Production of anti-rM14 and anti-rM42 antibodies and their application in the study of expression of metalloproteases M14 and M42 of keratinolytic strain of Bacillus paralicheniformis
Main Article Content
Authors
A. Maduakhassova
National Center for Biotechnology, 13/5 Korgalzhyn Road, Astana, 010000
L.N.Gumilyev Eurasian National University, 2 Kanysh Satpayev Street, Astana, 010008
A. Mussakhmetov
National Center for Biotechnology, 13/5 Korgalzhyn Road, Astana, 010000
Zh. Akishev
National Center for Biotechnology, 13/5 Korgalzhyn Road, Astana, 010000
S. Aktayeva
National Center for Biotechnology, 13/5 Korgalzhyn Road, Astana, 010000
L.N.Gumilyev Eurasian National University, 2 Kanysh Satpayev Street, Astana, 010008
B. Khassenov
National Center for Biotechnology, 13/5 Korgalzhyn Road, Astana, 010000
Abstract
In the present study, two metalloproteases M14 and M42 from a Kazakh strain of Bacillus paralicheniformis T7 with high proteolytic activity were successfully cloned, expressed and purified. Protease genes were amplified by polymerase chain reaction (PCR) using specific primers selected on the basis of full genome sequencing data of B. paralicheniformis strain N7 (GenBank accession #CP124861). The amplified fragments were cloned in the expression vector pET-28c(+). Recombinant analogs of the metalloproteases M14 and M42 were obtained by plasmid expression in Escherichia coli cells of the strain ArcticExpressRP(DE3). Using metal affinity chromatography, rM14 and rM42 proteins were purified and polyclonal antibodies were produced by immunization of rabbits. Using anti-rM14 and anti-rM42 antibodies, the expression level of native metalloproteases M14 and M42 in cell lysate and culture fluid of B. paralicheniformis T7 strain was examined after culturing the strain on keratin-containing media: chicken feather, horn, hoof, hide and wool of cattle. Substrate-dependent expression of metalloprotease genes of B. paralicheniformis strain T7 was established. The obtained results indicate the prospective use B. paralicheniformis strain T7 as a producer of metalloproteases M14 and M42.
Keywords
proteases, antibodies, Bacillus paralicheniformis, recombinant protein, metalloproteases, M14, M42
Article Details
References
Rao M. B., Tanksale A. M., Ghatge M. S., Deshpande V. V. Molecular and biotechnological aspects of microbial proteases // Microbiology and molecular biology reviews : MMBR. ‒ 1998. ‒ Vol. 62, №. 3. ‒ P. 597-635. 10.1128/MMBR.62.3.597-635.1998.
Contesini F. J., Melo R. R., Sato H. H. An overview of Bacillus proteases: from production to application // Crit Rev Biotechnol. ‒ 2018. ‒ Vol. 38, №. 3. ‒ P. 321-334. 10.1080/07388551.2017.1354354.
Jagadeesan Y., Meenakshisundaram S., Saravanan V., Balaiah A. Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management // Int J Biol Macromol. ‒ 2020. ‒ Vol. 163. ‒ P. 135-146. 10.1016/j.ijbiomac.2020.06.219.
Ward O. P. 3.49 - Proteases // Comprehensive Biotechnology (Third Edition) / Moo-Young M. ‒ Oxford: Pergamon, 2011. ‒ C. 604-615.
Abdelgalil S. A., Soliman N. A., Abo-Zaid G. A., Abdel-Fattah Y. R. Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO // Sci Rep. ‒ 2021. ‒ Vol. 11, №. 1. ‒ P. 6071. 10.1038/s41598-021-85207-4.
SanthaKalaikumari S., Sivakumar R., Gunasekaran P., Rajendhran J. Whole-genome Sequencing and Mining of Protease Coding Genes in Bacillus paralicheniformis MKU3, and its Degradomics in Feather Meal Medium // Curr Microbiol. ‒ 2021. ‒ Vol. 78, №. 1. ‒ P. 206-217. 10.1007/s00284-020-02271-1.
Fang Z., Yong Y. C., Zhang J., Du G., Chen J. Keratinolytic protease: a green biocatalyst for leather industry // Appl Microbiol Biotechnol. ‒ 2017. ‒ Vol. 101, №. 21. ‒ P. 7771-7779. 10.1007/s00253-017-8484-1.
Papadopoulos M. C. The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal // Animal Feed Science and Technology. ‒ 1986. ‒ Vol. 16, №. 1. ‒ P. 151-156. Crossref.
Rehman R., Ahmed M., Siddique A., Hasan F., Hameed A., Jamal A. Catalytic Role of Thermostable Metalloproteases from Bacillus subtilis KT004404 as Dehairing and Destaining Agent // Appl Biochem Biotechnol. ‒ 2017. ‒ Vol. 181, №. 1. ‒ P. 434-450. 10.1007/s12010-016-2222-5.
Cai C. G., Lou B. G., Zheng X. D. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis // J Zhejiang Univ Sci B. ‒ 2008. ‒ Vol. 9, №. 1. ‒ P. 60-7. 10.1631/jzus.B061620.
Korniłłowicz-Kowalska T., Bohacz J. Biodegradation of keratin waste: Theory and practical aspects // Waste Manag. ‒ 2011. ‒ Vol. 31, №. 8. ‒ P. 1689-701. 10.1016/j.wasman.2011.03.024.
Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding // Anal Biochem. ‒ 1976. ‒ Vol. 72. ‒ P. 248-54.
Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 // Nature. ‒ 1970. ‒ Vol. 227, №. 5259. ‒ P. 680-5.
Daroit D. J., Brandelli A. A current assessment on the production of bacterial keratinases // Crit Rev Biotechnol. ‒ 2014. ‒ Vol. 34, №. 4. ‒ P. 372-84. 10.3109/07388551.2013.794768.
Wang B., Yang W., McKittrick J., Meyers M. A. Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration // Progress in Materials Science. ‒ 2016. ‒ Vol. 76. ‒ P. 229-318. Crossref.
Brandelli A., Daroit D. J., Riffel A. Biochemical features of microbial keratinases and their production and applications // Appl Microbiol Biotechnol. ‒ 2010. ‒ Vol. 85, №. 6. ‒ P. 1735-50. 10.1007/s00253-009-2398-5.
Babalola M. O., Ayodeji A. O., Bamidele O. S., Ajele J. O. Biochemical characterization of a surfactant-stable keratinase purified from Proteus vulgaris EMB-14 grown on low-cost feather meal // Biotechnology Letters. ‒ 2020. ‒ Vol. 42, №. 12. ‒ P. 2673-2683. 10.1007/s10529-020-02976-0.
Fraser R. D., Parry D. A. Molecular packing in the feather keratin filament // J Struct Biol. ‒ 2008. ‒ Vol. 162, №. 1. ‒ P. 1-13. 10.1016/j.jsb.2008.01.011.
Gupta R., Sharma R., Beg Q. K. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology // Crit Rev Biotechnol. ‒ 2013. ‒ Vol. 33, №. 2. ‒ P. 216-28. 10.3109/07388551.2012.685051.
Gupta R., Ramnani P. Microbial keratinases and their prospective applications: an overview // Appl Microbiol Biotechnol. ‒ 2006. ‒ Vol. 70, №. 1. ‒ P. 21-33. 10.1007/s00253-005-0239-8.
Ramnani P., Singh R., Gupta R. Keratinolytic potential of Bacillus licheniformis RG1: Structural and biochemical mechanism of feather degradation // Canadian journal of microbiology. ‒ 2005. ‒ Vol. 51. ‒ P. 191-6. 10.1139/w04-123.