Preparation Of Cross Linked Lipase Aggregates For The Synthesis Of Starch Laurate

The organic solvents such as dimethyl sulfoxide(DMSO)were mainly used as reaction system for the enzymatic synthesis of starch esters,which has a undesirable impact on the activity of free lipase.Therefore,the development high stability and reusability immobilized lipase has become the key technology for the enzymatic synthesis of starch esters.Cross-linked enzyme aggregates(CLEAs)has gradually attracted more and more attention because of its simple preparation,high activity retentivity,stability and reusability.Based on this,the preparation conditions,properties,and structure of lipase-CLEAs were investigated in this study.Furthermore,the catalytic synthesis of starch laurate was also investigated to provide a new way for the enzymatic synthesis of starch esters.The cross-linked enzyme aggregate technology was used in the immobilization of lipase,the enzyme was aggregated first then cross-linked by glutaraldehyde.Effects of precipitation agent,glutaraldehyde concentration,crosslinking time and enzyme concentration on the activity of lipase-CLEAs were discussed.The optimum immobilization conditions were: precipitant,ammonium sulfate;crosslinker concentration,16 mM;enzyme concentration,25 mg/m L;crosslinking time,1.5 h.The maximum activity was 11.48 U/mg for the lipase-CLEAs.And the activity of CLEAs reached 13.24 U/mg with the addition of BSA.The enzymatic properties and structure characteristics of lipase-CLEAs were characterized.Compared with free lipase,the optimal temperature of lipase-CLEAs was increased from 40°C to 50°C.The optimal pH value of both the immobilized enzyme and the free enzyme was 7.5.The temperature,pH and storage stability of lipase-CLEAs were superior to the free enzyme.And the reusability was significantly improved when considering the fact that 50.26% of their initial activity remained after 10 recycles of usage.The Km of the lipase-CLEAs with p-NPP as substrate was 13.78 mM.The higher Km of the immobilized enzyme suggested that its affinity to the substrates became weaker.Fourier transform infrared spectroscopy(FT-IR)showed that the lipase was cross-linked with glutaraldehyde.The changes of infrared spectrum signal in amide?band showed that the rigid structure was increased,which improved the stability of free enzyme.The scanning electron microscope(SEM)showed that CLEAs was irregular and there are many small holes on the surface,which was beneficial to the combination of substrate and enzyme active center.Then starch laurate were prepared in DMSO solvent systems by using lipase-CLEAs as catalyst.Effects of reaction temperature,reaction time,molar ratio of lauric acid to starch and lipase dosage on the degree of substitution(DS)of starch laurate were discussed,respectively.Results showed that the optimum conditions of starch laurate preparation were: reaction temperature,60°C;reaction time,4 h;lauric acid-starch ratio of 3:1(n/n);lipase-starch ratio of 12%(w/w).Under the optimum conditions,the DS of starch laurate reached 0.151.After 3 times of the esterification reaction with lipase-CLEAs as catalyst,the DS of starch laurate was still up to 0.067.Structure characteristics of starch laurate prepared by enzymic method were characterized.FT-IR analysis showed that a new peak at 1713 cm-1 for C=O stretching in the ester group was observed,which confirmed the formation of starch laurate.It could be seen from the SEM that the morphology of starch granules was severely damaged and the surface became rough and porous,which indicated the alteration of starch structure.X-ray diffraction(XRD)revealed that starch laurate had a VH type crystalline pattern.Furthermore,the thermal stability of the starch laurate decreased indicated by thermal gravimetric analysis(TGA).The physicochemical properties of starch laurate were studied.The physicochemical properties included transparency,solubility,freeze-thaw stability,capability of antiretrogradation,emulsifying activity and emulsion stability.Enzymatic esterification of starch made the transmittance increased from 13.21% to 54.56% and the solubility increased from 1.63% to 20.34%.With the increase of starch laurate substitution degree,the transmittance and solubility decreased.Compared with native starch,the capability of anti-retrogradation of starch was significantly increased.Esterification contributed to the improvement in starch emulsifying activity and emulsion stability,while the freeze-thaw stability got worse.