Department of Molecular Biology, Centre for Molecular Biology Severo Ochoa (CSIC-UAM), University Autonomous from Madrid. C/Nicolás Cabrera, 1. Cantoblanco, 28049, Madrid, Spain.
Institute of Catalysis and Petrochemistry, CSIC, C/Marie Curie, 2. Cantoblanco, 28049, Madrid, Spain.
Microb Cell Fact. 2020 Jul 11;19(1):140. doi: 10.1186/s12934-020-01397-y.
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.
Mg/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.
α-葡萄糖苷酶是一种分布广泛的酶，具有多样化的底物特异性，传统上被用于以寡糖和多糖为起始原料的生物技术产业。根据氨基酸序列同源性，α-葡萄糖苷酶被分为两个主要家族，GH13 和 GH31。家族 GH13 的成员包含几种已被证实具有蔗糖水解活性的α-葡萄糖苷酶。此前，从花蜜定殖酵母 Metschnikowia reukaufii 中描述了一种蔗糖分解活性，该酵母能够从蔗糖中产生具有 α-(1→1)、α-(1→3)和 α-(1→6)糖苷键的罕见糖。
本研究首次对来自花蜜酵母 M. gruessii 和 M. reukaufii 的 α-葡萄糖苷酶基因进行了特征描述和异源表达。纯化并对重组蛋白（Mg-αGlu 和 Mr-αGlu）进行了生化分析。这两种酶主要对蔗糖、麦芽糖和对硝基苯-α-D-吡喃葡萄糖苷表现出水解活性。对这些蛋白的结构分析鉴定了来自 α-淀粉酶家族的共同特征，特别是属于 GH13 家族的糖苷水解酶。鉴定了包含催化三联体的三个酸性残基，并通过定点突变证实了它们对蛋白水解机制的重要性。用初始底物蔗糖生产了天然存在于蜂蜜中的低聚糖，并产生了与以前用 M. reukaufii 细胞提取物获得的相同产物谱（异麦牙糖、海藻糖、艾杜糖、蜜二糖、曲二糖和松二糖）的混合物。此外，还检测到了与其同源物 Mg-αGlu 相同的酶活性。有趣的是，由重组蛋白介导的反应中获得的异麦牙糖量高达 170g/L，是迄今为止报道的最高值。
异源过表达了 Mg/Mr-αGlu，并对其生化和结构特征进行了分析。重组 α-葡萄糖苷酶在温和的反应条件下具有优异的性质，此外还具有 pH 和热稳定性。此外，该酶通过转糖基作用产生了一种罕见的异葡糖寡糖混合物，主要是异麦牙糖和海藻糖。这些化合物是蜂蜜的天然成分，从这种天然来源中纯化是不可行的，这使得这些酶对生物技术产业非常有吸引力。最后，值得注意的是，由于这些糖具有合适的甜度、黏度和保湿能力，因此它们具有作为食品添加剂的潜在应用。
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