Application Of ?-galactosidase In Preparing Galactooligosaccharides And Preparation Of Cross-linked Enzyme Aggregates

In this paper, the industrial applications in preparation of galacto-ooligosaccharides (GOS) and preparation of cross-linked enzyme aggregates (CLEAs)of ?-galactosidase were investigated. In order to improve the yield of GOS, addingmetal ions and sodium borate were developed in the process of lactose hydrolysis.Furthermore, the preparation method of CLEAs was studied and evaluated to solvethe problem of recycling ?-galactosidase. As preparing of-galactosidase CLEAs,Fe3O4magnetic nanoparticle was used to improve the reusability of CLEAs. Theconclusions can be summarized as follows:Synthesis of GOS by ?-galactosidase: The optimal conditions for enzymaticpreparation of GOS were determined as37°C, pH8, the intial lactose concentration of400g/L, and the enzyme concentration of25?L/g lactose. Under these conditions, themaximum GOS concentration and lactose conversion were achieved58.22g/L and60.28%for reaction of2h, respectively.The effect of metal ions on properties of ?-galactosidase and its applecation:The certain concentration (<1mol/L) of K+, NH4+would active the transglycosidaseand hydrolase activity of ?-galactosidase. However, Na+would active thetransglycosidase activity and inhibit the hydrolase activity. High concentrations ofmetal ions (>1mol/L) could inhibit ?-galactosidase activity and did not affect thesecondary structure of ?-galactosidase. The inactivation was reversible. Furthermore,the optimal conditions for enzymatic synthesis of GOS were determined as37°C, pH8,0.08mol/L of K+, the intial lactose concentration of500g/L, and the enzymeconcentration of10?L/g lactose. Under these conditions, the maximum GOSconcentration was achieved94.74g/L for reaction of5h. Compared with the systemwithout metal ions, lactose conversion increased by8.06%, and the yield of GOSincreased by62.71%.Synthesis of GOS by ?-galactosidase with Na2B4O7: Na2B4O7was added to thereaction mixture for increasing the lactose conversion and GOS yield. For the reaction,the optimal conditions were pH8.0at37?for12h with12.5?L/g (Enzyme/Lactose)?-galactosidase,400g/L lactose and0.05mol/L Na2B4O7. Under these conditions, the lactose conversion and the GOS concentration were72.01%and115.43g/L,respectively. Na2B4O7significantly improved the GOS yield.Preparation of ?-galactosidase CLEAs: The optimal conditions for preparationof ?-galactosidase CLEAs were determined as20times dilution of original enzymeliquid,1:1of2-propanol (v/v) precipitation for30min at4°C, and cross-linking of30min by0.125%(v/v) glutaraldehyde. Thus45.77%of the reservation activity couldbe otained. Compared with the free enzyme, the ?-galactosidase CLEAs had widerrange and higher value of pH and temperature, and excellent thermal stability.Preparation of magnetic ?-galactosidase CLEAs: The optimal conditions forpreparation of magnetic ?-galactosidase CLEAs were determined as follows:100?L0.042g/mL Fe3O4magnetic nanoparticle and2mL20mg/mL BSA were mixed andstirred for1.5h;20times dilution of original enzyme liquid,1:1of2-propanol (v/v)precipitation for60min at4°C, and cross-linking of60min by0.125%(v/v)glutaraldehyde. Thus58.67%of the reservation activity could be otained. Themagnetic ?-galactosidase CLEAs could be easily recycled, so even if reused by fourtimes, the yield was still20%of the starting value.