Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

Abstract

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of ␣-1,4 glucans to produce cyclic ␣-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

Large-ring Cyclodextrins

“…This suggests that a yield of LR-CDs with a high degree of polymerization would be improved if only the coupling reaction was inhibited. Fujii et al made amylomaltase mutants by introducing random mutations into the gene coding for this enzyme as proof (71,72). In 2002, it was found that glycogen debranching enzyme (GDE, EC 2.4.1.25/EC 3.2.1.33) from Saccharomyces cerevisiae was also able to produce a LR-CD mixture (73).…”

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

“…This second binding site was proposed to help form a CA by the hydrophobic interaction of Tyr-54 and Tyr-101 with the substrate (37). The mutation of these residues affected the hydrolysis activity of the TaAM enzyme, which played an important role in product formation (8,9). A novel AM from the mesophilic bacterium Corynebacterium glutamicum ATCC 13032 (CgAM) has recently been reported, and interestingly, it has only a 28% amino acid sequence similarity to TaAM (36).…”

The Concept of the .ALPHA.-Amylase Family: A Rational Tool for Interconverting Glucanohydrolases/Glucanotransferases, and Their Specificities

“…Although the thermostability was high enough to be used for the industrial scale, the yield of cycloamylose using Thermus aquaticus (Taq) amylomaltase decreased to 60% at the end point of the reaction since the enzyme significantly exhibited hydrolytic activity. 47,48) In such a case, we have two ways to overcome the problems. One is enhancing the thermostability of potato D enzyme, and the other is erasing the hydrolytic activity of Taq amylomaltase.…”

“…Indeed, we successfully enhanced the thermostability of potato type L α glucan phosphorylase 49) and Streptococcus sucrose phosphorylase. 50) We chose the latter strategy for Taq amylomaltase 47,48) (Fig. 3).…”

“…The yield of cycloamylose using the engineered enzyme remained at more than 90% until the end of reaction, while the reducing power was not significantly increased. 47,48) We are going further to improve enzymes based on the concept of the α amylase family as a rational tool for designing and engineering industrially useful enzymes.…”