| D-Tagatose is a ketohexose rarely found in nature. D-tagatose has a great potential for use in a variety of applications, such as food, medicine, health care products. It is important to develop a method for its mass production.1. The laboratory isolated a thermophilic strain possessing L-AI gene from hot spring sludge. The results of physiological and biochemical analysis and16S rDNA homology comparison identified it as Anoxybacillus flavithermus TC-06. The results showed that the strain could be cultivated in a wide range of growth temperature (45-70℃) and pH (5.5-9.5) conditions, while its growth optimum temperature and pH values were55℃and8.5, respectively.2. The gene encoding L-AI from A. flavithermus (AFAI) was cloned by PCR and expressed at a high level in E. coli BL21(DE3). SDS-PAGE results show that the molecular weight of the recombinant protein must be56.3kDa, the recombinant protein comprised of22.5percent of total proteins in the cell.3. The cells were harvested by centrifugation at4℃for10min at12,000rpm. They were disrupted by sonication, and the suspension was centrifuged at4℃for10min at12,000rpm. The supernatant was heated at80℃for5min and centrifuged at4℃for10min at12,000rpm to remove host proteins. The enzyme was purified using an Ni-NTA affinity column after heat treatment. It had more than90%purity and a specific activity of26.4U/mg.4. The optimum pH and temperature is9.5-10.5and95℃, respectively. The results indicate that the recombinanted L-AI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for D-galactose. The activity of the enzyme and thermostability were totally independent for metallic ions. Its activity was enhanced by borate, as the catalytic efficiency (kcat/Km) for D-galactose with borate was twice as much as that without borate.5. Under optimum conditions the recombinanted L-AI could convert D-galactose into D-tagatose, and the highest conversion yield was more than60%for60min at95℃. |
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