Study Of Conformation And Enzymatic Characteristics Of Biluochun Lipoxygenase

Lipoxygenase （LOX） is an essential enzyme in tea leaves. On one hand, it can promotearoma formation, on the other hand, if the residue in tea is too large, it might lead to quantitydecreased. Based on the infrared matched absorption theory, materials possess the character ofselective absorption, with this character, by using infrared of certain wavelength to processLOX, efficiency of inactivating LOX could be enhanced. Meanwhile, with heat effect,infrared could dry tea leaves during processing LOX, and achieve the infrared dry-branching.In this thesis, author used Biluochun as raw material, explored enzymological characteristicsof LOX in tea leaves and the effect on the LOX caused by infrared and the conformationtransformation of LOX also detected. Main content as followed:（1） The effects of extraction condition on the extraction of LOX in tea leaves werestudied, and Response Surface Methodology was used to optimize extraction conditions;ammonia sulfate fractional precipitation was used to achieve preliminary separation, andDEAE Sepharose Fast Flow ion exchange column and Superdex G-200gel column were usedto further purify. Result shows, using the disodium hydrogen phosphate-citric acid bufferwhich pH value is6.2, the triton concentration is0.21％and contains0.18%polyvinylpyrrolidone （w/v） can get the highest activity in the extraction. Underabovementioned conditions, enzyme activity of extraction was47352.3U high. Using10%saturation ammonium sulfate can remove the unwanted protein, and using50%saturationammonium sulfate can precipitate the LOX. After Superdex G-200gel column purification,abovementioned enzymes are purified by86fold, with molecular weights of105kDa bySDS-PAGE.（2） The free LOX is not statable, after storage for7days, it’s activity that have basicallylosted. The best concentration of linoleic acid is0.594mmol/L; Ca²⁺may improve the activityof LOX, but with the concentration improving, the degree of improving have decreased;Co²⁺、K⁺、Mg²⁺、Na⁺that all have negative effects of LOX, and the effects become obviouslywith the concentration improving; with the concentration of Cu²⁺、Mn²⁺、Fe3+improving, theirnegative effects have decreased, and when the concentration reached20mM, these metal ionsshowing positive effects, only when the concentration of Zn²⁺is2mM can showing positiveeffects, otherwise the effects seems to be negative; organic reagent usually shows negativeeffects, and with the concentration improving, the effect become obviously; but the lysine isjust the opposite. It’s thermostability is not perfect, under90℃, after30min, it’s activity maylargely disappear.（3） Biluochun LOX have better absorptivity of3μm and6μm infrared wavelength.（4） It’s activity have some relationship with the secondary and the three-level confirmation, which give us the microcosmic result.