Study On Preparation And Properties Of Cross-linked Enzyme Aggregates (CLEAs) Of β-galactosidase

The cross-linked enzyme aggregates (CLEAs) of Kluyveromyces lactis andAspergillus oryzae β-galactosidase were preparaed by polyethylene glycol (PEG)400as precipitant and dialdehyde starch (DAS) as cross-linker. The addition of bovineserum albumin (BSA) was used to protect the enzyme. The influence of concentrationof PEG400and DAS, oxidation degree of DAS, amount of BSA, cross-linkingtemperature and time on the CLEAs activity were discussed. The structure of CLEAswas analysised by SEM. The optimum temperature and optimum pH of CLEAs wereobtained. The thermal stability and kinetics of CLEAs were determined. The processof lactose hydrolysis and galactooligosaccharides (GOS) synthesis by CLEAs werealso inverstigated. Furthermore, β-galactosidase CLEAs was applied in oral enzymepreparations, and the effect of preparative processes on the acidoresistance andactivity of CLEAs were researched in this paper. The experimental results follow as:The optimum preparative processes of K. lactis and A. oryzae β-galactosidaseCLEAs were determined by single factor method, and the high activity residues of53.84%and55.25%respectively were presented by75%(v/v) PEG400,10%(w/w)DAS with80%oxidation degree, BSA with the weight ratio of8to enzyme,cross-linking at room temperature for1h.The CLEAs especially A. oryzae CLEAs have higher thermal stability than thefree enzyme. The affinity interaction between enzymes and substrates of CLEAs hadbeen weakened, however A. oryzae CLEAs appeared a better affinity than K. lactisCLEAs. There was nearly no change in kcatof K. lactis CLEAs while the kcatof A.oryzae CLEAs decreased. The difference in properties of K. lactis and A. oryzaeβ-galactosidase CLEAs is due to the structural distinction of CLEAs.The CLEAs of A. oryzae β-galactosidase showed two times higher lactosehydrolysis from solution in batch process at60℃than the free enzyme after10h, andhad a better reusability than K. lactis β-galactosidase CLEAs. It was indicated that thevelocity of GOS synthesis was growing with the increase of enzyme concentration,but meanwhile the inhibition by products was strengthened.The alginate (ALG) beads of K. lactis β-galactosidase CLEAs were prepared and coated by Eudragit○R. The activity residue of CLEAs released after incubation ingastro-intestinal fluid at37℃was43.29%, and the lactose hydrolysis in intestinalfluid showed6.3times higher than that of A. oryzae β-galactosidase CLEAs whichhad the same activity initially without any treatment.