The production, purification and catalytic utility of lignin peroxidase from "sporotrichum pulverulentum"

Abstract

Production, purification and catalytic utility of lignin peroxidase (LiP) from Sporotrichum pulverulentum. The study of Lip has been hampered by the difficulty in producing this enzyme in sufficient quantities. Several strains of Phanerochaete chrvsosporium and Sporotrichum Pulverulentum were screened for LiP expression under different culture conditions to find a method of producing adequate supplies of the enzyme for the proceeding work in this thesis. A reliable method of Lip production was achieved using 750m1 agitated cultures of S. pulverulentum containing the detergent tween 80. Lip from S. pulverulentum was purified by HPLC and was found to consist of up to 14 isozymes which varied in molecular weight, pH optimum and specific activity for veratryl alcohol. However, their catalytic spectra were similar. The isozymes from S. pulverulentum had higher molecular weights and lower pI values than those published for LiP from P. chrvsosporium, which suggested that they were not as closely related as had been assumed. Lip from S. pulverulentum was able to oxidise a range of methoxy-substituted benzyl alcohols to their respective aldehydes. The susceptibility of benzyl alcohol oxidation by LiP depended upon the amount and position of methoxyl group substitution. LiP oxidation of these substrates was dependent upon how electron-rich the molecular π-orbitals of the substrates were, but steric effects may also have been important. LiP oxidation of benzyl alcohols under aerobic conditions led to additional products such as quinones, ring-cleavage products and chloro-substituted aromatics. These latter products provided evidence for the existence of LiP-derived aryl radical cations for a range of benzyl alcohol substrates, which is consistent with the peroxidative one-electron oxidation theory of Lip degradation of lignin. In addition LiP was shown to catalyse the peroxidative oneelectron oxidation of phenolics such as p-cresol and catechol to produce dimers and polymers. Lyophilised LiP was shown to be catalytically active in organic solvents such as ether and propyl acetate. An increase in enzyme stability of up to 30 times of that in water and a broadening of its catalytic spectrum was observed. Lip was also found in C. versicolor demonstrating that LiP may be a common constituent of ligninolytic white-rot fungi. In addition, other. extracellular peroxidases were present in this fungus. These peroxidases were novel compared to the extracellular peroxidases from P. chrysosporium since at least one of these could not oxidise veratryl alcohol and neither of these peroxidases were manganese-dependent