| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 102 === Abstract
D-Allose is a C-3 epimer of D-glucose, which have only slightly amount existed in nature. It has many physiological activities including inhibiting cancer cell proliferations, anti-tumor, decreasing oxidative pressures and immunosuppression. Moreover, it possesses nearly no calories and toxicity, and therefore has a potential to be used as a food additive. L-Rhamnose isomerase (L-RhI) can catalyze sugar isomerization between aldoses and ketoses. Some L-RhIs have a broad range of substrate selectivity, they can be used in the presence of AsDPE to produce D-allose from D-fructose which is a cheap material, and the intermediate product D-psicose is also an expensive and functional sugar.
This study aims to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 L-RhI against D-allose by protein engineering. First, we performed the computer simulation to obtain modeling structures, and docked target substrates to the modeling structures. After analyzing relative sites of the substrate binding, residue Ile102 was mutated to other nine polar or charged amino acid residues, and residues Phe142 and His100 were mutated to six other polar or charged amino acid residues with similar side-chain lengths. Quick-screening result shows that mutant I102N Ts L-RhI has higher relative specific activity against L-rhamnose, mutant I102N and I102R Ts L-RhIs have higher relative specific activities against D-allose. Comparing to wild-type Ts L-RhI, I102N and I102R Ts L-RhIs have more preference against D-allose, while I102Y Ts L-RhI has more preference against L-rhamnose. Mutations on both Phe142 and His100 residues have resulted poor relative specific activities and only mutant H100D Ts L-RhI has more preference against D-allose than wild-type. We also have performed several partial sequence replacements around Leu59 which is located in a lid-like loop region, and the quick-screening result shows that double mutant L59M/S60G Ts L-RhI has more preference against D-allose. In a consequence of their lower KM values, mutant I102Y, I102Q and L59M/S60G Ts L-RhIs have higher catalytic efficiencies against L-rhamnose, and I102N, I102R, I102Q, L59M/S60G Ts L-RhIs have higher catalytic efficiencies against D-allose comparing to wild-type Ts L-RhI. The wild-type and mutant Ts L-RhIs were used in the presence of AsDPE to produce D-allose from 10% (w/v) D-fructose, there are no significantly differences in final predicted yields between wild-type and mutant Ts L-RhIs, and the final predicted yields can reach 22% D-psicose and 15% D-allose. Mutant I102Q Ts L-RhI consumed less time to reached the final predicted yields suggesting that this enzyme has the potential to be used in D-allose production.
Keywords : Protein engineering, L-rhamnose isomerase, Substrate specificity.
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