| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 103 === D-Allose has only rare amounts in nature, and it belongs to high price rare sugars. It has many physiological activities including inhibiting cancer cell proliferations, anti-tumor and immunosuppression. L-Rhamnose isomerase (L-RhI) can catalyze sugar isomerization between aldoses and ketoses. L-RhIs can be used in the presence of D-Psicose-3-epimerase to produce D-allose from D-fructose, and the intermediate product D-psicose is also an expensive sugar. To reuse the enzyme by immobiling on chitin, this study aims to fuse signal peptide and chitin binding domain sequences with Thermoanaerobacterium saccharolyticum NTOU1 L-RhI (TsRhI) gene by gene engineering then transform it into Bacillus subtilis WB800N to express CBD-fused TsRhI. SDS-PAGE analysis and activity assays were used to investigate the CBD-fused TsRhI expression and secretion. But the expression is so rare and the extracelluar activity of enzyme is only between 0.05 and 0.08 U/mL. We speculate that the restriction recognition sequence between start codon and ribosome binding site might cause less expression and secretion. By way of gene deletion to remove restriction recognition sequence, the secretion has still no improvement. Thereafter, the immobilized cells of Cleancoli BL21 (DE3) carrying pHT254-TsRhI prepared by entraping cells with sodium alginate were studied for reusing the enzyme. The optimal conditions of immobilization are 3% alginate, 142.8 g/L cell mass, 0.3 M CaCl2, and 0.1% CTAB. Since Cleancoli BL21 (DE3) cells carrying both pET21b-TsRhI and CodonPlus-RIL plasmids can express TsRhI very well, the cells have been immobilized and characterized. The immobilized cells have been stored in 4˚C and room temperature for 12 days, and still have 80% activity. The TsRhI in the immobilized cells has the optimal temperature at 80˚C, and the activity was fully retained after 2 h incubation at 40 - 75˚C. The enzyme in the immobilized cells is stable at pH range 5 – 9, and still has 80% activity after repeatedly used 18 times.
The TsRhI-containing immobilized cells were used in the presence of AsDPE-containing immobilized cells at the ratio of activity AsDPE : TsRhI = 1 : 14 to produce D-allose from 30% (w/v) D-fructose in 50 mM Tris-HCl buffer (pH 7.0) plus 0.25 mM Co2+ at 60˚C. HPLC was used to analyze the amounts of sugars in the converted reaction mixture. The reaction can reach equilibrium with yields of 30.1% D-psicose and 9.2% D-allose after 6 h.
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