Department of Molecular Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
PLoS One. 2018 Apr 23;13(4):e0196250. doi: 10.1371/journal.pone.0196250. eCollection 2018.
Enzymatic functionalization of cross-β structured protein nanofibrils has hitherto resulted in a severe reduction of the catalytic efficiency of high turnover biocatalysts. It has been speculated that steric restrictions and mass transport pose limits on the attached enzymes, but detailed kinetics analyzing this have not yet been reported. For a more comprehensive understanding, we studied protein nanofibrils endowed with TEM1, a β-lactamase from Escherichia coli. The packing density of TEM1 along the fibrils was controlled by co-fibrillation; in other words, the N-terminal ureidosuccinate transporter Ure2(1-80) from Saccharomyces cerevisiae was simultaneously aggregated with the chimeric proteins TEM1-Ure2(1-80). The mature fibrils were trapped in a column, and the rate of ampicillin hydrolysis was recorded using a continuous substrate flow. The turnover rate was plotted as a function of substrate molecules available per enzyme per second, which demonstrated that an elevated substrate availability counteracts mass transport limitations. To analyze this data set, we derived a kinetic model, which makes it possible to easily characterize and compare enzymes packed in columns. The functional TEM1 nanofibrils possess 80% of the catalytic turnover rate compared to free TEM1 in solution. Altogether, we have created protein nanofibrils that can effectively hydrolyze β-lactam antibiotic contaminations and provided a groundwork strategy for other highly functional enzymatic nanofibrils.
迄今为止，将交叉-β 结构蛋白纳米纤维进行酶功能化处理导致高转化生物催化剂的催化效率严重降低。有人推测，空间位阻和质量传递对附着的酶施加了限制，但尚未有详细的动力学分析对此进行报道。为了更全面地了解这一现象，我们研究了具有 TEM1 的蛋白纳米纤维，TEM1 是一种来自大肠杆菌的β-内酰胺酶。通过共纤维化控制 TEM1 在纤维上的堆积密度；换句话说，来自酿酒酵母的 N 端脲酰琥珀酸转运蛋白 Ure2(1-80)与嵌合蛋白 TEM1-Ure2(1-80)同时聚集。成熟的纳米纤维被捕获在柱子中，使用连续底物流记录氨苄青霉素水解的速率。将周转率绘制为每秒钟每个酶可用的底物分子数的函数，结果表明，提高底物可用性可以抵消质量传递限制。为了分析这个数据集，我们推导出了一个动力学模型，该模型使得对填充在柱子中的酶进行简单的特征描述和比较成为可能。与游离在溶液中的 TEM1 相比，功能性 TEM1 纳米纤维具有 80%的催化周转率。总之，我们已经成功制备了能够有效水解β-内酰胺抗生素污染的蛋白纳米纤维，并为其他具有高功能的酶纳米纤维提供了一种基础策略。
