Abstract
Backgroundα-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.ResultsIn 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.ConclusionsMg/Mr-αGlu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant α-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.
Highlights
The already proven relationship between food and health has promoted a growing biotechnological interest in functional foods and nutraceutical ingredient markets, where bioactive oligosaccharides are gaining relevance among compounds such as dietary fibres, peptides, polyols or unsaturated fatty acids [1,2,3]
Isolation and heterologous expression of α‐glucosidases from two Metschnikowia species As mentioned before, we have recently reported a potential α-glucosidase activity in M. reukaufii cell extracts that was able to hydrolyse sucrose and produced, by transglucosylation, different honey oligosaccharides, mainly isomelezitose and trehalulose [32]
In order to isolate the gene which encodes the α-glucosidase that could be responsible for the activity previously detected in M. reukaufii cell extracts, oligonucleotides directed to the ID CM010598.1 sequence ends were designed and used in PCR reactions containing genomic DNA from M. gruessii and M. reukaufii
Summary
The already proven relationship between food and health has promoted a growing biotechnological interest in functional foods and nutraceutical ingredient markets, where bioactive oligosaccharides are gaining relevance among compounds such as dietary fibres, peptides, polyols or unsaturated fatty acids [1,2,3]. In GlcOS, α-glucose units can be connected by α-(1→6), α-(1→4), α-(1→3) or α-(1→2) links giving rise to isomalto-oligosaccharides (IMOs), malto-oligosaccharides (MOS), nigero-oligosaccharides and koji-oligosaccharides, respectively [6]. Several bioactive oligosaccharides including FOS (inlulin and 1F-FOS serie), GOS, GlcOS (basically IMOS), lactulose, lactosucrose and raffinose have been recognized as prebiotics agents promoting the growth of beneficial bacteria, mainly Bifidobacterium spp. and Lactobacillus spp. [12, 13] Due to their technofunctional properties, bioactive oligosaccharides have applications in food technology as bulking and moisture retaining agents, fat and sugar substitutes, textural enhancers, and non-cariogenic ingredients, which give them great biotechnological utility [14, 15]
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