ISOLATION PARTIAL PURIFICATION AND STABILITY STUDIES OF MANGANESE PEROXIDASE FROM WHITE ROT BASODIOMYCETES, PLEUROTUS TUBER-REGIUM

ABSTRACT

Manganese peroxidase (MnP) enzyme is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter- decomposing fungi. Multiple forms of this glycosylated haeme protein with molecular weight normally at

40 to 50 kDa are secreted by ligninolytic fungi into their micro-environment. In the present study, a ligninolytic fungus was able to produce manganese peroxidase. The organism was identified as Pleurotus tuber-regium. Parameter such as pH,  temperature, carbon source, nitrogen source wereassayed and characterized for better production of manganese peroxidase. The mass production of the enzyme was carried out using optimized conditions and the activity and specific activity was found to be 1.50U/ml and 0.76U/mg, respectively. Seventy percent ammonium sulphate precipitation was found suitable to precipitate protein with higher peroxidase activity. After ammonium sulphate precipitation, the specific activity was found to be 0.77U/mg. After gel filtration, two peaks were obtained with specific activities

2.75U/mg and 2.09U/mg specifically for peak A and B. The optimum pH for the two peaks were 4.5 and

5.0, respectively with optimum temperature of 40oC. The pH and temperature stability studies showed that the purified enzyme had a residual activity within the range of 80 to 70% after pre-incubation for 120 min  for pH of 4.5 and temperature of 40oC.The kinetic parameters, maximum velocity (Vmax)  and Michaelis Menten constant(Km)obtained from Lineweaver-Burk plot of initial velocity data and V0  at different substrate concentrations [S] were found to be 0.08mg/ml and 0.69µmol/min, respectively using H2O2 as substrate for peak A. After using phenol red, Km and Vmax were 0.08mg/ml and 1.49µml/min. More so, for peak B, Km  and Vmax  were 0.18mg/ml and 0.96µmol/min using H2O2 as substrate while 0.08mg/ml and 1.46µmol/min were obtained using phenol red as substrate, respectively.

CHAPTER ONE

INTRODUCTION

At present lignocellulose is a major raw material for forestry, pulp and paper industry and the emerging second generation biofuel production. Among cellulose and hemicellulose, lignin is a major component of lignocellulosic biomass and largely responsible for its strength. Inside the Northern coniferous forest belt the importance of lignin utilization is stressed in wood-based biorefineries due to high amounts of lignin in softwoods (Li et al., 2009). Lignin is a heterogeneous, branched and complex polymer consisting of phenylalanine-derived aromatic subunits (Whetten and Sederoff, 1995). Because of its recalcitrance,  lignin complicates the utilization of biomass polysaccharides in biorefineries and increases the energy consumption in mechanical pulping (Jiang et al., 2008). In nature one group of organisms, the basidiomycetous fungi are able to effectively degrade lignin by employing a family of lignin degrading enzymes. These  organisms  can  be  divided  into  wood-colonizing  white-rot  fungi  and  soil  litter- decomposing fungi. Fungal attack on lignin is attributed to certain secreted nonspecific oxido- reductases, which produce low molecular weight mediators able to intrude recalcitrant biopolymers. The family of extracellular ligninolytic enzymes typically includes lignin peroxidases (LiP,  EC  1.11.1.14),  laccases  (EC  1.10.3.2), manganese  peroxidase (MnP,  EC

1.11.1.13), versatile peroxidase (VP, EC 1.11.1.16) and other accessory enzymes. Out of these enzymes MnP is thought to play the most crucial role in lignin degradation, as it is found in all lignin degrading fungi white-rot fungi (Jarvinen et al., 2012)

1.1      Peroxidase

Peroxidase (POXs) (E.C.1.11.1.7) are  among the  most  ubiquitous enzymes  in  plant species. Peroxidases are also found in some animal tissues and microorganisms where they play a role in protection against toxic peroxides (Wong, 2009). In plants they participate in the lignification processand in the mechanism of defense in physically damaged or infected tissues. Peroxidases are heme-containing enzymes that use H2O2 to oxidize a large diversity of hydrogen donors such as phenolic compounds, aromatic amines, ascorbic acid, auxin and certain inorganic ions  (Wong,  2009).  The  family  of  plant  POXs  comprise  yeast  cytochrome  cPOXs,  plant ascorbate POXs, fungal POXs and classical plant secretory POXs. The group of mammalian POXs include myeloPOX, lactoperoxidase, thyroid POX and prostaglandinH synthetase.They

are ubiquitous in nature and are involved in various physiological processes in plants. Studies have suggested that peroxidases play a role in lignification, suberization, cross-linking of cell wall structural proteins, auxin catabolism, self-defense against pathogens and senescence (Hiraga et al., 2001).Plant peroxidases contain two-calcium ions (Ca2+), which are essential for the structural stability and thermal stability of the enzyme as well as its in vitro activation during analysis (Manu and Prasada-Rao, 2009). Peroxidases are widely used in clinical laboratories, industries and in environmental conservation.

1.2Wood Composition and Degradation by Fungi

Wood is a porous plant material made up of various types of xylem cells. Softwoods (in gymnosperm trees) consist mainly of long tracheids and smaller ray parenchyma cells. Water transport and stem strength are mainly sustained by the dead tracheid cells. Hardwoods (in angiosperm plants and deciduous trees) have more diverse types of xylem cells including fibers, vessels, and ray parenchyma cells. These cells are responsible for support and nutrient storage as well as water and nutrient transport between plant roots and the photosynthesizing leaves or needles (Eriksson et al., 1990). Wood cell walls, in particular the long tracheids and fibers, consist of several layers which differ in their structure and chemical composition (Fig. 1a). The major   components   of   the   wood   cell   walls   are   three   biopolymers,   namely   cellulose, hemicellulose, and lignin (Harris and Stone, 2008).

Lignin is an aromatic and amorphous polymer present in all layers of woody cell walls. In fibers and tracheids, the thin middle lamella has the highest lignin content, whilst most of the lignin exists in the thick secondary wall layers embedded with cellulose microfibrils and hemicellulose (Eriksson et al., 1990). Lignin mechanically strengthens vascular plants and aids in water transportation since it physically attaches the xylem cells together. At the same time, lignin protects the more easily degradable cellulose and hemicellulose polymers from microbial attack. The heterogenic lignin polymer is synthesized in the plant xylem cells from phenylpropanoid precursors i.e. p-coumaryl, coniferyl, and sinapyl alcohol. During lignin biosynthesis these monolignols are polymerized to p-hydroxyphenyl, guaiacyl, and syringyl type of lignin subunits by the action of laccases and peroxidases (Higuchi, 2006). Lignin subunits are joined together by diverse carbon-carbon and ether bonds of which the β-aryl-ether (β-O-4) bond is the most common (Sjöström, 1993). The composition and amount of lignin varies between

softwood and hardwood, and between plant species. The lignin content of softwoods (25-33% of xylem dry weight) consists mainly of guaiacyl subunits while hardwood lignins (20-25% of xylem dry weight) contain both guaiacyl and syringyl subunits (Adler, 1977; Higuchi, 2006). In grass plants, xylem cell wall lignin also contains p-hydroxyphenyl subunits and esterified ferulic acid (Eriksson et al., 1990, Hatfield et al., 1999). Knowledge of the chemical structure of diverse plant lignins is still incomplete, although several lignin models have been presented (Fig. 1b).

Most wood species contain approximately 40-45% (as dry weight) cellulose which is the main component of wood (Eriksson et al., 1990). In the linear cellulose polymer, repeating glucose units are linked together by β-1,4-glucosidic bonds and the degree of polymerization is up to about 15000 glucose units within one polymeric chain (Kuhad et al., 1997). In wood cell wall the long, contiguous cellulose chains are stabilized by hydrogen bonds into microfibrils and further into cellulose fibers. The majority of cellulose is situated in the thick S layer of xylem secondary wall (Fig.  1a)  where  its  fibrillous structure gives  mechanical strength to  wood. Usually, the highly organized crystalline cellulose dominates whereas only a small portion exists as amorphous non-organized cellulose  which  is  more  susceptible to  enzymatic degradation (Kuhad et al. 1997).

Hemicelluloses are  a  diverse group of branched heteropolysaccharides consisting of different hexose, pentose, and sugar acid units. Most of the hemicelluloses act as a supporting material and usually comprise 20-30% of wood dry weight (Sjöström, 1993). Hemicelluloses are amorphous and have a moderate degree of polymerization (100-200 units) and thus are more easily biodegradable than cellulose. The composition and structure of hemicelluloses differ in softwood and hardwood. Galactoglucomannans are the main hemicelluloses in softwood while glucuronoxylan dominates  in  hardwood  (Eriksson  et  al.,  1990).  In  the  woody  cell  walls, hemicelluloses are  bound to  cellulose  microfibrils via  hydrogen bonds. Hemicelluloses and lignins are covalently linked forming the so called lignin-hemicellulose matrix Fig. 1.a ( Kuhad et al., 1997; Harris and Stone, 2008). Depending on the wood species, 2-5% of the wood dry weight  is  made  up  of  extractives  (Sjöström and  Westermark,  1998).  Extractives  are  non- structural constituents of wood. They may be broadly divided into terpenes, resins, and phenols (Kuhad et al., 1997). These various organic compounds have several roles, acting as a nutrition reserve for the living wood cells as well as giving protection against microbial degradation

(Sjöström, 1993). In addition, low amounts of proteins and inorganic compounds are present in the wood (Fengel and Wegener, 1989).

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