Overexpression of Thermal and pH Stable Alginate Lyase of P. aeruginosa 293 and In silico Study of algL Gene

Document Type : Research Article

Authors

1 Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

2 Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

3 Department of Microbiology, Faculty of Biological Sciences, University of Tehran, Tehran, Iran

Abstract

P. aeruginosa is an opportunistic bacterium that produces a capsule-like polysaccharide called alginate in response to various stimuli. The mucoid strain of Pseudomonas aeruginosa produces alginate which is an exopolysaccharide and is involved in the pathogenicity and persistence of these bacteria in infections. The alginate lyase gene is required for alginate synthesis. The enzyme can also degrade this polymer. This enzyme has a polymorphism in different bacteria even in one species and finding an enzyme with tremendous characters is very important. In this study, alginate lyase from P. aeruginosa strain 293 which was previously isolated from the sputum, and the encoding gene was characterized, and thermal and pH stability, as well as the substrate specificity of the partially purified alginate lyase, were determined. The amount of 70% activity of the enzyme was maintained after incubation at 80 ˚C for 6 hrs and 50% activity retained after incubation in alkaline and acidic pH. Moreover, it showed activity towards guluronic acid blocks, mannuronic acid blocks, and alginate blocks with both of them. Due to the unique properties of the alginate lyase that are useful in medicinal, and industrial applications, the gene encoding the enzyme was expressed in pET-28a (+)/E. coli BL21 (DE3) system to produce 371 -amino acid alginate lyase protein, the molecular weight of which was estimated by Sodium Dodecyl Sulfate- Polyacrylamide gel electrophoresis to be about 40 kDa. Bioinformatic analysis of P. aeruginosa strain 293 algL gene revealed that G225A point mutation can improve its thermostability. Therefore, the P. aeruginosa 293 alginate lyase is proposed as an appropriate candidate for the evaluation of potential therapeutic and industrial applications.

Keywords

References

Ahmed N. 2007. Genetics of bacterial alginate: alginate genes distribution, organization and biosynthesis in bacteria. Curr Genomics 8:191-202.
Bayer AS, Park S, Ramos MC, Nast CC, Eftekhar F, and Schiller NL. 1992. Effects of alginase on the natural history and antibiotic therapy of experimental endocarditis caused by mucoid Pseudomonas aeruginosa. Infect Immun 60: 3979-3985.
Bayer AS, Speert DP, Park S, Tu J, Witt M, Nast CC, et al. 1991. Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonas aeruginosa. Infect Immun 59: 302-308.
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. 
Britton HTS, Robinson RA. 1993. CXCVIII. Universal buffer solutions and the dissociation constant of veronal. J Chem Soc 1931: 1456-1462.
Chen P, Zhu Y, Men Y, Zeng Y, Sun Y. 2018. Purification and characterization of a novel alginate lyase from the marine bacterium Bacillus sp. Alg07. Mar Drugs  16: 86-99.
Del Mar Lleò M, Pierobon S, Tafi MC, Signoretto C, Canepari P. 2000. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Applied and Environmental Microbiology. Appl Environ Microbiol  66: 4564-4567.
Eftekhar F, Schiller NL. 1994. Partial purification and characterization of a mannuronan-specific alginate lyase from Pseudomonas aeruginosa. Curr Microbiol 29: 37-42.
Farrell EK, Tipton PA. 2012. Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa. Biochemistry 51:10259-10266.
Franklin MJ, Nivens, DE, Weadge JT, Howell PL. 2011. Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl. Front Microbiol 2: 167-182.
Gol Fakhrabadi H, Ghadam P, Mohammadi, P, Abdi-Ali A. 2015. Purification and Characterization of Alginate Lyase from Mucoid Pseudomonas aeruginosa Strain 214. J Genet Resour 1: 55-60.
Ghadam P, Akhlaghi F, Abdi Ali A. 2017. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm. Iran J Basic Med Sci 20: 467-473.
Haug A, Larsen B, Smidsrød O. 1966. A study of the constitution of alginic acid by partial acid hydrolysis. acta chem Sc 20: 183-190.
Hoshino T, Kageyama M. 1980. Purification and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa. J Bacteriol 141: 1055-1063.
Huang G, Wang Q, Lu  M, Xu C, Li F, Huang S. 2018. AlgM4: A new salt-activated alginate lyase of the PL7 family with endolytic activity. Mar Drugs 16: 120- 133.
Inoue A, Takadono K, Nishiyama R, Tajima K, Kobayashi T, Ojima T. 2014. Characterization of an alginate lyase, FlAlyA, from Flavobacterium sp. strain UMI-01 and its expression in Escherichia coli. Mar Drugs 12: 4693-4712.
Jain S, Ohman DE. 2005. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa. Infect Immun 73: 6429-6436.
Kim HS, Lee CG, Lee EY. 2011. Alginate lyase: structure, property, and application. Biotechnol Bioprocess Eng  16: 843-851.
Laemmeli UK. 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227:680-685.
Li JW, Dong S, Song J, Li CB., Chen XL, Zhang YZ. 2011. Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524. Mar Drugs  9: 109-123.
Li S, Wang L, Jung S, Lee B, He N, Lee M. 2020. Biochemical Characterization of a New Oligoalginate Lyase and Its Biotechnological Application in Laminaria japonica Degradation. Front Microbiol 11: 1-12.
Moradali MF, Donati I, Sims IM, Ghods S, Rehm BH. 2015. Alginate polymerization and modification are linked in Pseudomonas aeruginosa. MBio 6: e00453-15.
Nguyen LK, Schiller NL. 1989. Identification of a slime exopolysaccharide depolymerase in mucoid strains of Pseudomonas aeruginosa. Curr Microbiol 18: 323-329.
Park HH, Kam N, Lee EY, Kim HS. 2012. Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol 14:189-202.
Peciña A, Pascual A, Paneque A. 1999. Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme. J Bacteriol 181:1409-1414.
Piroozmand F, Ghadam P, Zarrabi M, Abdi Ali A. 2020. Biochemical and computational study of an alginate lyase produced by Pseudomonas aeruginosa strain S21. Iran J Basic Med Sci 23: 454-460.
Sambrook J, Fritsche F, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.New York: Cold Spring Harbor Laboratory Press.
Sambrook J, Russel DW. 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press.
Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467.
Schiller NL, Monday SR, Boyd CM, Keen NT, Ohman DE. 1993. Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol 175: 4780-4789.
Sharma G, Rao S, Bansal A, Dang S, Gupta S, Gabrani R. 2013. Pseudomonas aeruginosa biofilm: potential therapeutic targets. Biologicals 42: 1-7.
Shimokawa T, Yoshida S, Kusakabe I, Takeuchi T, Murata K, Kobayashi H. 1997. Some Properties and Action Mode of (1→4)-α-L-Guluronan Lyase from Enterobacter cloacae M-1. Carbohydr Res 304: 125-132.
Swift SM, Hudgens JW, Heselpoth RD, Bales PM, Nelson DC. 2014. Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A. PLoS One 9: e112939-e112949.
Tavafi H, Abdi-Ali A, Ghadam P, Gharavi S. 2016. Screening of alginate lyase-producing bacteria and optimization of media compositions for extracellular alginate lyase production. Iran Biomed J 21: 48-56.
Tavafi H, Abdi-Ali A, Ghadam P, Gharavi S. 2019. Evaluation of the synergistic effect of bacterial recombinant alginate lyase and therapeutic antibiotics on the growth of planktonic Pseudomonas aeruginosa. JMW 12: 160-171
Wang Y, Chen X, Bi X, Ren y, Han Q, Li S. 2019. Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property. Mar Drugs 17: 308-322.
Wang Y, Guo EW, Yu WG, Han F. 2013. Purification and characterization of a new alginate lyase from marine bacterium Vibrio sp. Biotechnol Lett 35: 703-708.
Weissbach A, Hurwitz J. 1958. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli. J Biol Chem 234: 705-709.
Wong TY, Preston LA, Schiller NL. 2000. Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 54: 289-340.
Xiao L, Han F, Yang Z, Lu XZ, Yu WG. 2006. A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03. World J Microbiol Biotechnol 22: 81-88.
Zhu BW, Huang LSX, Tan HD, Qin YQ, Du YG, Yin H. 2015. Characterization of a new endo-type polyM-specific alginate lyase from Pseudomonas sp. Biotechnol Lett 37: 409-415.
Zhu B, Ni F, Ning L, Sun Y, Yao Z. 2018. Cloning and characterization of a new pH-stable alginate lyase with high salt tolerance from marine Vibrio sp. NJ-04. Int J Biol Macromol 115: 1063–1070.
Zhu B, Ni F, Sun Y, Ning L, Yao Z. 2019. Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides. Biotechnol Biofuels 12:1-3.
Journal of Genetic Resources
Volume 7, Issue 1
2021
Pages 49-58

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