11 July, 2020

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp


α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo- and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described.

In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-D-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far.

Ref.:"Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugarsMartin Garcia-Gonzalez, Marina Minguet-Lobato, Francisco J. Plou & Maria Fernandez-Lobato Microbial Cell Factories volume 19, Article number: 140 (2020) https://doi.org/10.1186/s12934-020-01397-y