Abstract
The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC–MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120 h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates.
Key points
• A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases.
• This actinomycete efficiently converts bristle meal to a protein hydrolysate.
• Proteome analysis identified 57 proteases in its secretome.
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Acknowledgements
Lene Blicher is thanked for expert assistance with mass spectrometry and Karina Jansen for general technical assistance.
Funding
This work was funded by the Innovation Fund Denmark | The Program Commission on Health, Food and Welfare to the project Keratin2Protein (grant 1308-00015B) and joint (1/3) PhD stipends from DTU to RE and FCF. Center for Advanced Food Studies (LMC) is thanked for supporting the acquisition of the Bruker Daltonics Ultraflex II MALDI-TOF/TOF mass spectrometer. The Orbitrap Fusion Tribrid Mass Spectrometer was granted by the Villum Foundation.
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RE and BS conceived the review. YH and LL performed the PPR analysis. FCF and KVG contributed by biological processing experiments. RE, PH, and BS conducted proteome analysis. RE and BS wrote the paper with input from LL, YH, FCF, and KVG. All authors have read and approved the manuscript.
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Espersen, R., Huang, Y., Falco, F.C. et al. Exceptionally rich keratinolytic enzyme profile found in the rare actinomycetes Amycolatopsis keratiniphila D2T. Appl Microbiol Biotechnol 105, 8129–8138 (2021). https://doi.org/10.1007/s00253-021-11579-2
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DOI: https://doi.org/10.1007/s00253-021-11579-2