Research On The Biosynthesis Method By Immobilized Enzyme And Stability Of Theaflavins

Aiming at the low efficiency of extraction method, great amount of industrial wastewater produced by chemical oxidation method, discontinuous producing catalyzed by dissociated enzyme in TF production methods for the moment and the un-carried on work about production TF by immobilization enzyme in our research team, in this thesis, we mainly studied the embed material which applied in immobilized enzyme to catalyze tea polyphenols oxidation and to produce TFs, the permeability of objection which was catechin in the embed material, as well as the reaction solvent system and the stability of theaflavin. The main results of every part work were as followings.In Chapter One, firstly, we reviewed the research on biological activity and the production methods of theaflavins in latest decade, the research procession of polyphenol oxidase (PPO) and the utilization of immobilization enzyme were discussed as well.In Chapter Two matrixs sodium alginate and chitosan were compared for immobilizing PPO. The content range of catechins in sodium alginate was 19.6～7.5 mg/ml in aqueous phase system, while in chitosan was 17.7～15.5 mg/ml; the permeability of chatechin component in two carriers was also different. The permeability difference between sodium alginate and chitosan of four ester-catechin (GC,EGC,C and EC) was more significant than four nonester-catechin.In Chapter Three, we studied the immobilization parameter of immobilization enzyme, biochemistry characteristic of IPPO and effect of ions on IPPO. We confirmed the optimum parameter such as the ratio of IPPO and sodium alginate was 1:1, the content of glutaraldehyde and CaCl₂ was separately 0.25% and 2%. The effect of metal ions on enzyme catalyzation was carried out. The activity of dissociated PPO was higher than that of control by 23.4% when the Cu²⁺ content was 0.1mM. However, the Cu²⁺ content higher than 0.1 mM the activity of PPO was restrained. Al³⁺restrained the activity of dissociated PPO through the whole reaction. The kinetic constants K_m and V_max of IPPO whose value were 3.0 mg/ml and 84.2 mg/ml respectively were calculated according to Lineweaver-Burk plot. Further more, different solvents effect the catalyze ability obviously. Aqueous phase was selected as the reaction solvent after being compared with ten organic solvents which were n-hexane, chloroform, dichloromethane, ethyl acetate, acetonitrile, ethanol, butanol, acetone, methanol and methylbenzene. The yield of TF in aqueous phase was the highest, which was 0.6 mg/ml.In Chapter Four, the stability of IPPO was discussed. The remained activity arrived at 80.1% after 80 times repeat using. The activity of immobilized enzyme was 72.9% of crude activity after stored in 0.1M citric acid buffer solution (pH5.6, 4℃) for 80 days.In Chapter Five, the effect of different pH value on stability of TF was performed. TF was much more stable in pH value extension which was from 2.2 to 5.6 than in the pH value from 6.5 to 8.0. The stability order of TF monomers in pH2.2～5.6 citric acid buffer solution was TF₁, TF₂A, TF₂B >TF₃. While in pH 6.6 to 8.0 citric acid buffer solution, the stability of TF monomer was as followings: TF₃> TF₁, TF₂A, TF₂B, which meanings TF₃ degraded more slowly in the alkaline condition than other TF monomers. The effect of five factors on the stability of TF were designed here, five factors included packing material, light condition, temperature, humility and the reaction time was illuminated. The relative analysis of TF in different packing material and reaction time was significant, while the other factors did not had significant effect in this study.