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Azo dying of α-keratin material improves microbial keratinase screening and standardization.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2020-02-28 , DOI: 10.1111/1751-7915.13541
Milena Gonzalo 1 , Roall Espersen 2 , Waleed A Al-Soud 1 , Francesco Cristiano Falco 3 , Per Hägglund 2 , Søren J Sørensen 1 , Birte Svensson 2 , Samuel Jacquiod 1
Affiliation  

Microbial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.

中文翻译:


α-角蛋白材料的偶氮染色改善了微生物角蛋白酶的筛选和标准化。



通过酶促反应的微生物转化作为从角蛋白材料中回收氨基酸和短肽的一种经济有效且环境友好的方法受到了广泛的关注。然而,准确评估微生物角蛋白酶活性并不简单,目前可用的方法缺乏敏感性和标准化。在这里,我们建议一种优化的偶氮角蛋白测定,其底物直接由偶氮染色的角蛋白原料产生。我们在方案中引入了上清液过滤,以实现角蛋白酶反应的最佳停止,而不是广泛使用的三氯乙酸 (TCA),因为它会产生偏差并影响灵敏度。我们还提出了一种使用蛋白酶 K(一种众所周知的角蛋白酶)对角蛋白酶活性信号进行标准化的方法,作为参考,以实现研究之间的可重复性。最后,我们通过针对商业测定(角蛋白天青)的基准评估了我们开发的几种细菌分离物的方法。在不同的设置下,Azokeratin 方法比常用的基于 Keratin Azure 的测定法更灵敏(3 倍)。我们认为,这种方法可以应用于任何类型的角蛋白基质,从而获得更稳健和更灵敏的结果,可用于与其他研究进行进一步比较,从而代表微生物角蛋白降解领域的重要进展。
更新日期:2020-02-28
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