Research Article | Volume: 6, Issue: 1, Jan-Feb, 2018

Biochemical and molecular characterization of a new pullulan producer Rhodosporidium paludigenum PUPY-06

RS Singh Navpreet Kaur   

Open Access   

Published:  Jan 17, 2018

DOI: 10.7324/JABB.2018.60106

Pullulan is a random coil glucan polymer produced by Aureobasidium pullulans. It is one of the commercially promising biopolymers frequently used in pharmaceutical, food and cosmetics industries. The aim of present study was to isolate an efficient fungal strain producing melanin-free pullulan. The fungal strains were isolated from fresh and decaying leaves of plants and screened for pullulan production. Fungal isolate PUPY-06 was the best producer of pullulan (2.12%, w/v). Morphological studies revealed the oval and cylindrical shape of its cells, budding and presence of septate as well as pseudohyphae. Colonies of the isolate on agar plate cultures were beige-to-orange colored with smooth, mucoid, and butyrous texture. Morphological and biochemical characterization established the fungal isolate as Rhodosporidium sp. 18s rRNA sequencing, and phylogenetic analysis revealed the isolate to be Rhodosporidium paludigenum. Fourier-transform infrared spectroscopy and nuclear magnetic resonance of crude pullulan confirmed its structural characteristics. Maximum production of pullulan was achieved after 7 days of cultivation in shake-flask fermentations. Agitation mode of cultivation supported the higher production of pullulan in comparison to the stationary mode. R. paludigenum PUPY-06 was found as a novel pullulan-producing strain. This is a first report on pullulan production by R. paludigenum PUPY-06.

Keyword:     18s rRNA Nuclear magnetic resonance Phylogenetic tree Pullulan Rhodosporidium paludigenum Shake-flask fermentations.


Singh RS, Kaur N. Biochemical and molecular characterization of a new pullulan producer Rhodosporidium paludigenum PUPY-06. J App Biol Biotech. 2018;6(1):28-37. DOI: 10.7324/JABB.2018.60106

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. Fraser CG, Jennings HJ. A glucan from Tremella mesenterica NRRL-Y6158. Can J Chem 1971;49:1804-7.

2. Waksman N, Lederkremer RM, Cerezo AS. The structure of an α-D-glucan from Cyttaria harioti fischer. Carbohydr Res 1977;59:505-15.

3. Oliva EM, Cirelli AF, Lederkremer RM. Characterization of a pullulan in Cyttaria darwinii. Carbohydr Res 1986;158:262-7.

4. Chi Z, Zhao S. Optimization of medium and new cultivation conditions for pullulan production by a new pullulan-producing yeast strain. Enzyme Microb Technol 2003;33:206-11.

5. Corsaro MM, Castro C, Evidente A, Lanzetta R, Molinaro A, Parrilli M, et al. Phytotoxic extracellular polysaccharide fractions from Cryphonectria parasitica (Murr) Barr strains. Carbohydr Polym 1998;37:167-72.

6. Reis RA, Tischer CA, Gorin PA, Iacomini M. A new pullulan and a branched (1→3)-, (1→6)-linked beta-glucan from the lichenised ascomycete Teloschistes flavicans. FEMS Microbiol Lett 2002;210:1-5.

7. Delben F, Forabosco A, Guerrini M, Liut G, Torri G. Pullulans produced by strains of Cryphonectria parasitica-II. Nuclear magnetic resonance evidence. Carbohydr Polym 2006;63:545-54.

8. Forabosco A, Bruno G, Sparapano L, Liut G, Marino D, Delben F. Pullulans produced by strains of Cryphonectria parasitica-I. Production and characterization of the exopolysaccharides. Carbohydr Polym 2006;63:535-44.

9. Singh RS, Saini GK, Kennedy JF. Pullulan: Microbial sources, production and applications. Carbohydr Polym 2008;73:515-31.

10. Singh RS, Saini GK. Pullulan-hyperproducing color variant strain of Aureobasidium pullulans FB-1 newly isolated from phylloplane of Ficus sp. Bioresour Technol 2008;99:3896-9.

11. Sugimoto K. Pullulan production and applications. J Ferment Assoc Jpn 1978;36:98-108.

12. Tsujisaka Y, Mitsuhashi M. Pullulan. In: Whistler RL, BeMiller JN, editors. Industrial Gums, Polysaccharides and their Derivatives. San Diego, California: Academic Press; 1993. p. 447-60.

13. Shingel KI. Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan. Carbohydr Res 2004;339:447-60.

14. Singh RS, Saini GK. Pullulan as therapeutic tool in biomedical applications. In: Singh RS, Pandey A, Larroche C, editors. Advances in Industrial Biotechnology. India: IK International Publishing House Pvt. Ltd.; 2014. p. 263-91.

15. Singh RS, Saini GK. Biosynthesis of pullulan and its applications in food and pharmaceutical industry. In: Satyanarayana T, Johri BN, Prakash A, editors. Microorganisms in Sustainable Agriculture and Biotechnology. Part 2. New Delhi: Springer Science + Business Media B.V; 2012. p. 509-53.

16. Leathers TD. Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol. 2003;62:468-73.

17. Singh RS, Kaur N. Microbial biopolymers for edible film and coating applications. In: Nawani NN, Khetmalas M, Razdan PN, Pandey A, editors. Advances in Biotechnology. India: IK International Publishing; 2015. p. 187-216.

18. Singh RS, Saini GK, Kennedy JF. Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system. Carbohydr Polym 2010;81:252-9.

19. Singh RS, Saini GK, Kennedy JF. Maltotriose syrup preparation from pullulan using pullulanase. Carbohydr Polym 2010;80:401-7.

20. Singh RS, Saini GK, Kennedy JF. Continuous hydrolysis of pullulan using covalently immobilized pullulanase in a packed bed reactor. Carbohydr Polym 2011;83:672-5.

21. Singh RS, Kaur N, Kennedy JF. Pullulan and pullulan derivatives as promising biomolecules for drug and gene targeting. Carbohydr Polym 2015;123:190-207.

22. Singh RS, Kaur N, Rana V, Kennedy JF. Pullulan: A novel molecule for biomedical applications. Carbohydr Polym 2017;171:102-21.

23. Singh RS, Kaur N, Rana V, Kennedy JF. Recent insights on applications of pullulan in tissue engineering. Carbohydr Polym 2016;153:455-62.

24. Kageyama S, Wada H, Muro K, Niwa Y, Ueda S, Miyata H, et al. Dose-dependent effects of NY-ESO-1 protein vaccine complexed with cholesteryl pullulan (CHP-NY-ESO-1) on immune responses and survival benefits of esophageal cancer patients. J Transl Med 2013;11:246.

25. Kong IG, Sato A, Yuki Y, Nochi T, Takahashi H, Sawada S, et al. Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumonia. Infect Immun 2013;81:1625-34.

26. Kong SH, Noh YW, Suh YS, Park HS, Lee HJ, Kang KW, et al. Evaluation of the novel near-infrared fluorescence tracers pullulan polymer nanogel and indocyanine green/γ-glutamic acid complex for sentinel lymph node navigation surgery in large animal models. Gastric Cancer 2015;18:55-64.

27. Kulicke WM, Heinze T. Improvements in polysaccharides for use as blood plasma expanders. Macromol Symp 2006;231:47-59.

28. Sawada SI, Sasaki Y, Nomura Y, Akiyoshi K. Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase. Colloids Polym Sci 2011;289:685-91.

29. Chatap VK, Maurya AR, Deshmukh PK, Zawar LR. Formulation and evaluation of nisoldipne sublingual tablets using pullulan and chitosan for rapid oromucosal absorption. Adv Pharmacol Pharm 2013;1:18-25.

30. Bauer R, Begerow D, Sampaio JP, Weiss M, Oberwinkler F. The simple-septate basidiomycetes: A synopsis. Mycol Prog 2006;5:41-66.

31. Toome M, Roberson RW, Aime MC. Meredithblackwellia eburnea gen. et sp. nov., kriegeriaceae fam. nov. and kriegeriales ord. nov.-- toward resolving higher-level classification in microbotryomycetes. Mycologia 2013;105:486-95.

32. Kirk PM, Cannon PF, Minter DW, Stalpers JA. Dictionary of the Fungi. 10th ed. Wallingford, UK: CAB International; 2008.

33. Moore RT. Taxonomic proposals for the classification of marine yeasts and other yeast-like fungi including the smuts. Bot Mar 1980;23:361-73.

34. Pollock TJ, Thorne L, Armentrout RW. Isolation of new Aureobasidium strains that produce high-molecular-weight pullulan with reduced pigmentation. Appl Environ Microbiol 1992;58:877-83.

35. Fell JW, Tallman AS. Rhodosporidium paludigenum sp. nov., a basidiomycetous yeast from intertidal waters of South Florida. Int J Syst Bacteriol 1980;30:658-9.

36. Wickerham LJ. Taxonomy of yeasts. Tech Bull U S Dep Agric 1951;1029:1-55.

37. Custers MT. Onderzoekingen Over het Gistgeslacht Brettanomyces. PhD Thesis. The Netherlands: Technische Hoogeschool te Delft; 1940.

38. Seeliger HP. Use of a urease test for the screening and identification of cryptococci. J Bacteriol 1956;72:127-31.

39. Christensen WB. Urea decomposition as a means of differentiating proteus and paracolon cultures from each other and from Salmonella and Shigella types. J Bacteriol 1946;52:461-6.

40. Van Der Walt JP, Hopsu-Havu VK. A colour reaction for the differentiation of Ascomycetous and hemibasidiomycetous yeasts. Antonie Van Leeuwenhoek 1976;42:157-63.

41. Whiffen AJ. The production, assay, and antibiotic activity of actidione, an antiobiotic from Streptomyces griseus. J Bacteriol 1948;56:283-91.

42. Yarrow D. Zygosaccharomyces barker. In: Rij NJ, editor. The Yeasts - A Taxonomic Study. Amsterdam: Elsevier; 1984. p. 449-65.

43. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-20.

44. Singh RS, Saini GK. Production, purification and characterization of pullulan from a novel strain of Aureobasidium pullulans FB-1. J Biotechnol 2008;136:S506-7.

45. Gaur R, Singh R, Tiwari S, Yadav SK, Daramwal NS. Optimization of physico-chemical and nutritional parameters for a novel pullulan-producing fungus, Eurotium chevalieri. J Appl Microbiol 2010;109:1035-43.

46. Kurtzman CP, Fell JW. The yeasts. A Taxonomic Study. 4th ed. Amsterdam: Elsevier; 1998.

47. Yimyoo T, Yongmanitchai W, Limtong S. Carotenoid production by Rhodosporidium paludigenum DMKU3-LPK4 using glycerol as the carbon source. Kasetsart J (Natural Science) 2011;45:90-100.

48. Lee JH, Kim JH, Zhu IH, Zhan XB, Lee JW, Shin DH, et al. Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans. Biotechnol Lett 2001;23:817-20.

49. Lazaridou A, Roukas T, Biliaderies CG, Vaikousi H. Characterization of pullulan produced from beet molasses by Aureobasidium pullulans in a stirred tank reactor under varying agitation. Enzyme Microb Technol 2002;31:122-32.

50. Singh RS, Singh H, Saini GK. Response surface optimization of the critical medium components for pullulan production by Aureobasidium pullulans FB-1. Appl Biochem Biotechnol 2009;152:42-53.

51. Gibbs PA, Seviour RJ. Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture? Appl Microbiol Biotechnol 1996;46:503-10.

52. Punnapayak H, Sudhadham M, Prasongsuk S, Pichayangkura S. Characterization of Aureobasidium pullulans isolated from airborne spores in Thailand. J Ind Microbiol Biotechnol 2003;30:89-94.

53. Prasongsuk S, Berhow MA, Dunlap CA, Weisleder D, Leathers TD, Eveleigh DE, et al. Pullulan production by tropical isolates of Aureobasidium pullulans. J Ind Microbiol Biotechnol 2007;34:55-61.

54. Prasongsuk S, Sullivan RF, Kuhirun M, Eveleigh DE, Punnapayak H. Thailand habitats as sources of pullulan-producing strains of Aureobasidium pullulans. World J Microbiol Biotechnol 2005;21:393-8.

55. Madi NS, Harvey LM, Mehlert A, McNeil B. Synthesis of two distinct exopolysaccharide fractions by cultures of the polymorphic fungus Aureobasidium pullulans. Carbohydr Polym 1997;32:307-14.

56. Yurlova NA, Hoog GS. A new variety of Aureobasidium pullulans characterized by exopolysaccharides structure, nutritional physiology and molecular features. Anton Leeuw J Microbiol 1997;72:141-7.

57. Kacurakova M, Capek P, Sasinkova V, Wellner N, Ebringerova A. FT-IR study of plant cell wall model compounds: Pectic polysaccharides and hemicellulose. Carbohydr Polym 2000;43:195-203.

58. Kacuráková M, Mathlouthi M. FTIR and laser-Raman spectra of oligosaccharides in water: Characterization of the glycosidic bond. Carbohydr Res 1996;284:145-57.

59. McIntyre DD, Vogel HJ. Nuclear magnetic resonance studies of homopolysaccharides related to starch. Starch Stärke 1991;43:69-76.

60. McIntyre DD, Vogel HJ. Structural studies of pullulan by nuclear magnetic resonance spectroscopy. Starch Stärke 1993;45:406-10.

61. Youssef F, Roukas T, Biliaderis CG. Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture. Process Biochem 1999;34:355-66.

62. Singh RS, Saini GK, Kennedy JF. Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1. Carbohydr Polym 2009;78:89-94.

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