| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
The aim of this project was to establish new methods to synthesize alpha-linked oligosaccharides using engineered carbohydrate-hydrolysing enzymes. I focused the following two points : 1. establishment the new reaction suitable for oligosaccharide synthesis using catalytic residue-mutated "inactive" hydrolases. 2. changing specificities of the enzyme and screening enzymes showing novel specificities. In the point-1, a new basic method effective for oligosaccharide synthesis was developed, and through the point-2, wide varieties of oligosaccharide would be prepared.
Point-1, catalytic residues mutants of dextran glucosidase from Streptococcus mutans oligo-1,6-glucosidase from Bacillus subtilis, and other enzymes were generated, and applied for oligoshaccharide synthesis. The conditions were optimized. One example was panose produced from alpha-glucosyl fluoride and maltose in high yield of 71% by the "inactive" hydrolase, while the wild-type "active" enzyme also produced panose, but subs
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equently hydolrolysed it, resulting in low yield of 16%.
Point-2. Substrate specificity was changed in some enzymes. Dextran glucosidase from S. mutans was the mostly engineered enzyme. Dependency of activity on chain length of substrates was modulated, and a mutant dextran glucosidase lost activity for dextran, but preferred trisaccharide. The strictly restricted alpha-1,6 specificity was also altered into alpha-1,4 specificity without significant decrease in reaction velocity. E. coli alpha-xylosidase was also successfully engineered to be an alpha-glucoside hydrolase. Point-2B, the wide variety of enzymes were screened. Alpha-glucosidases from some varieties of rice and honeybee were purified, characterized, and sequenced, as well as recombinant protein production. Particularly, we found that some rice enzymes were able to act on starch granules. Alpha-glucosidase I from European honeybee exhibited very high transglucosylation even though high amino acid identity of 40% was retained with alpha-glucosidase II and III from the honeybee. Less
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