One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

Document Type: Original Article


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

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


Objective(s): Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl) is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated.
Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR.
Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1.
Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.


1. Pinard MA, Lotlikar SR, Boone CD, Vullo D, Supuran CT, Patrauchan MA, et al. Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa. Bioorg Med Chem 2015; 23:4831–4838.

2. O'Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol 2000; 45:49-79.

3. Høiby N, Johansen HK, Moser C, Song ZJ, Ciofu O, Kharazmi A. Pseudomonas aeruginosa and the biofilm mode of growth. Microb Infect 2001; 3:1–13.

4. Prasanna SS, Doble M.  Medical biofilms–Its formationand prevention using organic molecules. J Indian Inst Sci 2008; 88:27-35.

5. Gacesa P. Bacterial alginate biosynthesis-recent progress and future prospects. Microbiology 1998; 144:1133-1143.

6. Govan JR,  Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia .  Microbiol Rev 1996; 60:539–574.

7. Farrell EK, Tipton PA. Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa. Biochemistry 2012; 51:10259–10266.

8. Hatch RA, Schiller NL. Alginate Lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents Chemother 1998;42:974-977.

9. Alipour M, Suntres ZE, Omri A. Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa. J Antimicrob Chemother 2009; 64:317-325.

10. Lamppa JW, Griswolda KE. Alginate lyase exhibits catalysis-independent biofilm dispersion and antibiotic synergy. Antimicrob Agents Chemother 2013; 57:135-147.

11. Eftekhar F, Schiller NL. Partial purification and characterization of a mannuronan-specific alginate lyase from Pseudomonas aeruginosa. Curr Microbiol 1994; 29:37-42.

12. Weissbach A, Hurwitz J. The formation of 2-keto-3- deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J Biol Chem 1959; 234:705-709.

13. Nguyen LK, Schiller NL. Identification of a slime polysaccharide depolymerase in mucoid strain of Pseudomonas aeruginosa. Curr Microbiol 1989; 18:323-329.

14. Xiao L, Han F, Yang Z, Lu X, Yu W. A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03. World J Microbiol Biotechnol 2006; 22:81-88.

15. Miyake O, Ochiai A, Hashimoto W, Murata K. Origin and diversity of alginate lyases of families PL-5 and -7 in sphingomonas sp. strain A1. J Bacteriol 2004; 186:2891-2896.

16. Haug A, Larsen B, Smidsrød O. A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem Scand 1966; 20:183-190.

17. Sepehr Sh, Rahmani-Badi A, Babaie-Naiej H, Soudi MR. Unsaturated fatty acid, cis-2-decenoic acid, in combination with disinfectants or antibiotics removes pre-established biofilms formed by food-related bacteria. Plos One 2014; 9:1-9.

18. SkjaK-BrK G, Larsen B. Purification of mannuronan C5-epimerase by affinity chromoatography on alginate-sepharose. Carbohydr Res 1982; 103:137-140.

19. Laemmeli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nat Pub Group(NPG) 1970; 227:680-685.

20. Caetano-Anolles G, Bassam BJ. DNA amplification finger printing using arbitrary oligonucleotide primers. Appl Biochem Biotechnol 1993; 42:189-200.

21. Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R. Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using micro titer plate method. J Microbiol Methods  2014; 105:134-140.

22. Li Y, Hanna MN, Svensater G, Ellen RP, Cvitkovitch DG. Cell density modulates acid adaptation in streptococcus mutans: implications for survival in biofilms. J Bacteriol 2001; 183:6875-6884.

23. Wilson K. Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol 2001;  2:2.4.

24. Bakkevig K, Sletta H, Gimmestad M, Aune R, Ertesvåg H, Degnes K, et al. Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment. J Bacteriol 2005; 187:8375–8384.

25. Li JW, Dong S, Song J, Li CB, Chen XL, Xie BB, et al. Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524. Mar Drugs 2011; 9:109-123.

26. Fu XT, Lin H, Kim SM. Purification and characterization of a Na+/ K+ dependent alginate lyase from turban shell gut vibrio sp. YKW-34. Enzyme Microb Technol 2007; 41:828-834.

27. Jain S, Ohman DE. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.  Infect Immun 2005; 73:6429-6436.