Authors: Garcia-Gonzalez, M; Minguet-Lobato, M; Plou, FJ; Fernandez-Lobato, M
Microb. Cell. Fact.. vol: 19. page: .
Date: jul-11. 2020.
Background: alpha-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, alpha-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several alpha-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with alpha-(1 -> 1), alpha-(1 -> 3) and alpha-(1 -> 6) glycosidic linkages from sucrose was described. Results: In this study, genes codifying for alpha-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-alpha Glu and Mr-alpha Glu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-alpha-d-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the alpha-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, the and erose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-alpha Glu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, similar to 170 g/L, were the highest reported so far. Conclusions: Mg/Mr-alpha Glu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant alpha-glucosidases displayed excellent properties in terms of mild reaction conditions, in addition to pH and thermal stability. Besides, the enzymes produced a rare mixture of hetero-gluco-oligosaccharides by transglucosylation, mainly isomelezitose and trehalulose. These compounds are natural constituents of honey which purification from this natural source is quite unviable, what make these enzymes very interesting for the biotechnological industry. Finally, it should be remarked that these sugars have potential applications as food additives due to their suitable sweetness, viscosity and humectant capacity..