Construction Of Biocatalytic Colloidosome And Research Of The Catalytic Performance

Colloidosome,an interesting microcapsule that was produced from Pickering emulsion template,has attracted a surge of interest for enzyme immobilization.In this paper,a promising approach to prepare colloidosome with solid cores was developed by polymerization the inner emulsion phase catalyzed by the Grubbs’catalyst and the properties of the immobilized enzymes were also investigated.The details were summarized as follows:（1）Synthesis of Fe3O4-based colloidosome（CALB@CM-Fe）where Lipase B from Candida antarctica（CALB）was covalently bond on the surface of Fe3O4 nanoparticles and the study of catalytic properties and stabilities about CALB@CM-Fe.The enzyme loading and the specific hydrolytic activity of CALB@CM-Fe were 76 mg/gsupport.and 13U/mgsupport,respectively.The stability test indicated that CALB@CM-Fe can retain 60.54%of original activity after incubation at 50 ^oC for 6 h,while N435 and the free lipase only retained 33.66%and 8.01%of original activity respectively under the same condition.These findings showed that CALB@CM-Fe had the enhanced thermal stability.The stability of free lipase,CALB@CM-Fe,and N435 in organic reaction system were measured.Results showed that whether in the aprotic non-polar solvent（cyclohexane）or in the aprotic polar solvent（t-butanol）,CALB@CM-Fe exhibited better organic solvent resistance.Meanwhile,the operational stability and storage stability of CALB@CM-Fe were measured.the CALB@CM-Fe showed excellent properties.CALB@CM-Fe was successfully used to catalyze the esterification of fatty acids and alcohols with different length in organic solvent,showing the versatility of CALB@CM-Fe.（2）Synthesis of silica-based colloidosome（CALB@CM-Si）where CALB was physically adsorbed on the silica material and the study of catalytic properties and stabilities about CALB@CM-Si.The enzyme loading and the specific hydrolytic activity of CALB@CM-Si were 116.3 mg/g_supportupport and 209.6 U/mg_support,respectively.Compared with CALB@CM-Fe,various stability of CALB@CM-Si has been significantly improved.CALB@CM-Si can retain 105.6%of original activity after incubation in t-butanol for 96 h,while CALB@CM-Fe was 94.07%under the same condition.CALB@CM-Si can retain67.65%of original activity after constantly shaking for 196 h,comparing to 49.02%of CALB@CM-Fe.Comparing the storage stability,after 30 days,CALB@CM-Si retained84.32%of enzyme activity,slightly higher than that of CALB@CM-Fe（82.15%）.The yields of the esters which were catalyzed by CALB@CM-Si were significantly higher than those catalyzed by CALB@CM-Fe in a series of esterification.Since that,CALB@CM-Si was conduced as an effective improvement to the CALB@CM-Fe.（3）Synthesis of glycerol carbonate catalyzed by CALB@CM-Si.A series of experimental investigation on on the conversion of glycerol and the producr selectivity revealed that the optimum conditions were summarized as follows:In solvent-free reaction system,a 40-fold of DMC excess and 1 wt%of CALB@CM-Si were considered as the optimal molar ratio and the optimum biocatalyst conversion,respectively.At the optimal reaction temperature（50 ^oC）,molecular sieve didn’t need to be added additionally,while,the addition of Triton X-100（10 v/v%）can slightly enhanced the synthesis of glycerol carbonate.Maximum yield of glycerol carbonate（78.08%）was established after 24h reaction,while,the glycerol conversion was 94.25%.In order to simplify the downstream reaction,surfactant was not added during the cycle experiments.After seven cycles,CALB@CM-Si could still catalyze this reaction efficiently and about 58.82%of production yield was obtained,which was much higher than catalyzed by N435（21.08%）.Therefore,CALB@CM-Si seems to be a promising biocatalyst for enzymatic synthesis of glycerol carbonate.