Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering.
Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering.
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通过基因突变和蛋白质工程修饰甜味蛋白莫内林的甜度和稳定性。
DOI：
10.1155/2016/3647173
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发表时间：
2016
影响因子：
--
通讯作者：
Liu B
中科院分区：
文献类型：
--
作者：
Liu Q;Li L;Yang L;Liu T;Cai C;Liu B
天然甜蛋白Monellin具有高甜度和低卡路里的特点，表明其在食品应用中的潜力。然而，由于其耐热性和耐酸性较低，限制了其应用。在本研究中，我们通过测序、结构分析和定点突变的方法证明了Monellin的热稳定性可以在不降低甜度的情况下得到改善。我们分析了位于α-螺旋上的残基以及一个可电离的残基C41。在所研究的突变体中，E23A和C41A突变体的效果最为显著。前者热稳定性显著提高，甜度不变。突变蛋白在85℃下孵育30 m in后稳定，甜度增加，热稳定性略有改善。此外，我们还发现，大多数提高蛋白质热稳定性的突变体分布在α-螺旋的两端。分子生物物理分析表明，可电离残基的埋藏状态可能解释了突变蛋白质的调制性质。我们的结果证明，甜蛋白Monellin的性质可以通过生物信息学分析、基因操作和蛋白质修饰来进行修饰，这突显了基于结构-功能关系设计新型有效甜蛋白的可能性。
Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in the α-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of α-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships.