Conversion of .ALPHA.-Amylase into Transglycosylation Enzyme.

DOI Web Site Web Site

Bibliographic Information

Other Title
  • タンパク質工学によるα‐アミラーゼの糖転移酵素化
  • タンパクシツ コウガク ニ ヨル アルファ アミラーゼ ノ トウ テンイ コウ

Search this article

Description

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 (G4), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G4. 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 (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) 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.

Journal

Details 詳細情報について

Report a problem

Back to top