Influence Of Surfactants Toward Soybean Coat Peroxidase's Enzymatic Activity And Structure

Naturally, lignin degradation is the restrictive step that determines the overall degradation of lignocellulose. At the same time, initial degradation fractionlets restrict further and thorough degradation. In chemistry, surfactant behaves remarkable solubilization ability. A surfactant has hydrophilic head and hydrophobic tail; meanwhile enzymatic protein displays electrolytic character in different ionic solutions; so in forth, there exists interaction.Soybean coat peroxidase belongs to class III of the plant peroxidase superfamily; it includes Fe(III) protoprophyrin K(heme) as the prosthetic group. SBP has broad resources, abundant quantity, and wide substrate reaction range; it is thermal stable and tolerates wide pH span. Hence, it demonstrates wonderful application potential.The enzyme is purified through consecutive treatments consisting of ammonium sulfate fractionation, column chromatography by direct elution from CM-Sepharose, gradient elution from DEAE-Sepharose and gel filtration on Bio-Gel P-100; then purified enzyme is detected by SDS-PAGE gel electrophoresis and demonstrates a single band.Purified SBP is desiccated by freezing; I choose three solutions: 20mmol/L pH2.6 citrate acid-Na₂HPO₄, 50mmol/L pH4.2 and 5.2 acetate buffers. Before experiments, add corresponding buffer to SBP powder to make its concentration be 250mg/ml. Under 30℃, set up gradient surfactants' concentrations until the dealt SBP's enzymatic activity remains stable under a special concentration and time. Then select the very surfactants' concentration to treat SBP for one hour (the chosen time), scan on the circular dichromatism spectrum and fluorescent spectrum; ultraviolet-visible spectrum scans SBP's variations within 2h after surfactants' adding to immediately.Experimental results indicate that SBP's activity is greatly related with its structural changes after surfactants' joining to. Remarkable deactivations appear when anionic surfactant SDS reacts with SBP under pH2.6 and 4.2 buffer condition: there exists strong static electricity, enzymatic activity loses, secondary structures change, fluorescent intensity increases, characteristic Soret band moves to shorter wavelength, breakup energy of porphyrin Ï€-Ï€^* resonance augments. Wheares under buffer pH5.2, there is no strong static electricity and instead being a longer wavelength movement; SDS's participation just restricts the relative movement of heme's ethylene; a new fluorescent peak forms and the reduction of enzymatic activity is due to the packing of micella's formation. Contrarily, cationic surfactant CTAB and nonionic surfactant (AEO)₉ have the same effects: the two substances' enterings cause the superficial enzymatic activity to decrease rather than the total loss in whatever the buffers. There is no Soret movements except that a longer wavelength movement by (AEO)₉'s treatment under pH5.2 buffer condition. Fluorescent intensity increases a little as well as a new appearance of fluorescent peak. All these indicate that hydrophobic tails of the two substances penetrate into the inner structure of SBP, hence lead to the decomposition of its tertiary and secondary structures. Endogenesis fluorescent amino acids expose and locate in a new ionic environment.Select those states that SBP remains its half enzymatic activity, comparing and contrasting SBP's fluorescent spectrum results in three different buffers under three different surfactants' treatments. Though being of lower concentration, SDS makes fluorescent amino acids' quenching phenomenon which was caused by heme, disappears, fluorescent intensity intensified considerably; in contrast, much higher concentrations of CTAB and (AEO)₉ change the quenching efficiencies slightly. However, the penetration of hydrophobic tails which results in the exposure of inner fluorescent amino acid gives rise to the change of ionic environments and initiates new fluorescent peak.