Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.
Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.
复制标题
DOI：
10.1021/acs.analchem.1c03633
复制
发表时间：
2022-01-18
影响因子：
7.4
通讯作者：
Reuel, Nigel F.
中科院分区：
文献类型：
--
作者：
Agarwal, Sparsh;Kallmyer, Nathaniel E.;Vang, Dua X.;Ramirez, Alma, V;Islam, Md Monirul;Hillier, Andrew C.;Halverson, Larry J.;Reuel, Nigel F.
降解生物膜胞外聚合物（EPS）的水解酶共治疗允许抗生素更好的扩散和更有效的治疗;目前用于定量测量EPS的酶促降解的方法不适用于高通量筛选。在此，我们提出了生物膜EPS功能化的单壁碳纳米管（SWCNT）探针的快速筛选水解酶的选择性和活性的EPS。通过监测SWCNT荧光的淬灭来量化生物膜EPS降解的程度。我们使用这个平台来筛选十六种水解酶，这些水解酶对一组野生型铜绿假单胞菌和一种或多种EPS缺陷或改变的突变体具有不同的键断裂选择性。接下来，我们对囊性纤维化（CF）环境中发现的两种常见菌株进行了六种酶的浓度依赖性研究，对于每种酶，通过拟合平行的多位点降解模型提取了三个一级速率常数及其相对贡献，模型拟合良好（R2为0.65至0.97）。反应速率（周转率）取决于酶的浓度，范围从6.67 e-11到2.80 e-3 *s-1/mg/ml酶。最后，我们使用已建立的结晶紫染色试验对水解酶组的一个子集进行了确认。总之，我们的工作表明，这种模块化传感器是可扩展的EPS降解的高通量筛选，从而提高了新的水解酶的发现和开发的速度。
Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring quenching of the SWCNT fluorescence. We used this platform to screen sixteen hydrolases with varying bond breaking selectivity against a panel of wild type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in Cystic Fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on enzyme concentration and range from 6.67e-11 to 2.80e-3 *s−1 per mg/ml of enzyme. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of the hydrolase panel. In summary, our work shows that this modular sensor is amendable to high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.
登录
查看更多内容
DOI：
10.1099/00221287-13-3-572
发表时间：
1955-01-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
HOLLOWAY, BW
通讯作者：
HOLLOWAY, BW
影响因子：
4.9
作者：
Gu, LR;Elkin, T;Sun, YP
通讯作者：
Sun, YP
影响因子：
--
作者：
Chatzinikolaou, I;Abi-Said, D;Samonis, G
通讯作者：
Samonis, G
影响因子：
3.7
作者：
Fanaei Pirlar, Rima;Emaneini, Mohammad;Jabalameli, Fereshteh
通讯作者：
Jabalameli, Fereshteh
影响因子：
--
作者：
Ambriz-Aviña V;Contreras-Garduño JA;Pedraza-Reyes M
通讯作者：
Pedraza-Reyes M