| Sugar fatty acid esters (SFAEs) are biodegradable surfactants, possessing a wide range ofcritical micelle concentration (CMC) and hydrophilic-lipophilic balance (HLB) values, thuswidely used in food, cosmetic, pharmaceutical and detergent industries. The recently-reportedantimicrobial and insecticidal activities of SFAEs further broadened their applications inagriculture and medicines. In industry, sugar esters were manufactured by chemical synthesiswhich has poor selectivity, many by-products and is harmful to the environment.Lipase-catalyzed synthesis of sugar esters has gained much attention in the past decades, dueto their high regioselectivity, mild reaction conditions and environmental friendliness.However, the main hurdle to the industrial application of enzymatic approaches in thesynthesis of sugar esters is the high cost of the procedure and low stability of some freeenzymes in organic solvents. As one of the biocatalysis, whole cells are widely existed innature and are preferred to isolated enzymes because they eliminate the needs for enzymepurification and immobilization which account for a large part of the enzyme cost. In addition,the whole cells can provide a natural environment for enzyme location which protects theenzymes from a rapid deactivation in non-aqueous solvents. Till then the biocatalyticsynthesis of sugar esters by wild microbial cells has so far remained unexplored. In thisdissertation, the possibility of biocatalytic synthesis of glucose esters by whole cells fromdifferent sources were investigated. The influences of the culture components on the catalyticactivities of the cells in the synthesis of glucose esters were discussed and comparativeanalysis of different kinds of microbial cells catalyzed transesterification of glucose withdifferent fatty acid vinyl esters were studied. In order to improve the efficiency of whole cellscatalyzed synthesis of glucose ester, the influences of various nonaqueous solvents andreaction conditions on whole-cell catalyzed transesterification in organic solvents and ionicliquids were studied. Based on these, the optimal reaction conditions for whole cells catalyzedtransesterification of glucose with various vinyl esters were established.Results showed that, the catalytic activity on the synthesis of glucose esters of differentPseudomonas strains and fungi could be affected by culture components, this might becausecell-bound enzymes that could catalyze the tranesterification of glucose are induced enzymes.Whole cells from Pseudomonas stutzeri cultured in SM-3containing glucose had the highestcatalytic activity on the synthesis of glucose propionic ester, while whole cells fromAspergillus oryzae2cultured in SM-1containing soybean oil had the highest catalytic activity on the synthesis of glucose laurate, and glucose conversion were96.6%and50.5%,respectively, which showed that different microbial cells had different selectivity towarddifferent carbon chain vinyl ester donors.Results about the effects of various reaction conditions on the whole cells of P. stutzericatalyzed transesterification of glucose with vinyl propionate showed that the lyophilizedcells showed an evident solvent dependence in the reaction. Among the tested solvents bothpure and binary, except for acetonitrile-pyridine and tetrahydrofuran-pyridine, the substrateconversion and initial rate clearly increased with increasing hydrophobicity of the organicsolvents used. The best results of the whole-cell catalyzed reaction were observed inisooctane-pyridine system. The optimum isooctane concentration, water content, molar ratioof acyl donor to glucose, biocatalyst dosage and reaction temperature for thetransesterification performed in pyridine-isooctane were30%(v/v),2%(v/v),10:1,80mg mL-1and35℃, respectively, under which the initial rate and conversion of the reactionfor24h were29.7mmol/L h and97.2%, respectively. A preparative-scale production of6-O-propionic glucose ester without significant loss of initial rate and substrate conversionfurther demonstrated that whole-cell of P. stutzeri was an efficient alternative to enzymes for“gWee”X_YheXiX f g Zc Xe eXYeWX.Whole cells of lyophilized P. stutzeri could also catalyze the regioselectivetransesterification of glucose with long chain fatty acid esters in ionic liquids (ILs). In pureILs, whole cells of P. stutzeri catalyzed synthesis of glucose ester could only be performed inC4MIm TfO. The addition of tetrahydrofuran (THF) or pyridine into ILs significantlyincreased product yield. It also showed that, among the tested ILs-pyridine binary solvents,the product yield clearly increased with increasing hydrophobicity of the ionic liquids usedand the best results was observed in C4MIm TfO-pyridine system, followed byC8MIm BF4-pyridine with the product yield being of59.6%and54.4%, respectively. Themost suitable ILs content in pyridine-C4MIm TfO system and pyridine-C8MIm BF4was60%while in THF-C4MIm TfO system, the best result was observed when the content of ILs was20%. In pyridine-C4MIm TfO system, except for vinyl stearate, the product yield decreasedwith the increasing carbon chain of fatty acid ester used and the time needed to reach theequilibrium balance was also increased. Within0-168h, the highest product yield for wholecells of P. stutzeri catalyzed transesterification of glucose with vinyl laurate, vinyl myristate,vinyl palmitate or vinyl stearate were90.9%,79.5%,41.0%and60.7%, respectively. Inaddition, the study of operational stability showed that the addition of ILs in pyridine could significantly increase the stability of lyophilized whole cells of P. stutzeri. The residue relativeactivity of whole cells of P. stutzeri after reuse of one time in pyridine-C4MIm TfO systemand pyridine-C8MIm BF4system were51.6%and38.2%, respectively, much higher than inpure pyridine system.Within this study, results of HPLC, UHR-TOF-MS and13C NMR analysis showed that6-O regiomer (>99%) was formed in the transesterification of glucose with various vinylesters, which confirmed that Pseudomonas stutzeri cells exhibited high regioselectivitytoward the6-O-hydroxy group of glucose. |
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