AIMS Bioengineering, 2017, 4(3): 366-375. doi: 10.3934/bioeng.2017.3.366

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Enhanced extracellular chitinase production in Pseudomonas fluorescens: biotechnological implications

Faculty of Science and Engineering, Laurentian University, Sudbury, ON, Canada

Chitin is an important renewable biomass of immense commercial interest. The processing of this biopolymer into value-added products in an environmentally-friendly manner necessitates its conversion into N-acetyl glucosamine (NAG), a reaction mediated by the enzyme chitinase. Here we report on the ability of the soil microbe Pseudomonas fluorescens to secrete copious amounts of chitinase in the spent fluid when cultured in mineral medium with chitin as the sole source of carbon and nitrogen. Although chitinase was detected in various cellular fractions, the enzyme was predominantly localized in the extracellular component that was also rich in NAG and glucosamine. Maximal amounts of chitinase with a specific activity of 80 µmol NAG produced mg–1 protein min–1 was obtained at pH 8 after 6 days of growth in medium with 0.5 g of chitin. In-gel activity assays and Western blot studies revealed three isoenzymes. The enzyme had an optimal activity at pH 10 and a temperature range of 22–38 ℃. It was stable for up to 3 months. Although it showed optimal specificity toward chitin, the enzyme did readily degrade shrimp shells. When these shells (0.1 g) were treated with the extracellular chitinase preparation, NAG [3 mmoles (0.003 g-mol)] was generated in 6 h. The extracellular nature of the enzyme coupled with its physico-chemical properties make this chitinase an excellent candidate for biotechnological applications.
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References

1. Khoushab F, Yamabhai M (2010) Chitin research revisited. Mar Drugs 8: 1988–2012.    

2. Cheba BA (2011) Chitin and chitosan: marine biopolymers with unique properties and versatile applications. Global J Biotech Biochem 6: 149–153.

3. Thiagarajan V, Revathi R, Aparanjini K, et al. (2011) Extra cellular chitinase production by Streptomyces sp.PTK19 in submerged fermentation and its lytic activity on Fusarium oxysporum PTK2 cell wall. Int J Curr Sci 1: 30–44.

4. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biot 71: 773–782.    

5. Haggag WM, Abdallh E (2012) Purification and characterization of chitinase produced by Endophytic ptomyceshygroscopicus against some phytopathogens. J Microbiol Res 2: 145–151.    

6. Reissig JL, Strominger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 21: 959–966.

7. Kamil Z, Saleh M, Moustafa S (2007) Isolation and identification of rhizosphere soil chitinolytic bacteria and their potential in antifungal biocontrol 1. Global J Mol Sci 2: 57–66.

8. Bhattacharya D, Nagpure A, Gupta RK (2007) Bacterial chitinases: properties and potential. Crit Rev Biotechnol 27: 21–28.    

9. Hamid R, Khan MA, Ahmad M, et al. (2013) Chitinases: an update. J Pharm Bioallied Sci 5: 21–29.

10. Narayanan K, Chopade N, Raj PV, et al. (2013) Fungal chitinase production and its application in biowaste management. J Sci Ind Res 72: 393–399.

11. Appanna VD, Pierre MS (1996) Aluminum elicits exocellular phosphatidylethanolamine production in Pseudomonas fluorescens. Appl Environ Microb 62: 2778–2782.

12. Auger C, Han S, Appanna VP, et al. (2013) Metabolic reengineering invoked by microbial systems to decontaminate aluminum: implications for bioremediation technologies. Biotechnol Adv 31: 266–273.    

13. Appanna VD, Gazsó LG, Pierre MS (1996) Multiple-metal tolerance in Pseudomonas fluorescens and its biotechnological significance. J Biotechnol 52: 75–80.    

14. Bignucolo A, Appanna VP, Thomas SC, et al. (2013) Hydrogen peroxide stress provokes a metabolic reprogramming in Pseudomonas fluorescens: enhanced production of pyruvate. J Biotechnol 167: 309–315.    

15. Middaugh J, Hamel R, Jean-Baptiste G, et al. (2005) Aluminum triggers decreased aconitase activity via Fe-S cluster disruption and the overexpression of isocitrate dehydrogenase and isocitrate lyase: a metabolic network mediating cellular survival. J Biol Chem 280: 3159–3165.    

16. Yanes ML, Bajsa N (2016) Fluorescent Pseudomonas: a natural resource from soil to enhance crop growth and health, In: Microbial models: from environmental to industrial sustainability Springer Singapore, 323–349.

17. Chenier D, Beriault R, Mailloux R, et al. (2008) Involvement of fumarase C and NADH oxidase in metabolic adaptation of Pseudomonas fluorescens cells evoked by aluminum and gallium toxicity. Appl Environ Microb 74: 3977–3984.    

18. 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.    

19. Mailloux R, Lemire J, Kalyuzhnyi S, et al. (2008) A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity. Extremophiles 12: 451–459.    

20. Alhasawi A, Auger C, Appanna VP, et al. (2014) Zinc toxicity and ATP production in Pseudomonas fluorescens. J Appl Microbial 117: 65–73.    

21. Sharp RG (2013) A review of the applications of chitin and its derivatives in agriculture to modify plant-microbial interactions and improve crop yields. Agronomy 3: 757–793.    

22. Neiendam NM, Sørensen J (1999) Chitinolytic activity of Pseudomonas fluorescens isolates from barley and sugar beet rhizosphere. FEMS Microbiol Ecol 30: 217–227.    

23. Negrulescu A, Patrulea V, Mincea MM, et al. (2012) Adapting the reducing sugars method with dinitrosalicylic acid to microtiter plates and microwave heating. J Braz Chem Soc 23: 2176–2182.    

24. Auger C, Lemire J, Cecchini D, et al. (2011) The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens. PloS One 6: e28469.    

25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685.

26. Liu CL, Shen CR, Hsu FF, et al. (2009) Isolation and identification of two novel SDS‐resistant secreted chitinases from Aeromonas schubertii. Biotechnol Prog 25: 124–131.    

27. Loni PP, Patil JU, Phugare SS, et al. (2014) Purification and characterization of alkaline chitinase from Paenibacillus pasadenensis NCIM 5434. J Basic Microb 54: 1080–1089.    

28. Velusamy P, Ko HS, Kim KY (2011) Determination of antifungal activity of Pseudomonas sp. A3 against Fusarium oxysporum by high performance liquid chromatography (HPLC). Agric Food Annal Bacteriol 1: 15–23.

29. Zou X, Nonogaki H, Welbaum GE (2002) A gel diffusion assay for visualization and quantification of chitinase activity. Mol Biotechnol 22: 19–23.    

30. Karunya SK (2011) Optimization and purification of chitinase produced by Bacillus subtilis and its antifungal activity against plant pathogens. Intl J Pharma Biol Arch 2: 1680–1685.

31. Viarsagh MS, Janmaleki M, Falahatpisheh HR, et al. (2010) Chitosan preparation from Persian Gulf shrimp shells and investigating the effect of time on the degree of deacetylation. J Paramed Sci 1: 2–6.

32. Kuzu SB, Güvenmez HK, Denizci AA (2012) Production of a thermostable and alkaline chitinase by Bacillus thuringiensis subsp. kurstaki strain HBK-51. Biotechnol Res Int 2012: 1–6.

33. Park SH, Lee JH, Lee HK (2000) Purification and characterization of chitinase from a marine bacterium, Vibrio sp. 98CJ11027. J Microbiol 38: 224–229.

Copyright Info: © 2017, Vasu D. Appanna, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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