| Co-immobilization is a novel technology developed in recent years. Due to the synergism effect of the enzymes, they are able to accomplish the"one-step"conversion of substrates to products. Coimmobilized enzymes offer an extraordinary application protential in future. Currently, co-immobilization ofβ-galactosidase and glucose isomerase for preparation lactulose has not been reported. In this research, gelatin and porous glass were used to as the carrier for co-immobilization ofβ-galactosidase and glucose isomerase. These co-immobilized enzymes were then applied to provide a new way for preparation lactulose.A thin-layer chromatography method for determinating lactulose was established, in which the mobile phase was butanol-alcohol-water (5:3:2, v/v/v). Lactulose was developed by ascending technique on the silica gel G plate, and visualized by aniline–diphenylamine–phosphoric acid. To determine the chromatographic peak areas of lactulose, mottles with thin-layer scanner in the sawtooth scanning mode is described. The detective wavelength was 526 nm. This method is simple and cost effective. It was suitable for batch determination of lactulose. HPLC was employed to analyze the products of enzymatic reaction, and it showed higher sensitivity and precision compared with TLC method. However, the accuracy of TLC method, was similar with that of HPLC method. Since HPLC method can determine only one sample at one time and its cost is higher, TLC method was still used in some cases in this research.Immobilization efficiency of gelatin, open pore gelatin cube andκ-carrageenan were compared and gelatin was selected as the carrier. Using the method of embedding -crosslinking,β-galactosidase and glucose isomerase were co-immobilized. The optimal immobilization conditions were optimized as that the pH was 8.6, gelatin concentration was 27% (w/w), glutaraldehyde concentration in the first and second cross-linking was 0.15% (v/v), the temperature and time of the second cross-linking were 15℃and 10 min. Under these conditions, the activity recoveries ofβ-galactosidase and glucose isomerase were 30.85% and 83.48%, respectively.The porous glass was prepared by sol-gel method. This method was simple and the size of particles was uniform. The preparation procedure wasas followed. The sol pH was adjusted to 3 by HCl and dehydrated at 60℃in order to transform to gel. With this process, the sol solution was clear and the gel solid was transparent. The porous glass prepared was used as carrier andβ-galactosidase and glucose isomerase were co-immobilized with covalent coupling method. Considering the activity recoveries, the optimum conditions were investigated through single factor experiments. After mixing porous with toluene andγ-APTES ((3-aminopropyl) trietoxysilane), the mixture was placed in 110℃oil bath for 3 h. The medium was dropped into 0.5% glutaraldehyde solution and reacted for another 90 min. The activity recoveries ofβ-galactosidase and glucose isomerase were 20.13% and 13.93%, respectively.The optimal temperature and pH for co-immobilized enzymes with gelatin were 45℃and 7.5, respectively. The remained activity of co-immobilized enzymes was no less than 80% after repetitive operation for six times. The efficiency with this mild method was satisfactory. The optimal temperature and pH of co-immobilized enzyme with porous glass as carrier were 45℃and 7.5, respectively. Furthermore, the thermal stability, pH stability and operating stability is better than that with gelatin as carrier. The co-immobilized enzyme activity still maintained 70% after repetitive operation for ten times. Although the co-immobilized efficiency of porous glass was lower, the co-immobilized enzyme has higher mechanical strength and could be reused more times.The conditions for lactulose production were pH 7.5, reaction temperature 45℃, reaction time 12 h, and lactose concentration 40%. The yield of lactulose production were 36.50 and 45.59 mg/mL, catalyzed by co-immobilized enzymes with gelatin and porous glass as carrier, respectively.
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