タンパク質工学によるα-アミラ-ゼの糖転移酵素化 (アミラ-ゼシンポジウム(1991)特集)
スポンサーリンク
概要
- 論文の詳細を見る
The 210 th lysine residue in Saccharomycopsis α-amylase (Sfamy) molecule was replaced by arginine and asparagine residues. The resulting K210R and K210N enzymes cleave mainly the first glycosidic bond from the reducinng end of maltotetraose (G<SUB>4</SUB>), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G<SUB>4</SUB>. We estimated the 8th subsite affinities of the mutant enzymes and compared them with that of the native enzyme. These facts suggest that the K210 residue composes the 8th subsite, one of the major subsites, and that a positively charged s-amino residue is necessary for the 8th subsite affinity. The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite. The 84th tryptophan residue was replaced by leucine residues. The resulting W84L enzyme showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G<SUB>7</SUB>), for example, maltooligosaccharide products larger than maltodecaose (G<SUB>10</SUB>) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. These large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the W84 residue located at subsite 5 plays an important role in the addition of a water molecule to a carbonium ion intermediate and/or in the liberation of the hydrolysis product from the substrate binding pocket. The doubly mutated enzymes, W84LK210 N, are expected to form the transglycosylation products different in size from those produced by the single mutant, W84L enzyme.
- 日本応用糖質科学会の論文
日本応用糖質科学会 | 論文
- 古細菌の産生する新規な糖質関連酵素およびそれらを用いたトレハロースの生産
- PCR法による米 (Oryza sativa L.) の品種判別およびその米加工品への応用
- モチ米のアミロペクチン鎖長分布と米粒の物理特性との関係
- ジャポニカ米とインデカ米の澱粉とプロテインボディの結合脂質について
- 促通拡散型糖輸送担体分子の構造機能相関