Molecular dynamic analysis of mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to γ-cyclodextrin

Abstract

Alpha-cyclodextrin (α-CD) glycosyltransferase (α-CGTase) can convert starch into α-CD blended with various proportions of β-cyclodextrin (β-CD) and/or γ-cyclodextrin (γ-CD). In this study, we verified the catalytic characteristics of purified Y195I α-CGTase and elucidated the mechanism of action with molecular dynamic (MD) simulations. We found that purified Y195I α-CGTase produced less α-CD, slightly more β-CD, and significantly more γ-CD than wild-type α-CGTase. Correspondingly, α-CD-based K m values increased, and β-CD- and γ-CD-based K m values decreased. MD simulation studies revealed that the dynamic trajectories of the substrate oligosaccharide chain in the mutant CGTase binding site were significantly different from those in the wild-type enzyme, with reduced hydrophobic interaction, finally resulting in different product specificity and more γ-CD formation.

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

“…In general, the products of CGTase reactions contain α-, β-, and γ-CD. , The purification of a single type of cyclodextrin from a CD mixture requires several steps, which can be time-consuming and disadvantageous for human health and the environment . Therefore, to enable an efficient purification process, CGTases with high product specificity have been developed. ,,− However, compared to other kinds of CGTases, α-CGTases show relatively low expression levels and their product ratio is usually lower than 90%, which limits the potential application of α-CD in many fields. ,,, To overcome this problem, another approach, “the solvent process”, has been developed. This method involves the addition of organic solvent as a complexing agent to extract target CDs selectively. − For example, in the presence of 1-butanol, the α:β CD formation ratio of the CGTase increased dramatically to 97:3 with a 42% (w/w) conversion rate.…”

Application of cyclodextrinase in non‐complexant production of γ‐cyclodextrin

“…As previously reported by Xie et al, the product specificity of α-CGTase is converted from α-CD to γ-CD by replacing the Tyr195 with Phe. 17 In addition, these results are further proved by Pei et al 18 However, in industrial scale production, complexant addition method is generally used to enhance γ-CD production and purification. 4 The complexant addition method requires organic complexants such as 5-cyclohexadecen-1-one, cyclohexadecen, or glycyrrhizin to extract γ-CD selectively and thus directing the enzymatic reaction to produce the γ-CD.…”

Engineering starch by enzymatic structure design for versatile applications in food industries: a critical review

“…Hydrolysis, in which water molecules act as receptors to glucose chains, causes the hydrolysis of the substrate. Disproportionation reaction involves the transfer of one linear molecule to another [69]. Coupling reaction is the reverse reaction of cyclodextrin, which can decompose cyclodextrin.…”