| Summary: | 博士 === 國立臺灣海洋大學 === 食品科學系 === 102 === L-Arabinose isomerase (EC 5.3.1.4, L-AI) mainly catalyzes the reversible aldose-ketose isomerization between L-arabinose and L-ribulose. L-AIs can also catalyze other reactions, such as the conversion of D-galactose to D-tagatose. In this study, the araA gene encoding L-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant L-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7−7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55 to 65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn2+ or 0.05 mM Co2+, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 L-AI has the lowest demand for metal ions among all characterized thermophilic L-AIs.
This thermophilic T. saccharolyticum NTOU1 L-AI can used to produce rare sugars, such as the production of D-tagatose from D-galactose and the producton of L-ribose from L-arabinose. The conversion yield of D-tagatose from D-galactose by using the L-AI at 60 oC was around 20% after 10 h, and around 41% when the reaction was approaching equilibrium (50 h). For the production of L-ribose, L-arabinose isomerization was first performed at 60 ºC for 6 h using T. saccharolyticum NTOU1 L-AI. After 6 h of the L-AI reaction, G. obscurus DSM43160 L-RI was added, and the reaction was continued at 30 ºC or 40 ºC for an additional 6 h. The conversion yields of L-arabinose to L-ribose by combining the L-AI and L-RI at 30 ºC and 40 ºC were 15.9% and 12.5% (mol mol-1), respectively.
To increase the activity of L-AI at lower reaction temperatures and lower pH values, we have constructed 6 mutant L-AIs, including N172H, V259K, V259E, V259Q, V259S, and I402A. Compare to wild-type L-AI, mutant N172H and I402A L-AIs have increased their avtivities against D-galactose at 40~80℃. N172H L-AI has an optimum temperature at 70℃ which is same as that of wild-type, while I402A L-AI has an optimum temperature at 65℃. And, mutant V259K L-AI has 21~33% increased activity in the pH range of 6.5~7.5.
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