Mikrobiol. Z. 2018; 80(5):36-47. Ukrainian.
Comparative Characteristics of Proteases of Bacillus thuringiensis IMV B-7324
and Bacillus thuringiensis var. israelensis ІМV В-7465
Dziubliuk N.A.1, Chernyshenko V.O.2, Brovarskaya O.S.1, Varbanets L.D.1
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
2Palladin Institute of Biochemistry, NAS of Ukraine
9 Leontovycha Str., Kyiv, 01601, Ukraine
The aim of this study was to characterize Bacillus thuringiensis ІМV В-7324 and B. thuringiensis var. israelensis ІМV В-7465 proteases with collagenase, elastase and fibrinolytic activities namely to measure their molecular weight, qualitative and quantitative amino acid and monosaccharide composition and compare these enzymes of the studied parameters. Methods. Homogeneity of the enzyme preparations was determined in denaturing (SDS-PAGE) and native (gel-filtration on Sepharose 6B) systems. The amino acid composition assay of the studied proteases was carried out using an automatic amino acid analyzer Т339 (Microtechna, Czech Republic). Identification of neutral monosaccharides of the preparations was carried out by the method of chromatographic mass spectrometry by Albershein et al. Results. For the first time has been shown that protease 1 and 2 of B. thuringiensis IMV B-7324 and B. thuringiensis var. israelensis IMV В-7465 protease have different values of molecular weight (about 25.8, 24.2 and 37.0 kDa respectively) and similar amino acid composition of molecules. The level of polar amino acids in protease 1 of B. thuringiensis IMV B-7324 is 1.5 times lower than in the rest of the tested enzymes. It has been shown for the first time that these proteases contain in their composition one or two monosaccharides. Conclusions. The obtained results demonstrate differences in the amino acid and monosaccharide composition of the proteases with collagenase, elastase and fibrinolytic activity within the B. thuringiensis species, which makes a significant contribution to its characterization.
Keywords: Bacillus thuringiensis, proteases, elastolytic, collagenolytic, fibrinolytic activity, molecular weight, amino acid and monosaccharide composition.
Full text (PDF, in Ukrainian)
- Rawlings ND, Salvesen GS. Handbook of Proteolytic Enzymes. 3rd ed. London: Academic Press, Elsevier; 2013. P. 1062-1064.
- Contesini FJ, Melo RR, Sato HH. An overview of Bacillus proteases: from production to application. Crit Rev Biotechnol. 2018; 38(3):321-334. https://doi.org/10.1080/07388551.2017.1354354
- Elleuch J, Zghal RZ, Lacoix MN, Chandre F, Tounsi S, Jaoua S. Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability. Microbiological Research. 2015; 176:48-54. https://doi.org/10.1016/j.micres.2015.04.007
- Ennouri K, Ayed RB, Triki MA, Ottaviani E, Mazzarello M, Hertelli F, Zouari N. Multiple linear regression and artificial neural networks for deltaendotoxin and protease yields modelling of Bacillus thuringiensis. Biotech. 2017; 7(3):187-199.
- Egorov NS, Yudina TG, Loriya JK, Kreyer VG. [About fibrinolytic activity of some variants of Bacillus thuringiensis]. Prikladnaya biokhimiya i mikrobiologiya. 1979; 15(3):416-420. Russian.
- Tenorio-Sánchez SA, Rojas-Avelizapa NG, Ibarra JE, Avelizapa LIR, Cruz-Camarillo R. Characterization of a Bacillus thuringiensis strain isolated from a highly polychlorinated biphenyls contaminated soil. Tecnóloga. 2010; 3(3):52-63.
- Matselyukh OV. [Obtaining of mutants of Bacillus sp. with enhanced elastase production]. Biotekhnolohiya. 2010; 3(2):42-47. Ukrainian.
- Matselyukh OV, Varbanets LD, Ivanitsa VO. Strain Bacillus thuringiensis IMV B-7324 – producer of the extracellular elastase. Pattent 97906UA. Publ. 26.03.2012, Bul. 6. Ukrainian.
- Nidialkova NA, Varbanets LD, Ivanitsa VO. Bacterial strain of Bacillus thuringiensis var. israelensis – producer of the extracellular collagenase. Pattent 96195UA. Publ. 26.01.2015, Bul. 2. Ukrainian.
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-275.
- Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-685. https://doi.org/10.1038/227680a0
- Varbanets LD, Borzova NV. [Glycosidases of microorganisms and methods of its analysis]. Kyiv: Naukova Dumka; 2010. Ukrainian.
- Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956; 28(3):350-356. https://doi.org/10.1021/ac60111a017
- Albershein P, Nevis DJ, English PD, Karr A. A method for analysis of sugars in plant cell wall polysaccharides by gasliquid chromatography. Carboh. Res. 1976; 5(3):340-345. https://doi.org/10.1016/S0008-6215(00)80510-8
- Lakin GF. [Biometriya]. Mocsow: Vysshaya shkola; 1990. Russian.
- Lapatch SN, Tchubenko AV, Babitch PH. [Statistical methods in biomedical research using Excel]. Kyiv: Morion, 2001. Russian.
- Nidialkova NA, Matselyukh OV, Varbanets LD. [Isolation of Bacillus thuringiensis IMV B-7324 fibrinolytic peptidase]. Mikrobiol Z. 2012; 74(5):9-15. Ukrainian.
- Matselyukh OV, Nidialkova NA, Varbanets LD. [Purification and physicochemical properties of Bacillus thuringiensis IМВ В-7324 peptidase with elastolytic and fibrinolytic activity]. Ukr Biokhim Z. 2012; 84(6):25-36. Ukrainian.
- Nidialkova NA, Varbanets LD, Chernyshenko VO. Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen. Ukr Biochem J. 2016; 88(3):18-28. https://doi.org/10.15407/ubj88.03.018
- Walls D, Loughran ST. Protein Chromatography: Methods and Protocols, Methods in Molecular Biology. New York: Humana Press: Springer; 2007.
- Yong-Gang Z, Hua LI, William XU, Jia L, Rui-An XU. An Overview of the Fibrinolytic Enzyme from Earthworm. Chin J Nat Med. 2010; 8(4):301-308. https://doi.org/10.3724/SP.J.1009.2010.00301
- Hammami A, Fakhfakh N, Abdelhedi O, Nasri M, Bayoudh A. Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing. Int J Biol Macromol. 2018; 108:56-68. https://doi.org/10.1016/j.ijbiomac.2017.11.148
- SinhaS, WatorekW, Karr S, Giles J, Bode W, Travis J. Primary structure of human neutrophil elastase. Proc Natl Acad Sci USA. 1987; 84(8):2228-2232. https://doi.org/10.1073/pnas.84.8.2228
- Takami H, AkibaT, Horikoshi K. Characterization of an alkaline protease from Bacillus sp. no. AH-101. Appl Microbiol Biotechnol. 1990; 33(5):519-523. https://doi.org/10.1007/BF00172544
- McKee T, McKee J. Biochemistry. The Molecular Basis of Life Updated Fifth Edition. New York: Oxford University Press; 2012.
- Nakajima N, Mihara H, Sumi H. Characterization of potent fibrinolytic enzymes in earthworm, Lumbricus rubellus. Biosci Biotechnol Biochem. 1993; 57(10):1726-1730. https://doi.org/10.1271/bbb.57.1726
- Goettig P. Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases. Int J Mol Sci. 2016; 17(12):1969. https://doi.org/10.3390/ijms17121969
- Russell D, Oldham NJ, Davis BG. Site-selective chemical protein glycosylation protects from autolysis and proteolytic degradation. Carbohydr. Res. 2009; 344(12):1508–1514. https://doi.org/10.1016/j.carres.2009.06.033
- Cesarman-Maus G, Hajjar KA Molecular mechanisms of fibrinolysis. British Journal of Haematology. 2005; 129(3):307–321. https://doi.org/10.1111/j.1365-2141.2005.05444.x
- Wendorf P, Geyer R, Sziegoleit A, Linder D. Localization and characterization of the glycosylation site of human pancreatic elastase 1. FEBS Letters. 1989; 249(2):275-278. https://doi.org/10.1016/0014-5793(89)80640-4