Hall B G
Biochemistry. 1981 Jul 7;20(14):4042-9. doi: 10.1021/bi00517a015.
Wild-type ebg enzyme, the second beta-galactosidase of Escherichia coli K12, does not permit growth on lactose. As part of a study of the evolution of new enzymatic functions, I have selected, from a lacZ deletion strain, a variety of spontaneous mutants that grow on lactose and other beta-galactoside sugars. Single point mutations in the structural gene ebgA alter the enzyme so that it hydrolyzes lactose or lactulose effectively; two mutations in ebgA permit galactosylarabinose hydrolysis, while three mutations are required for lactobionic acid hydrolysis. Wild-type ebg enzyme and 16 functional mutant ebg enzymes were purified and analyzed kinetically to determine how the substrate specificities had changed during the directed evolution of these new functions. The specificities for the biologically selected substrates generally increased by at least an order of magnitude via increased Vmax and decreased Km for the substrate. These changes were very specific for the selected substrate, often being accompanied by decreased specificities for other related substrates. The single, double, or triple substitutions in the enzymes did not detectably alter the thermal stability of ebg enzyme.
野生型ebg酶是大肠杆菌K12的第二种β-半乳糖苷酶，它不能使细菌在乳糖上生长。作为新酶功能进化研究的一部分，我从lacZ缺失菌株中筛选出了多种能在乳糖及其他β-半乳糖苷糖上生长的自发突变体。结构基因ebgA中的单点突变会改变该酶，使其能够有效水解乳糖或乳果糖；ebgA中的两个突变可使酶能够水解半乳糖基阿拉伯糖，而水解乳糖酸则需要三个突变。我们对野生型ebg酶和16种功能性突变ebg酶进行了纯化，并对其进行动力学分析，以确定在这些新功能的定向进化过程中底物特异性是如何变化的。对于通过生物筛选得到的底物，其特异性通常通过提高底物的Vmax和降低Km至少提高了一个数量级。这些变化对所选底物具有高度特异性，通常伴随着对其他相关底物的特异性降低。酶中的单、双或三重取代并未显著改变ebg酶的热稳定性。
