| Esculin is one of the main active ingredients of Qinpi(a traditional Chinese medicine). It belongs to flavonoids and contains coumarin structure with detoxification, treatment of cough and asthma and other functions. It was also found that esculin could inhibit tumor cell and LPS-induced acute lung inflammation. However, its application in the food and pharmaceutical industry was greatly limited by their poor lipophilic solubility. Reserarches showed that structural modification of esculin could improve its lipophilicity and solubility in oil, thereby enhancing their bioactivity. Both chemical and enzymatic methods have been investigated for esculin modification. Considering the low regioselectivity of the chemical methods and high cost of the free enzyme catalysts in previous researches, new whole-cell based stategies for esculin fatty acid ester synthesis were explored in this dissertation. Different whole cells were tested and screened from lipase-producing microbial strains; The influences of non-aqueous media, as well as reaction conditions, on microbial biomass and catalytic activity were researched; Besides, the effect mechanisim of acyl donor chain length on synthesis of aesculin fatty acid ester were discussed. Finally, the best reaction systems for synthesis of aesculin fatty acid esters were establish.Culture medium played an important role both in biomass and catalyzing ability. The biomass of bacteria cultivated by the culcure media containing glucose(SM-1) was greater than those cultivated by the culture media SM-2 containing soybean oil, while the fungi biomass in SM-2 were higher than those in SM-1. Among 13 strains tested, Pseudomonas stutzeri, Pseudomonas aeruginosa, Rhizopus oryzae, Aspergillus oryzae and Aspergillus niger showed relative catalytic activity. Pseudomonas stutzeri GIM 1.273 cultivated by SM-1 possessed the highest catalytic activity. Substrate conversion of esculin reactedd with vinyl acetate,vinyl octancate,vinyl laurate, vinyl myristate(VA/VO/VL/VM) was 71.67 %, 57.27 %, 40.80 % and 40.44 %, respectively. Regioselectivity increased with the growth of acyl donor chain length, there are monoester and a little diesters when acylation with VA and VO, while only generated monoester with long chain acyl donor.The P. stutzeri whole-cell catalyzed synthesis of esculin esters showed that cells were easy to be inactivited in strong polar solvents, although in those solvents acesulin had better solubility than hydrophobic solvents. Thus mixed solvents were tested. The initial rate and substrate conversion increased with the rise of logP values of the media, and pyridine-isooctane mixture was shown to be the best. The optimum water content, molar ratio of vinyl actate to esculin,whole cell catalysts dosage,reaction temperature and shaking speed were 1%, 20:1~25:1, 40 mg/mL, 40℃ and 180r/min. Under the optimal conditions, initial reaction rate and 24 h conversion were reached to 5.82 mmol/L·h and 92.70 %, respectively. The octanoylation conditions of esculin were optimized at 25:1 of substrate molar ratio, 40 mg/m L whole cell catalysts dosage, 40℃ and 180r/min. Under the optimal conditions, initial reaction rate and 24 h conversion were reached to 5.26 mmol/L·h and 85.86 %, respectively. Although the reaction optimal conditions were different, the influencing rule of different parameters to different aesculin fatty acid ester were similar, and the initial reaction rate and substrate conversion efficiency were decreased with the rise of acyl donor’s chain length.Pseudomonas stutzeri showed a good operational stability. The relative conversion of the the reaction from batch 2 to batch 5 were reached 70.66%, 65.49%, 48.19% and 45.35%, respectively, indicating the whole-cell catalyst could be used repeatedly.This research has its practical values, providing new strategies for biocatalyzed synthesis of a series of esculin fatty acid esters. |
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