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Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars.
Microbial Cell Factories ( IF 4.3 ) Pub Date : 2020-07-11 , DOI: 10.1186/s12934-020-01397-y
Martin Garcia-Gonzalez 1 , Marina Minguet-Lobato 1 , Francisco J Plou 2 , Maria Fernandez-Lobato 1
Affiliation  

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.

中文翻译:

两种蜜糖生产者Metschnikowia spp的两种α-葡萄糖苷酶的分子表征和异源表达。

α-葡糖苷酶是具有广泛底物特异性的广泛分布的酶,传统上以寡糖和多糖为原料,用于生物技术行业。根据氨基酸序列的同源性,α-葡糖苷酶被分为两个主要家族,GH13和GH31。GH13家族的成员含有几种α-葡糖苷酶,具有确定的蔗糖水解活性。以前,描述了来自花蜜定植的酵母Metschnikowia reukaufii的蔗糖分裂活性,其从蔗糖产生具有α-(1→1),α-(1→3)和α-(1→6)糖苷键的稀有糖。在这项研究中,首次对在蜜腺酵母格鲁埃斯氏菌和reukaufii菌中编码α-葡萄糖苷酶的基因进行了表征,并在大肠杆菌中进行了异源表达。重组蛋白(Mg-αGlu和Mr-αGlu)经过纯化和生化分析。两种酶均主要表现出对蔗糖,麦芽糖和对硝基苯基-α-d-吡喃葡萄糖苷的水解活性。这些蛋白质的结构分析允许从α-淀粉酶家族,特别是从属于GH13家族的糖苷水解酶中鉴定共同特征。确定了构成催化三联体的三个酸性残基,并通过定点诱变证实了它们与蛋白质水解机制的相关性。重组酶以蔗糖为初始底物产生天然存在于蜂蜜中的寡糖,并产生了混合物,其混合物具有先前用reukaufii细胞提取物获得的相同产品特征(异三聚糖,海藻糖,麦芽糖,三聚糖,金刚糖和艾草糖)。此外,用来自格鲁塞酵母的直系同源Mg-αGlu检测到相同的酶活性。有趣的是,在重组蛋白介导的反应中获得的异三聚糖量约为170 g / L,是迄今为止报道的最高水平。Mg /Mr-αGlu异源过量生产,并对其生化和结构特征进行了分析。除了pH和热稳定性外,重组α-葡萄糖苷酶在温和的反应条件下显示出优异的性能。此外,这些酶还通过转糖基化作用产生了稀有的杂葡萄糖寡糖混合物,主要是异三聚糖和海藻糖。这些化合物是蜂蜜的天然成分,从这种天然来源纯化是完全不可行的,这使得这些酶对于生物技术行业而言非常有趣。最后,
更新日期:2020-07-13
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