Reseacrh On The Enzymo Logical Characteristics Of Polyphenol Oxidase From Tea And The Effect Of Infrared On Its Activity And Conformation

Polyphenol Oxidase (PPO) is an essential enzyme in tea leaves. It can promote theformation of color, flavor and taste compounds in tea, but on the other hand, PPO’sparticipation in the enzymatic browning will affect the color of tea leaves and deteriorate thequality. Based on the infrared matched absorption theory, materials possess the character ofselective absorption, with this character, by using infrared of certa in wavelength to processPPO, efficiency of inactivating PPO could be enhanced. Meanwhile, with heat effect, infraredcould dry tea leaves during processing PPO, and achieve the infrared dry-branching. In thisthesis, author used Biluochun as raw material, explored enzymological characteristics of PPOin tea leaves and the effect on the PPO caused by infrared and the conformationtransformation of PPO also detected. Main content as followed:(1) The effects of extraction condition on the extraction of PPO 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 Superdex75gel column were used tofurther purify. Result shows, using the disodium hydrogen phosphate-citric acid buffer whichpH value is5.6and contains0.16%polyvinylpyrrolidone (w/v) can get the highest activity inthe extraction, when the solvent to materials ratio was4.5:1. Under abovementionedconditions, enzyme activity of extraction was9828.01U high. Using30%saturationammonium sulfate can remove the unwanted protein, and using70%saturation ammoniumsulfate can precipitate the PPO. The enzyme can be divided into2isozymes named PPO I andPPO II with the DEAE Sepharose Fast Flow ion exchange column. After Superdex75gelcolumn purification, abovementioned isozymes are purified by154fold and28.7fold, withmolecular weights of36kDa and42kDa by SDS-PAGE.(2) The enzymology characteristics of PPO in tea leaves were studied. The optimumsubstrates for PPO I and PPO II are catechol and pyrogallic acid, respectively; The optimalpH value is6.5both, and can steadily exist between pH7.0-9.0; The optimal temperature is30℃both, PPO II is slightly more stable than PPO I when appears to heat; Citric acid,Cysteine, Sodium sulphite, Ascorbic acid could inhibit the activity of PPO I and PPO II.(3) The effect of infrared process on enzyme activity of PPO I and PPO II was studied, itfigured out that under radiation of infrared with different wavelength, enzyme activity reduceswhen time and temperature accumulates, and the inactivation curve matches first-orderkinetics. PPO I is most sensitive to infrared with a3. μm wavelength, the inactivationconstant is0.7712min-1under80℃; PPO II is most sensitive to infrared with a6. μmwavelength, the inactivation constant is0.7625min-1under80℃.(4) By using circular dichroism spectra(CD) and fluorescence spectrum, the structure transformation of PPO I and PPO II under infrared was studied. Result shows, after infraredprocess, microcosmic structure of PPO I and PPO II would have significant changes. Circulardichroism spectra shows, after infrared process, α-helix in PPO I and PPO II graduallychanges into random form. This phenomenon is most obvious under infrared with3. μmwavelength for PPO I, and6. μm for PPO II. Fluorescence spectrum shows, after infraredprocess, peptide chains gradually unfold, and the fluorescent amino acid residue graduallyexposed, the largest emission wavelength has red shifted at the same time. Those all indicatethat the micro environment of amino acid residue has changed. Heat effect of infrared changesthe surface hydrophobicity of PPO, while wavelength merely has effect on hydrophobicity.