Studies On Stability And Inactivation/Activation Mechanism Of Chloroperoxidase Based On Additives

Chloroperoxidase (CPO) has halogenation, oxidation of alcohols,hydroxylation, epoxidation and sulfoxidation reactions and so on versatile catalyticactivities because of its unique active site structure, so it can be applied widely inorganic synthesis, biotransformation, pharmaceuticals, detoxication of environmentalpollutants and etc. However, due to their relative instability and inactivation in reactionsystem, the industrial application of native CPO has been limited. Moderately hightemperatures are often needed in industrial processes in order to accelerate reaction rate,increase solubility, reduce solution viscosity, and avoid microbiological pollution.However, thermoinactivation is often a dilemma in enzyme industrial applications.Therefore, improving thermostability of enzymes is a very important aim inenzymology.The effects of several polyhydroxy compounds (glucose, fructose, gumsugar,galactose, trehalose, dextran, xylose, PEG200, glycerin) and surfactant (dioctylsulfosuccinate sodium salt, AOT) on the catalytic activity and thermal stability of CPOin aqueous systems have been investigated at various temperatures using the oxidationof indole to oxindole as a well-functioning model reaction. The 25% superactivity wasfound in AOT solutions at 25℃and it could be maintained during the 882 hinvestigated. PEG200 and glycerin were proven to be the most efficient stabilizer forCPO in temperatures ranging from 25 to 60℃. It is interesting to note that the stabilityof CPO was prolonged from 10 h to 200 h in the presence of PEG200 and glycerin at 50℃. In trehalose solution, CPO stability was extended 2.6, 7, and 2.5-fold at 40℃, 50℃and 60℃, respectively. The protective mechanism of various additives on CPO wasdiscussed.We have surprising discovered that CPO was inactivated by many sugars (reducingand non-reducing) in the absence of H₂O₂, and furthermore native CPO was rapidlyconverted to a green species that it is stable. We research the interaction CPO andglucose by UV-Vis, Fluorescence, Laman spectroscopy, and compared with interactioncytochrome P-450, HRP, Myoglobin, Hemin and glucose. The results show that glucosecould enter into active center of CPO, and hydroxyl oxygen in glucose as electron donors would part coordinate with heme Fe³⁺ in CPO molecule, leading to distortstructure and increase in the non-planarity of the porphyrin ring. Reason was likely tohave two: (1) the CPO site is more open above the heme, and the catalytic base isglutamic acid rather than histidine as in other peroxidases. Futhermore base of glutamicis more than histidine, which increase eletron donor effect of hydroxyl in glucose and soeasy part coordinate with heme Fe³⁺ in CPO molecule. (2) A smaller channel of aboveheme in CPO activity centre has steroselectivity. However every kind of sugar isdifferent structure, for example four -OH of galactose is 1a2a3a4a, glucose is 1a2a3a4e;Xylose is a five-ring ketose and glucose is six-ring aldose. As result glucose couldoptimum enter into the channel.A 23.7% increase in the catalytic efficiency was obtained in the presence ofSHCH₂COOH. The phenomenon can be explained by increasing charge density oncentral metal ion of CPO, it will be favorable electron transfer to antibonding orbital ofoxygen in H₂O₂, promoting subsequent O-O bond cleavage and leading to the formationeasily of Fe^â…£=O-contained intermediates compound I so as to improve activity of CPO.