Generic placeholder image

Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

A Thermostable Aluminum-Tolerant Protease Produced by Feather-Degrading Bacillus thuringiensis Isolated from Tea Plantation

Author(s): Tianwen Wang*, Chen Liang, Sha Xiao, Li Li*, Hongju Xu, Yafei An, Mengyuan Zheng and Lu Liu

Volume 28 , Issue 5 , 2021

Published on: 03 November, 2020

Page: [563 - 572] Pages: 10

DOI: 10.2174/0929866527666201103153309

Price: $65


Background: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase.

Objective: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme.

Methods: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme.

Results: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca2+ and Mg2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn2+; DMSO <2%, v/v). The enzyme exhibited high resistance to Al3+, which might be explained by the soil properties of its host’s residence. Circular dichroism confirmed the contribution of ions to the structure and activity.

Conclusion: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties.

Keywords: Thermostability, serine protease, keratinase, Al3+ resistance, Mn2+ activation, organic activation, DMSO activation.

Graphical Abstract
Savickas, S.; Kastl, P.; Auf dem Keller, U. Combinatorial degradomics: precision tools to unveil proteolytic processes in biological systems. Biochim. Biophys. Acta. Proteins Proteomics, 2020, 1868(6), 140392.
[] [PMID: 32087360]
Gurumallesh, P.; Alagu, K.; Ramakrishnan, B.; Muthusamy, S. A systematic reconsideration on proteases. Int. J. Biol. Macromol., 2019, 128, 254-267.
[] [PMID: 30664968]
Bhari, R.; Kaur, M.; Singh, R.S. Thermostable and halotolerant keratinase from Bacillus aerius NSMk2 with remarkable dehairing and laundary applications. J. Basic Microbiol., 2019, 59(6), 555-568.
[] [PMID: 30900760]
Tian, J.; Xu, Z.; Long, X.; Tian, Y.; Shi, B. High-expression keratinase by Bacillus subtilis SCK6 for enzymatic dehairing of goatskins. Int. J. Biol. Macromol., 2019, 135, 119-126.
[] [PMID: 31125653]
Jardine, J.L.; Stoychev, S.; Mavumengwana, V.; Ubomba-Jaswa, E. Screening of potential bioremediation enzymes from hot spring bacteria using conventional plate assays and liquid chromatography - Tandem mass spectrometry (Lc-Ms/Ms). J. Environ. Manage., 2018, 223, 787-796.
[] [PMID: 29986326]
Vidmar, B.; Vodovnik, M. Microbial keratinases: enzymes with promising biotechnological applications. Food Technol. Biotechnol., 2018, 56(3), 312-328.
[] [PMID: 30510475]
Bordusa, F. Proteases in organic synthesis. Chem. Rev., 2002, 102(12), 4817-4868.
[] [PMID: 12475208]
Langeveld, J.P.; Wang, J.J.; Van de Wiel, D.F.; Shih, G.C.; Garssen, G.J.; Bossers, A.; Shih, J.C. Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. J. Infect. Dis., 2003, 188(11), 1782-1789.
[] [PMID: 14639552]
Ningthoujam, D.S.; Mukherjee, S.; Devi, L.J.; Singh, E.S.; Tamreihao, K.; Khunjamayum, R.; Banerjee, S.; Mukhopadhyay, D. In vitro degradation of β-amyloid fibrils by microbial keratinase. Alzheimers Dement. (N.Y.), 2019, 5, 154-163.
[] [PMID: 31193333]
Lin, X.; Kelemen, D.W.; Miller, E.S.; Shih, J.C. Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. Appl. Environ. Microbiol., 1995, 61(4), 1469-1474.
[] [PMID: 7747965]
Sangali, S.; Brandelli, A. Isolation and characterization of a novel feather-degrading bacterial strain. Appl. Biochem. Biotechnol., 2000, 87(1), 17-24.
[] [PMID: 10850670]
Werlang, P.O.; Brandelli, A. Characterization of a novel feather-degrading Bacillus sp. strain. Appl. Biochem. Biotechnol., 2005, 120(1), 71-79.
[] [PMID: 15640558]
Agrahari, S.; Wadhwa, N. Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site. Prikl. Biokhim. Mikrobiol., 2012, 48(2), 199-205.
[PMID: 22586913]
Ghaffar, I.; Imtiaz, A.; Hussain, A.; Javid, A.; Jabeen, F.; Akmal, M.; Qazi, J.I. Microbial production and industrial applications of keratinases: an overview. Int. Microbiol., 2018, 21(4), 163-174.
[] [PMID: 30810899]
Leveson-Gower, R.B.; Mayer, C.; Roelfes, G. The importance of catalytic promiscuity for enzyme design and evolution. Nat. Rev. Chem., 2019, 3(12), 687-705.
Jeong, J.H.; Jeon, Y.D.; Lee, O.M.; Kim, J.D.; Lee, N.R.; Park, G.T.; Son, H.J. Characterization of a multifunctional feather-degrading Bacillus subtilis isolated from forest soil. Biodegradation, 2010, 21(6), 1029-1040.
[] [PMID: 20454836]
Wang, T.; Liang, C.; Sun, Y.; Gao, W.; Luo, X.; Gao, Q.; Li, R.; Fu, S.; Xu, H.; He, T.; Yuan, H. Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample. Int. Microbiol., 2019, 22(2), 227-237.
[] [PMID: 30810985]
Qu, F.; Chen, Q.; Ding, Y.; Liu, Z.; Zhao, Y.; Zhang, X.; Liu, Z.; Chen, J. Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes. Mol. Biol. Rep., 2018, 45(6), 1681-1689.
[] [PMID: 30168098]
Su, C.; Gong, J.S.; Zhang, R.X.; Tao, L.Y.; Dou, W.F.; Zhang, D.D.; Li, H.; Lu, Z.M.; Xu, Z.H.; Shi, J.S. A novel alkaline surfactant-stable keratinase with superior feather-degrading potential based on library screening strategy. Int. J. Biol. Macromol., 2017, 95, 404-411.
[] [PMID: 27864058]
Fang, Z.; Zhang, J.; Du, G.; Chen, J. Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain. Sci. Rep., 2016, 6, 27953.
[] [PMID: 27298079]
Fang, Z.; Zhang, J.; Liu, B.; Du, G.; Chen, J. Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF. Microb. Biotechnol., 2016, 9(1), 35-46.
[] [PMID: 26552936]
Ruan, J.; Ma, L.; Shi, Y. Aluminium in tea plantations: mobility in soils and plants, and the influence of nitrogen fertilization. Environ. Geochem. Health, 2006, 28(6), 519-528.
[] [PMID: 16826449]
Fang, X-M.; Chen, F-S.; Hu, X-F.; Yuan, P-C.; Li, J.; Chen, X. Aluminum and nutrient interplay across an age-chronosequence of tea plantations within a hilly red soil farm of subtropical China. J. Soil Sci. Plant Nutr., 2014, 60(4), 448-459.
James, G.T. Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal. Biochem., 1978, 86(2), 574-579.
[] [PMID: 26289]
Si, J-B.; Jang, E-J.; Charalampopoulos, D.; Wee, Y-J. Purification and characterization of microbial protease produced extracellularly from Bacillus subtilis FBL-1. Biotechnol. Bioprocess Eng.; BBE, 2018, 23(2), 176-182.
Zhao, M.; Wang, H.B.; Ji, L.N.; Mao, Z.W. Insights into metalloenzyme microenvironments: biomimetic metal complexes with a functional second coordination sphere. Chem. Soc. Rev., 2013, 42(21), 8360-8375.
[] [PMID: 23881282]
Anson, M.L. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol., 1938, 22(1), 79-89.
[] [PMID: 19873094]
Morita, A.; Yanagisawa, O.; Maeda, S.; Takatsu, S.; Ikka, T. Tea plant (Camellia sinensis L.) roots secrete oxalic acid and caffeine into medium containing aluminum. Soil Sci. Plant Nutr., 2011, 57(6), 796-802.
Poopathi, S.; Thirugnanasambantham, K.; Mani, C.; Lakshmi, P.V.; Ragul, K. Purification and characterization of keratinase from feather degrading bacterium useful for mosquito control--a new report. Trop. Biomed., 2014, 31(1), 97-109.
[PMID: 24862049]
Park, G.T.; Son, H.J. Keratinolytic activity of Bacillus megaterium f7-1, a feather-degrading mesophilic bacterium. Microbiol. Res., 2009, 164(4), 478-485.
[] [PMID: 17459685]
Herlet, J.; Kornberger, P.; Roessler, B.; Glanz, J.; Schwarz, W.H.; Liebl, W.; Zverlov, V.V. A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes. Biotechnol. Biofuels, 2017, 10, 234.
[] [PMID: 29046720]
Rajput, R.; Sharma, R.; Gupta, R. Biochemical characterization of a thiol-activated, oxidation stable keratinase from Bacillus pumilus KS12. Enzyme Res., 2010, 2010, 132148.
[] [PMID: 21048858]
Yusuf, I.; Ahmad, S.A.; Phang, L.Y.; Syed, M.A.; Shamaan, N.A.; Abdul Khalil, K.; Dahalan, F.A.; Shukor, M.Y. Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-Alcaligenes sp. AQ05-001. J. Environ. Manage., 2016, 183, 182-195.
[] [PMID: 27591845]
Schwartz, A. Role of Ca and EGTA on stomatal movements in Commelina communis L. Plant Physiol., 1985, 79(4), 1003-1005.
[] [PMID: 16664518]
Lin, H.H.; Yin, L.J.; Jiang, S.T. Expression and purification of pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system. J. Agric. Food Chem., 2009, 57(17), 7779-7784.
[] [PMID: 19722707]
Wang, Y.; Xu, Q.; Lu, H.; Lin, L.; Wang, L.; Xu, H.; Cui, X.; Zhang, H.; Li, T.; Hua, Y. Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction. PLoS One, 2015, 10(3), e0122071.
[] [PMID: 25811789]
McLachlan, D.R.C.; Bergeron, C.; Alexandrov, P.N.; Walsh, W.J.; Pogue, A.I.; Percy, M.E.; Kruck, T.P.A.; Fang, Z.; Sharfman, N.M.; Jaber, V.; Zhao, Y.; Li, W.; Lukiw, W.J. Aluminum in neurological and neurodegenerative disease. Mol. Neurobiol., 2019, 56(2), 1531-1538.
[] [PMID: 30706368]
Tang, J. Competitive inhibition of pepsin by aliphatic alcohols. J. Biol. Chem., 1965, 240(10), 3810-3815.
[PMID: 5320642]
Bressollier, P.; Letourneau, F.; Urdaci, M.; Verneuil, B. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol., 1999, 65(6), 2570-2576.
[] [PMID: 10347045]
Thys, R.C.; Brandelli, A. Purification and properties of a keratinolytic metalloprotease from Microbacterium sp. J. Appl. Microbiol., 2006, 101(6), 1259-1268.
[] [PMID: 17105556]
Bach, E.; Sant’Anna, V.; Daroit, D.J.; Corrêa, A.P.F.; Segalin, J.; Brandelli, A. Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3. Process Biochem., 2012, 47(12), 2455-2462.
Saha, S.; Dhanasekaran, D.; Shanmugapriya, S.; Latha, S. Nocardiopsis sp. SD5: a potent feather degrading rare actinobacterium isolated from feather waste in Tamil Nadu, India. J. Basic Microbiol., 2013, 53(7), 608-616.
[] [PMID: 23864545]

Rights & Permissions Print Export Cite as
© 2022 Bentham Science Publishers | Privacy Policy