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A glucotolerant β-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds.
Microbial Cell Factories ( IF 4.3 ) Pub Date : 2020-06-10 , DOI: 10.1186/s12934-020-01386-1
Juan Antonio Méndez-Líter 1 , Manuel Nieto-Domínguez 1 , Beatriz Fernández de Toro 2 , Andrés González Santana 3 , Alicia Prieto 1 , Juan Luis Asensio 3 , Francisco Javier Cañada 2 , Laura Isabel de Eugenio 1 , María Jesús Martínez 1
Affiliation  

The interest for finding novel β-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris, purified, and characterized. The enzyme showed good efficiency on p-nitrophenyl glucopyranoside (pNPG) (Km= 3.36 ± 0.7 mM, kcat= 898.31 s−1), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the β-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-d-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different pNP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG). In this work, we report the characterization of a novel glucotolerant 1,2-β-glucosidase, which also has a considerable activity on 1,4-β-glucosyl bonds, that has been cloned in P. pastoris, produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications.

中文翻译:

一种来自真菌Talaromyces amestolkiae的糖耐量β-葡萄糖苷酶,其转化为用于酚类化合物糖基化的糖合酶。

寻找新型的β-葡萄糖苷酶可以提高第二代(2G)生物燃料产量的兴趣仍然很高。这些酶最需要的特征之一是葡萄糖耐量,这使得它们在高葡萄糖浓度下具有最佳活性。此外,将注意力集中在寻找用于糖苷合成的新型酶替代物中,近年来,糖苷酶的突变形式,称为糖合酶,引起了人们的极大兴趣。在这项工作中,来自子囊真菌Talaromyces amestolkiae的耐糖性β-葡萄糖苷酶(BGL-1)已在巴斯德毕赤酵母中异源表达,纯化和鉴定。该酶对对硝基苯基吡喃葡萄糖苷(pNPG)表现出良好的效率(Km = 3.36±0.7 mM,kcat = 898.31 s-1),但对纤维素寡糖有活性 这些酶的天然底物含量要低得多,这可能会限制其在木质纤维素降解应用中的利用。有趣的是,当检查BGL-1的底物特异性时,它对槐糖糖(葡萄糖的β-1,2二糖)的活性高于纤维二糖。此外,检查了BGL-1的转糖基化分布,并且为了扩大其合成能力,将其转化为糖合酶。该突变酶名为BGL-1-E521G,能够在糖基化反应中使用α-d-葡萄糖基氟化物作为供体,并以区域选择性方式合成不同pNP糖的糖基化衍生物,以及一些酚类化合物的合成。工业利益,例如表没食子儿茶素没食子酸酯(EGCG)。在这项工作中,我们报告了一种新型的耐糖1,2-β-葡萄糖苷酶的表征,它对已被克隆到巴斯德毕赤酵母中的1,4-β-葡萄糖基键也具有相当大的活​​性,产生,纯化和鉴定。另外,该酶被转化为有效的糖合酶,能够将葡萄糖分子转移至多种受体以获得所需的化合物。BGL-1及其糖合酶突变体在水解和合成方面均具有非凡的功能,这表明它可能是生物技术应用中一个有趣的工具。
更新日期:2020-06-10
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