/environmental biocatalysts”, it is inexpensive, available in large amounts (Stöcker, within the last years (Ruiz-Dueñas et al ., 2009a). 2008) (3,300 m 3 felled or otherwise removed roundwood Nonetheless, research work for delignification per year worldwide (Ek et al ., 2009)) and CO 2 neutral, concerning VPs are seldom, in particular kinetic studies thus contributing to the reduction of greenhouse gas with non-phenolic lignin model dimers (lignin model emissions (Lange, 2007). Furthermore, wood is rich in compounds (LMC)) containing a β-O-4 linkage. These both carbohydrates cellulose (40-50%) and hemicellulose components are the most frequent structural units in (24-35%) and in lignin ranging from 18% up to 35% lignin biopolymers ranging from 50% up to 65% (Howard et al., 2003) offering a variety of products (Adler, 1977). Moreover, the cleavage of such LMC is besides the biofuels. Nevertheless, much research and a substantial indicator for enzyme catalyzed lignin development is still needed for utilizing lignocellulosic depolymerization processes (Tien, 1987; Wong, 2009). biomass (Kamm et al ., 2008), such as wood, efficiently. β-O-4 dimers are therefore suitable for studying The lignin removal plays a considerable role in this delignification mechanisms in a more or less simplified connection, since this step has to be addressed before manner as it will be seen later in the text. polysaccharide bioconversion can be tackled. This process The present work focuses on kinetic investigations is hampered due to the lignin complexity (polydisperse 3D based on the degradation of the β-O-4 lignin model construct (Martínez et al ., 2005) causing difficulties in dimer adlerol by a new (Jena Bioscience, 2010) crude analysis) (Eriksson et al ., 1990) and as generally known, versatile peroxidase (VP) from Bjerkandera adusta as a its enormous recalcitrance to degradation. ligninolytic model peroxidase. All studies were H O -dependent ligninolytic heme peroxidases 2 2 conducted without the need of any mediator (e.g. the (POXs) and O -dependent laccases (Lac, EC 1.10.3.2), 2 non-phenolic monomer veratryl alcohol (VA) or Mn 2+ ) extracellular enzymes from Basidiomycetous white-rot or surfactant like Tween. In addition, the pH optimum of fungi (thus, Class II peroxidases), are the most efficient reaction was determined and enzyme deactivation by lignin degraders in nature (Kirk and Farrell, 1987). H O depending on adlerol amount was also examined. Compared to Lac, POXs have high redox-potentials (a good 2 2 Ligninolytic peroxidase assays are generally known overview is given by Torres and Ayala (2010)) receiving to be more complex and difficult to optimize “high interest as industrial biocatalysts” (Martínez, 2007), (Sinsabaugh, 2010; German et al ., 2011). Prior starting consequently. However, POXs have not yet been with the experimental description, the paper will implemented at large scale (Torres and Ayala, 2010). therefore be continued by a compact review of the POX The POXs include lignin peroxidases or ligninases reaction mechanism providing basic information for a (LiP, EC 1.11.1.14), manganese peroxidases (MnP, EC better understanding and in order to differentiate pure 1.11.1.13), and versatile peroxidases (VP, EC enzymatic from non-enzymatic reactions. For this 1.11.1.16), also known as hybrid peroxidases or lignin- purpose most background knowledge was received from manganese peroxidases, since those enzymes combine authors examining horseradish peroxidase (HRP) activity LiP and MnP catalytic properties. While LiP is from plants (Class III peroxidases) on appropriate probably the most famous biocatalyst studying phenolic substrates as well as LiP using mainly VA as delignification processes, VP is a relative new POX and substrate; β-O-4 lignin model dimers are also involved as was first interpreted as a MnP in 1996 by Martínez et LiP substrates. VA is a second metabolite of al . and in 2000 by Giardina et al . (Ruiz-Dueñas et al ., Phanerochaete chrysosporium and other white-rot fungi 2009a). In contrast to LiP and MnP, VPs are capable to (Schoemaker and Piontek, 1996) and thus the mediator direct degradation/oxidation of a broad spectrum of of choice for LiP enabling phenolic compound oxidation. persistent substrates (e.g. (non-) phenolic lignin Both, LiP and HRP are similar in many characteristics compounds, dyes, such as RB5 and others) meaning (e.g. the enzyme oxidation states as depicted in Fig. 1-3) without the requirement of mediators (Pogni et al ., (Schoemaker, 1990) and are so far the most popular and 2005); an important feature for biotechnological best studied peroxidases. Moreover, it will be expected applications. Although VPs are limited available (a that the used VP should be in compliance with a LiP major drawback for industrial utilization), they have from P. chrysosporium at least for ca. 60% Science Publications 366 AJBB Busse, N. et al. / American Journal of Biochemistry and Biotechnology 9 (4): 365-394, 2013 (Ruiz-Dueñas et al ., 1999). Consequently, LiP reactions (S, the actual electron donor “preferentially electron-rich are more concerned for comparison throughout this study aromatic compounds” (Lundell et al ., 1993a)). Both than those for MnP. Additionally, MnP is strongly Mn 2+ - reaction steps are pH-dependent (Pérez-Boada et al ., dependent resulting in Mn-mediated degradation 2005; Wong, 2009) and causing release of radical