Studies On Fermentation Conditions,Purification And Characteristics Of Lignin Peroxidases From Saccharomonospora Viridis

Lignin peroxidase was first isolated from white rot fungi in 1983,which is one kind of peroxidases with heme as its active center.LiP has been believed to play a key role in lignin biodegradation and recent studies have shown that it can degrade phenolic pollutants.Therefore the studies of LiP are of great significance to the world's resources and environment.Patterns of purification of peroxidase having lignin or manganese oxidation activities in white rot fungi have been extensively studied. However,there are very few data exist on the production and purification of Lip in Saccharomonospora viridis.In a 16L fermentor,production conditions of LiP from Saccharomonospora viridis were investigated.The optimal operation conditions were determined as follows:fermentor agitation rate 250r/min,ventilation amount 5L/min,the seeded volume10%,C/N ratio 1:3.The maximum enzyme activity could reach 0.41 U/mL at 96h while dissolution oxygen(DO) level maintained higher than 35% by enhancing aeration and changing agitation rate.And the growth curve and metabolism curve of this strain has been determined in the fermentor.The lignin peroxidases was separated and purified by ammonium sulfate precipitation and Sephacryl S-200 gel-filtration chromatography.The purified enzyme is demonstrated by SDS-PAGE to be a homogeneous protein and the molecular weight is estimated as 28.8KD,so purification multiple of LiP was enhanced 11.06.Furthermore,the present study was undertaken to investigate the enzyme characteristics of purified LiP.It exhibited the highest activity at 50℃and pH7.0.At pH 7.0～pH 10 and below 75℃,the enzyme showed high activity and good stability.At 75℃half life period of LiP is 0.5h.It was rather stable for a long time when stored at 4℃.The LiP was activated by Cu²⁺,Fe²⁺and Co²⁺,while inhibited by Ca²⁺,SDS.The LiP was a kind of low molecule mass glycoprotein containing 6.59%%carbohydrate.The apparent Michaelis constant of the LiP was 0.1057mmol/L for 2,4-DCP.The present paper deals with the production conditions in fermentor,the purification and characterization features relative to a Saccharomonospora viridis liquid culture medium.This work is helpful to understand the characteristics of Saccharomonospora viridis-LiP and to construct a higher efficiency LiP expression system,it has a theory meaning and production potential.