Preparation Of D-tagaotse Using Complexing Agent Or Immobilzed Enzyme

With the increasing incidences of diseases such as diabetes and obesity, people putmore emphasis on healthy diet and try to avoid foods with high quantity of fat and energy.As one kind of novel sweeteners, D-tagatose has less energy than sucrose-our traditionalsweetener. Besides, it is better than other low-calorie sweeteners on several properties,which bring it great potential for application. At present, the L-arabinose isomerase (L-AI)（EC5.3.1.4，L-AI）is the most effective enzyme for biotransformation of D-galactose toD-tagatose. Based on different affinities of complexing agents to D-galactose andD-tagatose, the existence of special complexing agents, such as boric acid (borate), couldregulate the isomerization process between substrates and products, resulting in anequilibrium shift toward D-tagatose product and a higher conversion yield. Besides,D-galactose can be obtained by hydrolysis of cheap lactose with lactase, along with manyadvantages of immobilized enzyme, D-tagatose can be synthetized directly from lactose bythe consecutive actions with immobilized lactase and L-AI.In this study, the function and regulation of complexing agents for the production ofD-tagatose were investigated. Besides, suitable carriers were selected to prepareimmobilized L-AI and lactase. Specific contents were as follows:The effects of boric acid and molybdate on the enzyme-catalyzed production ofD-tagatose were investigated. Results showed that only boric acid could promote theisomerization. In boric acid-borate buffer system, the effects of pH, temperature, molar ratioof boric acid ion and concentration of enzyme on conversion yield were studied. The resultsshowed the optimum conditions were65℃, pH9.0,1:2molar ratio of boric acid ion toD-galactose, concentration of enzyme0.39U/mL. Under these conditions, the enzymeconverted90g/L D-galactose to45g/L for16h with a yield of50%by the aid of boricacid(borate), while27%in the Tris-HCl buffer system without boric acid (borate). Resultsrevealed that boric acid (borate) did promote the formation of D-tagatose.Lactase was respectively immobilized on carriers of chitosan, diatomite, sodiumalginate-gelatin and one kind of mesoporous molecular sieves-SBA-15. Comparisons ofeffects on immobilization showed that SBA-15was the best carrier. On the basis ofadsorption by molecular sieve, glutaraldehyde-one kind of difunctional reagents was addedinto the immobilization system to form cross-linked enzyme aggregates. By means of singlefactor test and orthogonal design, the optimum immobilization conditions were determinedas: adsorption time2h,0.10%glutaraldehyde,4U/mg SBA-15, pH for adsorption7.0,crosslinking time1h. Under these conditions, the enzyme activity recovery ratio reached47.2%. Investigations of the properties and stability before and after immobilization showedthe thermostability and pH stability had been improved. When the immobilized lactase was used to hydrolyze90g/L lactose repeatedly, the hydrolyzation ratio was still more that60%after10times repeated operations. Besides, after storage for60days, the immobilizedlactase still remained more than a half of original activity, while free enzyme lost90%of itsactivity, indicating better storage stability.L-AI was immobilized by encapsulation with sodium alginate-gelatin, adsorption withSBA-15and diatomite respectively. The method of encapsulation with better effects waschosen as the immobilization method. The enzyme was further cross-linked withglutaradehyde to enhance the stability of immobilization system. By means of single factortests, optimum operation conditions were obtained:2.0%sodium alginate,2.0%gelatin,solidifying time6h,4.0%CaCl2,0.02%glutaraldehyde. Under the optimum conditions, therecovery ratio reached to78.2%. After seven times repeated operations, the loss of activityof immobilized L-AI was less than50%, showing good operation stability. Compared withfree L-AI, the pH stability and thermostability of immobilized L-AI all got someimprovements. Lactose and glucose could not restrain the isomerization of galactose, whichrevealed that it was an effective and rational way to consecutively produce D-tagatose withthese two kinds of immobilized enzyme.