Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: I45K, A357S, I20A, I20V, and I20E
Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: I45K, A357S, I20A, I20V, and I20E
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多粘类芽孢杆菌β-葡萄糖苷酶 B 突变体的数据设计：I45K、A357S、I20A、I20V 和 I20E
DOI：
10.1101/2020.10.07.330233
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Siegel, J.B.
中科院分区：
文献类型：
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作者：
Luong, J.A.;Vater, A.;Siegel, J.B.
酶中生物物理突变效应的大多数当前数据集的相对较小的大小和范围限制了开发数据驱动算法的能力，从而能够实现用于设计新酶功能的精确生成建模工具。本文对多粘类芽孢杆菌（Paenibacilluspolymyxa，BglB）β-葡萄糖苷酶B的5个新突变体的米氏常数（kcat，KM，kcat/KM）和热稳定性（TM）进行了表征。利用Foldit软件建立突变体的分子模型，构建突变体的合成基因，并弗罗姆中制备和纯化相应的蛋白。杆菌发现破坏活性位点附近预先存在的氢键的突变与不影响天然氢键的相同位点处的突变相比具有降低的表达。这与先前显示氢键和酶功能之间的关系的结果一致。这些突变体有助于一个不断增长的数据集>100突变体已被表征的表达，动力学，和热性能
The relatively small size and scope of most current datasets of biophysical mutation effects in enzymes limit the ability to develop data-driven algorithms enabling accurate generative modeling tools for designing novel enzyme function. Here, the Michaelis-Menten constants (kcat, KM, andkcat/KM) and thermal stability (TM) of five new mutations of β-glucosidase B fromPaenibacillus polymyxa(BglB) are characterized. Foldit software was used to create molecular models of the mutants, for which synthetic genes were constructed and the corresponding proteins produced and purified fromE. coli. It was found that mutations that disrupted pre-existing hydrogen bonds near the active site had reduced expression in contrast to mutations at the same site that did not affect native hydrogen bonding. This is consistent with previous results showing the relationship between hydrogen bonding and enzyme functionality. These mutants contribute to a growing data set of >100 mutants that have been characterized for expression, kinetic, and thermal properties