Volume 6, Issue 1

The CAZy database is a web-server for sequence-based classification of carbohydrate-active enzymes that has become the worldwide and indispensable tool for scientists engaged in this research field. It was originally created in 1991 as a classification of glycoside hydrolases (GH) and currently, this section of CAZy represents its largest part counting 172 GH families. The present Opinion paper is devoted to the specificity of α-amylase (EC 3.2.1.1) and its occurrence in the CAZy database. Among the 172 defined GH families, four, i.e. GH13, GH57, GH119 and GH126, may be considered as the α-amylase GH families. This view reflects a historical background and traditions widely accepted during the previous decades with respect to the chronology of creating the individual GH families. It obeys the phenomenon that some amylolytic enzymes, which were used to create the individual GH families and were originally known as α-amylases, according to current knowledge from later, more detailed characterization, need not necessarily represent genuine α-amylases. Our Opinion paper was therefore written in an effort to invite the scientific community to think about that with a mind open to changes and to consider the seemingly unambiguous question in the title as one that may not have a simple answer.

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.