Purification And Characterization Of Lignin Peroxidases From Lignite Degradation Strain-Penicillium Decumbens P6

Lignite is low-grade coal derived from plant debris over extended geological periods. Lignite also has an analogical structure similar to lignin, it has wide usages in the fields of industry, agriculture, medicine and so on. As for its hydrophobic characteristics and complexity of tridimensional structure, lignite is difficult to degrade. Lignin peroxidase (LiP) is one of the key enzymes on lignin degradation. Now many researches focued on LiP from white rot fungi, which has high lignite and lignin degradation ability, but our country just taked up in this field. Penicillium decumbens P6?which can degrade lignite effectively was isolated from coal mine and stored in our lab. In the present study, it was discovered that P6 could degrade aniline blue on the 4th days and could assemble tannic acid on the 10* days when grown in BM plate, which indicated it could excrete extracellular peroxidases and trace laccase. The peroxidases activities reached the highest on the 4th days in liquid culture. PAGE activities staining showed that P6 could excrete two major peroxidase isoenzymes and several esterase isoenzymes. No endoglucanases and xylanases were detected.The lignin peroxidase of Penicillium decumbens P6 was purified and characterized from the 4th days' liquid culture. The isolation procedure of enzyme involved 80% (NHO2SO4 precipitation, ion exchange chromatography on DEAE-cellulose and CM- cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. It exhibited a molecular weight of 46.3 KDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After purification, the specific activity improved from 0.05 U/mg to 7.52 U/mg, about 150.4 fold. Recovery of activity was 19.6%. When purified P6 LiP oxidased veratryl alcohol at 25℃, the Km of the LiP was 0.565 mmol/L and the Vmax was 0.088 mmol mg-1 protein min-1. It has wider pH ranges and higher temperature stability. The P6 LiP-catalyzed veratryl alcohol oxidase activity showed that the optimum pH at 25℃ was 4.0. The effect of temperature on enzyme activity was examined from 35℃ to 65℃ at optimum pH 4.0, with the optimum temperature at 45℃. The N-terminal of the 46.3KDa band was sequenced to the 10th amino acid residue and compared to other fungal peroxidase sequences obtained from Genbank. It had the conservative amino acid residue VLL with fungal MnP and other peroxidase, but no conservative amino acid residues with other fungal LiP.The lignite degradation ability of purified P6 lignin peroxidase was determined. The resultesshowed that the enzyme had obvious lignite degradation ability dependent on H2O2 activation. After degradation, the content of fulvic acids increased distinctly and the contents of H, O and N were higher than original lignite, but content of C decreased. Furthermore, the products of degradation could increase the germination rate and the speed of germination of pea.We designed two special primers according to N terminal amino acid sequences analyzed and got a 1956 bp cDNA fragment. Sequence comparison demonstrated that it had 45% homology with Piloderma croceum mnp2 gene. Results of the translated amino acids sequence comparison showed it had 6 main ORFs and 2 minor ORFs. It had conservative amino acids of catalase.Patterns of purification of peroxidase having lignin or manganese oxidation activities in white rot fungi have been extensively studied. However, very few data exist on the production and purification of Lip in Penicillium sp. The present paper deals with the purification and characterization features relative to a P6 liquid culture medium. This work is helpful to understand the characteristics of P6 LiP and to construct a higher efficency LiP expression system, it has a theory meaning and production potential.