Catalytic Properties And Steady State Kinetics Of Lignin Peroxidase In A Novel Reverse Micellar System

As an organic macromolecule polymer, lignin is extremely rich in nature andpossesses a very complex structure. There are hundreds of millions of tons of ligninemerging every year, which exists mainly in the form of the crop straw or printing anddyeing wastewater of paper and pulp industry. Such a huge amount of lignin is oftenreleased into the environment without being disposed effectively, which will result inserious environmental pollution. The molecular composition of lignin is very complex,so，as an alternative, most of the current researchers focus on the degradation of ligninmodel compounds. Lignin peroxidase, manganese peroxidase and laccase are the mostwidely used and most effectively three kinds of enzymes for lignin biodegradation,however lignin peroxidase plays a crucial role in lignin biodegradation process. Sincemost of the lignin model compounds have strong hydrophobicity with a lowersolubility in aqueous solution, which limits the efficiency of the enzyme catalyzingreaction in aqueous solution. Although an organic solvent can significantly improvethe solubility of the hydrophobic aromatic environmental pollutants, the existence of alarge amount of organic solvent will result in the deactivation of the lignin peroxidase.Therefore，it’s extremely important to seek a media system which not only maintainshigher activity of lignin peroxidase but also improves the solubility of hydrophobicsubstrate. Reverse micelle is such a media system, whose nano-scale’’pool’’ structurecan solubilize the water-soluble substances such as enzymes，proteins, etc., and theexternal organic solvents can dissolve hydrophobic substrates of the enzyme-catalyzed reaction. The reverse micellar system is the best media system ofwater-soluble enzyme catalyzing hydrophobic substrate, but the focus of previousstudies was tend to build reverse micellar systems using chemical surfactants,biosurfactants in this aspect were very rarely reported. As we all know, chemicallysynthesized surfactant have some characteristics such as toxicity, recalcitrance, easilycausing secondary pollution. Compared with the chemical surfactants, biosurfactantsproduced by microorganisms have been studied more and more becase of not havingthe pollution characteristics.In this paper, first, we investigated the degradation property of the non-phenolicsubstrates veratryl alcohol （VA） catalyzed by lignin peroxidase （Lip） in a positive and negative micellar systems built by biosurfactant monorhamnolipid （mono-RL）. Boththe main factors which affected the catalytic oxidation of veratryl alcohol by ligninperoxidase entrapped in reverse micellar or micellar systems and the activity andstability of LiP in different media conditions in reverse micellar system were studied.Meanwhile, H₂O₂played a dual role for catalytic reaction by LiP, namely, lowconcentrations was the activator of the reaction, and high concentration wouldbecome inhibitors. Therefore, we should first determine the activation concentrationrange of H₂O₂for the catalytic reaction.Second，we built a novel reverse micellar system structured by biosurfactant toexplore the catalytic kinetic mechanism that lignin peroxidase oxidates veratrylalcohol after determining the activation concentration range of H₂O₂. Because thereverse micellar system was a very complex non-homogeneous system composed byorganic solvent, surfactant and a small amount of water, it appeared more important todetermine the distribution coefficient of the hydrophobic substrate in the reversemicelles before discussing the catalytic kinetics mechanism.Experimental results showed that:1)In the positive and negative micellar system built by mono-RL, we studied themain factors affecting LiP catalytic degradation of VA. It was shown that Lip hostedin reverse micellar system could express higher catalytic activity under the followingoptimized media conditions: T=30。C，pH=3.8，[mono-RL]=10mM，胸=15.0，[H2〇2]=74jaM; With respect to the mono-RL micellar system, the optimum values in themicellar system were as follows: T=30。C，pH=3.4，[mono-RL]=0.012mM，[H2〇2]=2.45mM. In optimum catalytic conditions, the LiP expressed superactivity in thereverse micellar system.2)We discussed the stability of LiP in mono-RL reverse micellar system underdifferent reaction temperatures, the values of pH，water content m, the concentrationof the surfactant, or the concentration of H₂O₂and then drawed the conclusions asfollowing: LiP could maintain better stability at low temperatures, and its stabilitywould decrease with the increase of the water content m, or with increasingconcentrations of mono-RL and H₂O₂, and the effect of inactivation became more andmore obvious at high values. However, the stability of LiP decreased after increasingtrend with the increase of the values of pH and the value of pH which LiP kept betterstability was corresponding to the value of the optimum enzyme activity.3)Based on the biphasic model，a conclusion could be made that VA was mainlysolubilized in the pseudophase of the reverse micelle，and the partition coefficient of VA between the pseudophase and organic solvent phase was70.4in the reversemicellar system structured by mono-RL. The determination of partition coefficient isimportant for further discussing the kinetic mechanism of Lip-catalyed oxidation ofVA in the mono-RL reverse micellar system.4) The reaction that LiP catalyzed degradation of VA followed the ping-pongmechanism with the kinetic parameters vmax, Km,vA and Kmbei0.43,H202ng3jamol*L_、min₁，1.26jaM，and0.89jaM respectively in mono-RL/isooctane-/7-hexanol/water reverse micellar system. The reaction mechanism in the mono-RL reversemicellar system was the same as those in the aqueous solution. That was to say thepresence of the mono-RL would not change its catalytic kinetic mechanism. Thedifferences in the kinetic parameters were caused by microheterogeneity, the changesof the conformation of LiP and the reactivity of the substrates entrapped in mono-RLreverse micellar system.