Abstract
Keratinases have drawn increasing attention in recent decades owing to their catalytic versatility and broad applications from agriculture to medicine. In the present study, we isolated a highly keratinolytic and fibrinolytic bacterium from the campus soil and named it Stenotrophomonas sp. LMY based on genetic information. To identify the potential keratinase genes, the genome sequence of the strain was obtained and analyzed. Sequence alignment and comparison revealed that the protein 1_737 (KerZJ) had the highest sequence homology to a reported keratinase KerBL. We recombinantly expressed KerZJ in Escherichia coli Origami™ (DE) pLysS and purified it to homogeneity. KerZJ showed the highest activity at 40 °C and pH 9.0, and metal ions exhibited no significant effects on its activity. Although reducing agents would break the disulfide bonds in KerZJ and reduce its activity, KerZJ still exhibited the ability to hydrolyze feather keratin in the presence of β-ME. KerZJ could efficiently digest human prion proteins. In addition, KerZJ showed fibrinolytic activity on fibrin plates and effectively eliminated blood clots in a thrombosis mouse model without side effects. Our results suggest that KerZJ is a versatile keratinase with significant potential for keratin treatment, decontamination of prions, and fibrinolytic therapy.
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All data in this study could be obtained from the corresponding author upon reasonable request.
References
Al-Mamoori ZZ, Embaby AM, Hussein A, Mahmoud HE (2023) A molecular study on recombinant pullulanase type I from Metabacillus indicus. AMB Express 13:40. https://doi.org/10.1186/s13568-023-01545-8
Altaf F, Wu S, Kasim V (2021) Role of fibrinolytic enzymes in anti-thrombosis therapy. Front Mol Biosci 8:680397. https://doi.org/10.3389/fmolb.2021.680397
Anbesaw MS (2022) Bioconversion of keratin wastes using keratinolytic microorganisms to generate value-added products. Int J Biomater 2022:2048031. https://doi.org/10.1155/2022/2048031
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brooke JS (2012) Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 25:2–41. https://doi.org/10.1128/cmr.00019-11
Callegaro K, Brandelli A, Daroit DJ (2019) Beyond plucking: feathers bioprocessing into valuable protein hydrolysates. Waste Manag 95:399–415. https://doi.org/10.1016/j.wasman.2019.06.040
Cao ZJ, Zhang Q, Wei DK, Chen L, Wang J, Zhang XQ, Zhou MH (2009) Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme. J Ind Microbiol Biotechnol 36:181–188. https://doi.org/10.1007/s10295-008-0469-8
Embaby AM, Mahmoud HE (2022) Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study. AMB Express 12:135. https://doi.org/10.1186/s13568-022-01476-w
Fang Z, Zhang J, Liu B, Du G, Chen J (2013) Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1. Bioresour Technol 140:286–291. https://doi.org/10.1016/j.biortech.2013.04.091
Fang Z, Zhang J, Du G, Chen J (2016a) Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain. Sci Rep 6:27953. https://doi.org/10.1038/srep27953
Fang Z, Zhang J, Liu B, Du G, Chen J (2016b) Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF. Microb Biotechnol 9:35–46. https://doi.org/10.1111/1751-7915.12300
Fang Z, Sha C, Peng Z, Zhang J, Du G (2019) Protein engineering to enhance keratinolytic protease activity and excretion in Escherichia coli and its scale-up fermentation for high extracellular yield. Enzyme Microb Technol 121:37–44. https://doi.org/10.1016/j.enzmictec.2018.11.003
Hassan MA, Abol-Fotouh D, Omer AM, Tamer TM, Abbas E (2020) Comprehensive insights into microbial keratinases and their implication in various biotechnological and industrial sectors: a review. Int J Biol Macromol 154:567–583. https://doi.org/10.1016/j.ijbiomac.2020.03.116
Huang J, Wu C, Liu D, Yang X, Wu R, Zhang J, Ma C, He H (2017) C-terminal domains of bacterial proteases: structure, function and the biotechnological applications. J Appl Microbiol 122:12–22. https://doi.org/10.1111/jam.13317
Jankiewicz U, Larkowska E, Swiontek BM (2016) Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4. J Biosci Bioeng 121:614–618. https://doi.org/10.1016/j.jbiosc.2015.11.011
Khursade PS, Galande SH, Shiva Krishna P, Prakasham RS (2019) Stenotrophomonas maltophilia Gd2: a potential and novel isolate for fibrinolytic enzyme production. Saudi J Biol Sci 26:1567–1575. https://doi.org/10.1016/j.sjbs.2018.10.014
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Lasekan A, Abu Bakar F, Hashim D (2013) Potential of chicken by-products as sources of useful biological resources. Waste Manag 33:552–565. https://doi.org/10.1016/j.wasman.2012.08.001
López-Otín C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433–30437. https://doi.org/10.1074/jbc.R800035200
Matrawy AA, Khalil AI, Embaby AM (2022) Molecular study on recombinant cold-adapted, detergent- and alkali stable esterase (EstRag) from Lysinibacillus sp.: a member of family VI. World J Microbiol Biotechnol 38:217. https://doi.org/10.1007/s11274-022-03402-5
Nailufar F, Tjandrawinata RR, Suhartono MT (2016) Thrombus degradation by fibrinolytic enzyme of Stenotrophomonas sp. originated from Indonesian soybean-based fermented food on Wistar rats. Adv Pharmacol Sci 2016:4206908. https://doi.org/10.1155/2016/4206908
Okoroma EA, Purchase D, Garelick H, Morris R, Neale MH, Windl O, Abiola OO (2013) Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions. PLoS ONE 8:e68099. https://doi.org/10.1371/journal.pone.0068099
Pan X, Yang J, Xie P, Zhang J, Ke F, Guo X, Liang M, Liu L, Wang Q, Gao X (2021) Enhancement of activity and thermostability of keratinase from Pseudomonas aeruginosa CCTCC AB2013184 by directed evolution with noncanonical amino acids. Front Bioeng Biotechnol 9:770907. https://doi.org/10.3389/fbioe.2021.770907
Pei X, Wang Q, Meng L, Li J, Yang Z, Yin X, Yang L, Chen S, Wu J (2015) Chaperones-assisted soluble expression and maturation of recombinant co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature. J Biotechnol 203:9–16. https://doi.org/10.1016/j.jbiotec.2015.03.004
Peng Z, Zhang J, Du G, Chen J (2019) Keratin waste recycling based on microbial degradation: mechanisms and prospects. ACS Sustain Chem Eng 7:9727–9736. https://doi.org/10.1021/acssuschemeng.9b01527
Qu F, Chen Q, Ding Y, Liu Z, Zhao Y, Zhang X, Liu Z, Chen J (2018) Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes. Mol Biol Rep 45:1681–1689. https://doi.org/10.1007/s11033-018-4311-8
Rajput R, Tiwary E, Sharma R, Gupta R (2012) Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: altered substrate specificity and thermostability. Enzyme Microb Technol 51:131–138. https://doi.org/10.1016/j.enzmictec.2012.04.010
Sinha R, Khare S (2013) Thermostable proteases. In: Satyanarayana T, Littlechild J, Kawarabayasi Y (eds) Thermophilic microbes in environmental and industrial biotechnology. Springer, Dordrecht, pp 859–880
Suzuki Y, Tsujimoto Y, Matsui H, Watanabe K (2006) Decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria. J Biosci Bioeng 102:73–81. https://doi.org/10.1263/jbb.102.73
Tiwary E, Gupta R (2010) Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli. J Agric Food Chem 58:8380–8385. https://doi.org/10.1021/jf100803g
Trifonova A, Strateva T (2019) Stenotrophomonas maltophilia—a low-grade pathogen with numerous virulence factors. Infect Dis 51:168–178. https://doi.org/10.1080/23744235.2018.1531145
Tsiroulnikov K, Rezai H, Bonch-Osmolovskaya E, Nedkov P, Gousterova A, Cueff V, Godfroy A, Barbier G, Métro F, Chobert JM, Clayette P, Dormont D, Grosclaude J, Haertlé T (2004) Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies. J Agric Food Chem 52:6353–6360. https://doi.org/10.1021/jf0493324
Wang Z, Chen Y, Yan M, Li K, Okoye CO, Fang Z, Ni Z, Chen H (2023) Research progress on the degradation mechanism and modification of keratinase. Appl Microbiol Biotechnol 107:1003–1017. https://doi.org/10.1007/s00253-023-12360-3
Yamamura S, Morita Y, Hasan Q, Yokoyama K, Tamiya E (2002) Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. Biochem Biophys Res Commun 294:1138–1143. https://doi.org/10.1016/s0006-291x(02)00580-6
Yan F, Yan J, Sun W, Yao L, Wang J, Qi Y, Xu H (2009) Thrombolytic effect of subtilisin QK on carrageenan induced thrombosis model in mice. J Thromb Thromb 28:444–448. https://doi.org/10.1007/s11239-009-0333-3
Yi D, Xing J, Gao Y, Pan X, Xie P, Yang J, Wang Q, Gao X (2020) Enhancement of keratin-degradation ability of the keratinase KerBL from Bacillus licheniformis WHU by proximity-triggered chemical crosslinking. Int J Biol Macromol 163:1458–1470. https://doi.org/10.1016/j.ijbiomac.2020.08.021
Zhang J, Liang M, Wu L, Yang Y, Sun Y, Wang Q, Gao X (2023) Bioconversion of feather waste into bioactive nutrients in water by Bacillus licheniformis WHU. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-023-12795-8
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We would like to thank Editage (https://www.editage.cn) for the English language editing.
Funding
This work was supported by the Department of Science and Technology of Sichuan Province (2020YJ0129), the Collaborative Fund of Science and Technology Agency of Luzhou Government and Southwest Medical University (2020LZXNYDJ29), and the Science Fund Project of Southwest Medical University (2021ZKMS045).
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PH, ML, and JZ: designed the study, performed experiments, and analyzed data; WL, YY, YS, FK, SL, and YW: performed experiments and collected data; BX: performed review and writing; XG: acquired funding, wrote the manuscript, and supervised the study.
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Peng, H., Liang, M., Zhang, J. et al. Identification and characterization of a versatile keratinase, KerZJ, from Stenotrophomonas sp. LMY. World J Microbiol Biotechnol 40, 30 (2024). https://doi.org/10.1007/s11274-023-03836-5
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DOI: https://doi.org/10.1007/s11274-023-03836-5