Abstract
Abstract Sucrose-active enzymes belonging to the glycoside hydrolase (GH) family 70 are attractive tools for the synthesis of oligosaccharides, polysaccharides or glycoconjugates. However, their thermostability is an important issue for the development of robust and cost-effective enzyme-based processes. Indeed, GH70 enzymes are mesophilic and no thermophilic representatives have been described so far. Furthermore, structurally guided engineering is a challenge given the size of these proteins (120 to 250 kDa) and their organization in five domains. Herein, we have investigated the possible role of the domain C in the stability of GH70 enzymes. The alternansucrase (ASR) is the most stable enzyme of the GH70 family. Structural comparison of ASR to other GH70 enzymes highlighted the compactness of its domain C. We assumed that this atypical structure might be involved in the stability of this enzyme and decided to introduce this domain in another much less stable GH70 enzyme of known three-dimensional structure, the branching sucrase GBD-CD2. The chimeric GBD-CD2 exhibited a lower specific activity on sucrose substrate but its specificity was unchanged with the enzyme remaining specific for the branching of dextran via α-1,2 linkage formation. Interestingly, the chimera showed a higher melting temperature and residual activity than the wild-type enzyme after 10 min incubation at 30 °C showing that the domain C can affect GH70 enzyme stability and could be a potential target of both random or rational mutagenesis to further improve their stability.
Highlights
The alternansucrase (ASR) and the α-1,2 branching sucrase GBD-CD2 (GBD and CD stand for glucan binding domain and catalytic domain, respectively) are promising enzymes for the development of industrial applications including polysaccharide, oligosaccharide or glycoconjugate synthesis via the glucosylation of many different acceptors from sucrose substrate, an abundant and low-cost resource [1-5]
Four ionic interactions can be predicted in the domain C of ASR against two for DSR-M, GTF-SI and GTF180 and none for GTFA and GBD-CD2
Sequence alignment of the domain C of ASR and GBD-CD2 shows that the two domains share 49.64% of identity, while their extremities are highly conserved (Fig. 2)
Summary
The alternansucrase (ASR) and the α-1,2 branching sucrase GBD-CD2 (GBD and CD stand for glucan binding domain and catalytic domain, respectively) are promising enzymes for the development of industrial applications including polysaccharide, oligosaccharide or glycoconjugate synthesis via the glucosylation of many different acceptors from sucrose substrate, an abundant and low-cost resource [1-5]. These efficient α-transglucosylases belong to the family 70 of the glycoside hydrolases (GH) according to the CAZy classification [6]. It is this hypothesis that we wanted to study here by creating a chimeric protein comprising domains A, B, IV, V of GBD-CD2 grafted onto the C domain of ASR and by characterizing the biochemical properties of the chimera obtained
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