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Azo dying of ?-keratin material improves microbial keratinase screening and standardization.

ABSTRACT: 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.

SUBMITTER: Gonzalo M

PROVIDER: S-EPMC7264887 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Azo dying of α-keratin material improves microbial keratinase screening and standardization.

Gonzalo Milena M Espersen Roall R Al-Soud Waleed A WA Cristiano Falco Francesco F Hägglund Per P Sørensen Søren J SJ Svensson Birte B Jacquiod Samuel S

Microbial biotechnology 20200228 4

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 fi ...[more]

PMID: 32110845

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Project description:Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca(2+). The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry.

Dataset Information

Azo dying of ?-keratin material improves microbial keratinase screening and standardization.

Publications

Azo dying of α-keratin material improves microbial keratinase screening and standardization.

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