| Biocatalysis in anhydrous medium is an important subdivision of industrial biocatalysis.The improvement in the biocatalytic efficiency of enzyme is one of key challenges in biocatalytic technology.Propyl gallate ester(PG),usually synthesized by a chemical method,is a perfect food antioxidant and a medical intermediate.Presently,this chemical method cannot meet the requirements for green synthesis of PG due to such shortcomings as high energy consumption and serious corrosion.Fortunately in view of its unique advantages,biocatalysis in organic media has become an promising subdivision of PG green synthesis.Tannase(TAN)is a key enzyme catalyzing PG synthesis.But its apparent biocatalytic capability and stability can be reduced by organic media.In this thesis,one-pot biosynthesis of PG with tannic acid and n-propanol as substrates was regarded as a pattern reaction,where the effects of organic medium and the micro-environment of TAN were studied on the structure and activity of TAN.Based on these,biosynthesis of propyl p-hydroxybenzoate(PPHB)with bio-imprinted TAN was further explored.This work would provide a reference for the widespread application of TAN in the green synthesis of chemicals.In the transesterification,organic solvents as reaction media can enhance the solubility of substrate,and reduce the inhibiting effect resulted from water,and further inhibit the ongoing of the reverse reaction.In this thesis,the effects of six different organic solvents were investigated on the structure and activity of TAN.HPLC,UV-spectrum and FT-IR were applied to detect the variations in secondary structure of TAN.This results indicated that the molecular structure and group of orangic solvent have a significant effect on the catalytic performance of TAN.For example,the cyclic structure of organic solvent plays a negative role in the conformational stability of tannase.The alkyl group of organic solvent has an interaction with the groups of oxygen and nitrogen of TAN,and the interaction goes against the conformational stability.Micro-environment effectors such as buffer,metal ion,and surfactant are very crucial for enzymatic activity in organic reaction system.Firstly,toward this end,the effects of phosphate,acetate,citrate buffers and its concentration were studied on the structure and activity of TAN.The results indicated that the activity negatively links with the rate of change in the secondary structure,and acetate and phosphate buffers can enhance tannase activity by transforming ?-helix into ?-sheet.It has been revealed from a further study that a certain percentage of ?-helix and ?-sheet may be regarded as the structural base for the optimum activity of tannase.Secondly,it was found from the study on zinc ion that hyperchromic effect is increasing with increment of zinc ion concentration.Finally,it was found from the studies on surfactants that 0.24 mM span 20 is the optimum in activating TAN,andThe corresponding yield of PG increased by a factor of 7 relative to the control.Bio-imprinting technology can change the affinity of enzyme to the substrate.In this thesis,the TAN was imprinted by PHBA,and then it was applied to catalyze the esterification reaction from PHBA into PPHB.HPLC,FT-IR,and UV were utilized to investigate whether the product has appeared or not.Furthermore,the reaction medium was optimized,and the result showed that n-propanol is the optimum in which TAN had a maximum yield of approximate 860 mg/L.The conversional rate of substrate reached 26.5%.The study will be helpful for a deep insight into the relationship between the biocatalytic activity and conformational stability of enzymes.The study cannot only efficiently improve the compatibility of enzyme to organic media but also provide a novel technical support to enhance the biocatalytic performance of enzyme in organic media.Moreover,the used bio-imprinting technology has a significant meaning for enzyme modification,and gives an experience means to biosynthesize a few chemicals in organic media. |
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