AUTHOR: ANYAEGBUNAM, BEDE CHIKE
DEPARTMENT: APPLIED MICROBIOLOGY AND BREWING
AFFILIATION: NNAMDI AZIKIWE UNIVERSITY, AWKA
Results showed that Phanerochaete chrysosporium produced lignin peroxidase (LiP) and manganese peroxidase (MnP) and did not produce laccase. Extracellular lignin expression coincided with onset of idiophasic phase of growth and fell sharply after attaining the peak period. Medium supplementation with Tween 80, veratryl alcohol, and veratric acid resulted in considerable enhancement of LiP expression. The enhanced LiP activity by Tween 80 could in part be as a result of their protective effect on expressed enzyme activity. Ammonium tartrate and glucose were the best sources of nitrogen and carbon respectively for LiP expression. Favourable conditions for enhanced production of LiP by culture of P. chrysosporium was achieved with 56 mM (1% w/v) glucose, 0.05% Tween, 0.3 µm veratryl alcohol, 80 rpm of agitation, 2 µM, ammonium tartrate; 1.0 mM, veratric acid, added after 2 days of incubation, initial pH 4.5. The optimization resulted in approximately 9-fold increase in activity after 6 days of growth. Dye decolouration could be achieved by culture and application of the crude extracellular enzyme. Screening studies showed that P. chrysosporium could decolourize four distinct classes of dyes: triphenylmethane dyes, heterocyclic dyes, azo dyes, and anthraquinone dyes represented by bromophenol blue, methylene blue, methyl orange and remazol brilliant blue R (RBBR) respectively. RBBR was the most decolourized retaining only 25% of residual colour after six days of growth. Cultural dye decolouration experiments with RBBR showed that increasing dye concentration resulted in decreased degradation; pH and temperature optima were 4.5 and 40°C respectively; the best carbon source was glucose. In vitro dye decolouration on RBBR with crude enzyme resulted in 63% decolouration after 16 hours with pH and temperature optima of 3.0 and 40°C respectively. Purification of LiP resulted in purification factor of 23 with specific activity of 35.98 U/mg proteins and 30% retention of overall activity. The estimated molecular weight was 40 kDa. The xx purified LiP had Km of 65, 91 and 105 µM for veratryl alcohol, hydrogen peroxide and propanol respectively. The optimum pH activity of purified soluble LiP was 3.0 with optimum stability at pH 4.5. pH stability studies the soluble LiP showed that significant differences existed between levels of pH stability values (Tukey Test: P<0.05). From the result, although LiP was most active at pH 3 and below, it was not very stable at that pH range. Optimum temperature of activity was 40°C. Variability in temperature stability profile of soluble LiP between means of pairs of 4°C and 60°C was most significant (Tukey test: P<0.05). CuCl2 and FeCl2 activated LiP, while activities were suppressed by various inorganic compounds and EDTA. Cycloheximide and actinomycin D, protein synthesis and transcription inhibitors respectively, did not have apparent impact on in vitro LiP activity. Immobilization by entrapment in alginate beads resulted in highest yield with 4% sodium alginate, 1 M calcium chloride, 2.1 mm bead size and a curing time of 120 minutes. Significant change in optimum temperature values of the enzyme was recorded after immobilization. Immobilization resulted in greater resistance of LiP to temperature and pH effects. Thermoinactivation stability of immobilized LiP which retained 75% of its activity after 120 minutes of storage at 60°C was significant (T-Test: P<0.05) compared to free LiP which retained only 25%. The result of kinetic studies showed that after immobilization, there was decrease in substrate affinity and velocity of enzyme reaction for all the substrates tested. Immobilization allowed LiP to be used in repeated cycles of reactions. Topic: Production, Characterization and immobilization of Lignin Peroxidase from Phanerochaete Chrysosporium.
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Tags: Applied Microbiology and Brewing-PhD-2014. Ammonium Tartrate, Aromatic Pollutants, Biopulping, Biosciences, Cultural Decolouration-Dye, Depolymerization, Enzyme Production, Extracellular Enzymes, Ion Exchange-Chromatography, Kinetic Analysis, Lignin Degrading System, Ligninolytic Enzymes, Molecular Weigth Determination, Panus Tigrimus, pH Stability., Plant Biomass, White Rot Fungi