定性和定量测定为检测和蛋白质抗菌药物的表征
Summary
Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.
Abstract
Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.
Introduction
Antimicrobial agents play an essential role in targeting a wide range of microorganisms such as viruses, fungi, and bacteria. The antimicrobial activities produced by various bacteria range in target specificity from broad-spectrum to species-specific, including clinically relevant bacterial pathogens 1. In nature, protein antimicrobial compounds like bacteriocins and lysins represent a microbial arsenal of tools for self-defense, replication, and nutrient acquisition 2,3. Serving as mediators of population dynamics within microbial communities, protein antimicrobials provide a competitive advantage to the producing organism over nonproducing, sensitive species 4. As recombinant enzymes and probiotics, these proteins also have an economic and societal importance as the need for new sources of pathogen control increases with each new expansion of antimicrobial resistance within the bacterial population 5.
The first evaluations of newly discovered protein antimicrobials include determining the basic physical characteristics that are inherent to the enzyme. Methods for rapid screening of potential antimicrobials allow selection of candidates with hydrolytic activities against the desired target substrates. The microslide diffusion assay and the quantitative dye-release assay allow for the use of a variety of substrates in the detection of enzymatic hydrolysis. For protein antimicrobial enzymes with activity against the bacterial cell wall, the versatility of these assays allow rapid and effective, qualitative and quantitative screening against viable bacterial cells (microslide assay only), heat-killed whole bacterial cell substrates, or purified peptidoglycan from the target bacterium. These assays have utility in antimicrobial enzyme discovery for use in fields such as alternative therapeutics, food supplements, and inhibitors of biofilm production.
The microslide diffusion assay and the dye-release assay are demonstrated methods for detection and characterization of novel protein antimicrobials. The microslide diffusion assay provides a relative (qualitative) estimate of antimicrobial activity and allows for minimal inference of enzyme concentration and activity. Using this method, activity is readily visualized as the development of a zone of lysis with the absence of activity resulting in a lack of zone formation. The rate of zone development and the zone diameter are indicative of the amount and purity of the enzyme present in the sample; however, this rate and the quality of the substrate clearing within the zone can also be affected by the presence of contaminants, the completeness of the hydrolysis reaction, and the type of substrate. Interfering contaminants can affect the rate of enzyme diffusion from the well, while incomplete hydrolysis or variation of the substrate type can result in a turbid zone of lysis 6.
The dye-release assay quantitatively assesses the amount of covalently-linked Remazol brilliant blue R dye products released into the reaction supernatant from the enzymatically hydrolyzed substrate. The method has only slight variability associated with a difference in substrate, thus allowing greater sensitivity for detection of hydrolysis as compared to the microslide diffusion assay. These properties allow the method to have utility in the characterization of biochemical properties of the enzyme and in the standardization of the assay for comparison between different antimicrobial enzymes.
In this protocol, we provide methods to perform the basic microslide diffusion assay and dye-release assay, while demonstrating the scale of detection limits and variability for each. The assays are used to perform basic evaluations of an unknown protein antimicrobial purified from the culture supernatant of an uncharacterized soil bacterial isolate. Observed activities against bacterial cell substrates within the assays allow comparison of reaction activities between a previously uncharacterized protein antimicrobial and α-chymotrypsin, a control proteolytic enzymes. These protocols provide a foundation for the application of the two techniques that can be expanded and modified to accommodate varied applications.
Protocol
Representative Results
Discussion
该扩散的MicroSlide法和染料释放法检测和鉴定以前未知的蛋白质抗菌药物提供的优势。这些快速和灵敏的方法允许之前投入更大研究资源感兴趣的酶的鉴定有机体源库的高通量筛选。作为高调靶酶被识别，检测分析的变化可以用来快速检测酶或确定酶的生化特性。例如，一个多蛋白质纯化方案中，所述的MicroSlide扩散分析能够快速检测含有目的酶的级分。另外，对于酶反应最适可通过改变反应缓冲液和培养温度在染料释放测定法来确定。
用于检测在所述的MicroSlide扩散分析所需的酶物质的范围是酶characteri的函数STICS。该测定的检测限制一般要求使用较多量的酶比染料释放测定的。在这项研究中，所述的MicroSlide扩散分析（ 图1）的染料释放测定（ 图4）的检测限的比较示出，该的MicroSlide扩散试验比染料释放测定较不敏感的技术。当酶浓度不是一个问题，该法的MicroSlide允许用于生产抗菌蛋白对低劳动力和设备的需求目标底物文库的快速初步筛选。尽管需要更多的努力和设备，该染料释放测定法更灵敏，可重复的，得到定量结果，允许对于给定的基片相同或不同的酶的染料释放测定的比较。这两种方法都容易在大多数微生物实验室，无需高度专业化的仪器进行的。在的代理问题略去测定法，对照酶，α胰凝乳蛋白酶，在量低至使用的染料释放测定100纳克（ 图5）检测到的，而在的MicroSlide扩散分析检测出的最小的量为1微克（数据未示出）。
提供了可用作抗菌酶定性检测测定，一些扩散试验的变体已经报道11,13。在审查这些试验中，扩散的MicroSlide测定其相对较高的灵敏度作为初始屏幕，因为它容易反应遵守的开发。从Lachica 等人 11的工作，其中的琼脂糖覆盖被用于由金黄色葡萄球菌的菌株以检测生产核酸的修饰，所述的MicroSlide扩散试验的操作类似于径向免疫扩散法14作为蛋白质从井扩散到琼脂糖含有底物。径向immunodiffu当系统达到琼脂糖内漫射抗原和抗体之间的复合物形成的平衡锡永方法停止免疫沉淀的区的扩展。与此相反，的MicroSlide扩散分析的衬底最初由漫射蛋白抗微生物在包围井裂解的不断扩大区消化。区域的增大的直径继续，直到酶失去活性或衬底被耗尽。生产裂解的区的速率加快作为纯度，因而比活性，酶的或加入到孔中增加酶的浓度。
用于定量评估蛋白质抗微生物剂的抗热灭活B的酶活性枯草芽孢杆菌 ，被选择的染料释放测定。使用雷马素马斯亮蓝R染料（RBB）比色法标记的底物会使蛋白质抗菌活性状况的通用性和敏感性评价TY。 RBB标记的底物的结果中，可以用分光光度计在595nm处容易地测得的可溶性蓝色产品释放的酶水解。的RBB染料释放测定的几个变型中，开发了以表征其他蛋白质的抗菌酶，表明此测定应用与其它基材的灵活性。例如，在确定溶葡球菌酶溶菌活性的影响，周等人 ，报告了使用RBB-染色葡萄球菌细胞和葡萄球菌肽聚糖作为底12的染料释放测定。在此RBB染料释放测定的应用的变型，RBB标记藤黄微球菌衬底提出用作一个快速和灵敏的方法来诊断和筛选的疾病例如细菌感染和恶性肿瘤的存在，它可以在血液血清15相关，以人溶菌酶表达水平。此外，RBB标记细菌细胞衬底具有一个LSO在用于检测溶菌酶16的酶谱法中使用。
我们证明降低的检出限，方便和染料释放测定的再现性，从而允许快速文库筛选产酶。该测定的修饰允许下游定量生化表征测定法，包括温度，pH值，和盐度的影响以及对抗微生物蛋白6的活性的其它蛋白水解酶的影响。此外，定量的染料释放测定可用于多种酶的活性比较对于给定的衬底上。的RBB染料释放测定已报道用抗除表征和蛋白的抗微生物剂检测的多糖酶活性效用。卡尔 – 佩特森和埃里克森报告使用纤维素，木聚糖，甘露聚糖，以及保单的无定形，RBB染色多糖珠子检测内聚糖酶的活性的测定17。在这些研究结果的扩展，用于检测由苏云金芽孢杆菌的Bt-107产生的几丁质酶的酶的一个灵敏的方法使用标记的RBB 18胶体几丁质被开发。从这些和其他研究，RBB染色基板的市售来源已经出现在检测糖酵解活性，包括对角蛋白，支链淀粉，直链淀粉，糖原，海带多糖，D-木聚糖，偶氮大麦葡聚糖和淀粉的用途。
在这项研究中，我们证明在微量格式的染料释放测定的效用，降低以产生比色结果所需RBB标记的底物和酶的体积。如在图4和图5所示，该微板染料释放测定提供了比酶活性检测的MicroSlide扩散分析检测下限。对于快速的使用，节约劳动力，以及便于解释，麦克风roslide扩散分析提供了抗微生物活性的存在，以及抗微生物蛋白存在的下游蛋白质的分离和纯化步骤的指示初始的高通量筛选效用。这两项试验的组合，新的蛋白质抗菌药物初步筛选，并最终鉴定相得益彰
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was funded by The MITRE Corporation through the MITRE Innovation Program (Project 25MSR621-CA). The authors would like to thank Mark Maybury, Ph.D., Richard Games, Ph.D., James Patton, Ellen Mac Garrigle, Ph.D., Carl Picconatto, Ph.D., and Caroline Gary for their support and review of this work.
Materials
| 48-well flat-bottom microplate with low evaporation lid | Becton Dickinson | 3078 | |
| 96-well flat-bottom microplate (Costar 3595) | Corning, Inc. | 3595 | |
| agarose | Fisher Scientific | BP162-100 | |
| α-chymotrypsin | MP Biomedicals | 215227205 | |
| Bacillus subtilis 168 | American Type Culture Collection | 23857 | |
| C1000 Touch Thermal Cycler | Bio-Rad | ||
| cork borer | Humboldt | H-9661 | |
| lysozyme | Sigma-Aldrich | L6876-1G | |
| McFarland equivalence standard (2.0) | Fisher Scientific | R20412 | |
| microslides | Fisher Scientific | 22-38-103 | |
| nutrient broth | Fisher Scientific | S25959B | |
| peptidoglycan of Bacillus subtilis 168 | Sigma-Aldrich | 69554-10MG-F | |
| phosphate-buffered saline (1×) | Teknova | P0200 | |
| Pierce BCA Protein Assay Kit | Life Technologies | 23227 | |
| Synergy HT microplate spectrophotometer | BioTek Instruments, Inc. | ||
| Remazol brilliant blue R dye (RBB) | Sigma-Aldrich | R8001 | |
| Salmonella enterica subsp. enterica | American Type Culture Collection | 10708 | |
| sodium azide | Fisher Scientific | S227-100 | |
| sodium hydroxide (NaOH) | Fisher Scientific | S318-500 | |
| Titer Tops pre-cut adhesive polyethylene film | Diversified Biotechnology | T-TOPS-50 | |
| UltraPure distilled water | Invitrogen | 10977-015 | |
| Name | Company | Catalog Number | Comments |
| Solution | |||
| agarose solution 50 ml PBS 0.25 g agarose |
Add 10% sodium azide to the molten PBS/agarose solution | ||
| nutrient broth medium 4 g nutrient broth 500 ml Type I water |
|||
| 250mM sodium hydroxide (NaOH) solution 1 g NaOH 99 ml Type I water |
|||
| 200mM Remazol brilliant blue R dye (RBB) 1.25 g RBB 98.75 ml 250 mM NaOH solution |
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