The Purification Progress Of Lignin Peroxidase By Reversed Micelle System

Lignin is a widespread natural organic polymer compound, its degradation hasbecome a rate-limiting step of the carbon cycle on Earth. Lignin peroxidase enzymeplays a key role in the process of lignin degradation. Salting precipitation has been akey purification method of lignin peroxidase, but its effect is not obvious. The reversemicelles extraction is a novel biological separation and purification technology whichhas the advantages of simple operation, low cost, high efficiency of extraction, mildconditions and no secondary pollution to the environment, so it has gradually beenapplied to the separation of the enzyme. This study is divided into three parts to studythe production and purification of lignin peroxidase.The first part was to research and optimize the fermentation conditions of ligninperoxidase by Phanerochaete Chrysosporium. In both method one and stati methodtwo, the initial pH value and liquid volume on the effects of the amount of enzymeproduction was studied. The research results showed that when the initial pH valuewas4.5, the yield of enzyme was the highest. With the increasing of liquid volume,the lignin peroxidase activity was increasing ceaselessly. In both two methods, whenthe liquid volume was respectively100mL and60mL, the yield of enzyme reached themaximum value. In the optimum conditions, the LiP activity were respectively0.120U/mL and0.110U/mL, so the former was predominated by its higher activity.In the forward extraction processes, the effects of ionic species and concentration,RL concentration, the pH value of water phase and shaking time on extractionefficiency were studied in the second part. The research showed that the effect ofdifferent cations on extraction was K~+>Na~+in the reverse micelles which wasconstructed by rhamnolipid (RL). We could know that the best forward extractioncondition was: the concentration of RL and KCl was2.75mmol/L,0.04mol/L, pHvalue was3.0, the reaction time was30min. In this condition, the activity recovery ofLiP and purification fold was93%and2.9, respectively.In the backward extraction processes, the effects of ionic concentration, the pHvalue and ethanol concentration on extraction efficiency were studied in the three part.Through the study we could found that adding ethanol can improve the enzymeactivity recovery and purification multiples and made separae easier. The best backward extraction conditions were: the concentration of KCl was0.5mol/L, the pHvalue was6.0, the ethanol addition level was8%. Under this conditions, thepurification multiples was3.2. The SDS-PAGE electrophoresis analysis results furtherconfirmed that the purification of lignin peroxidase was successful.