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Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

Produção, purificação e aplicação da quitinase extracelular de Cellulosimicrobium cellulans 191

Abstracts

This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25ºC and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25ºC and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

chitinase; Cellulosimicrobium cellulans; fungal lysis; protoplasts


O presente estudo visou a produção, purificação e aplicação da quitinase extracelular da linhagem Cellulosimicrobium cellulans 191. A maior produção de quitinase em frascos agitados foi 6,9 U/mL após 72 h de fermentação a 25ºC e 200 rpm. Em fermentador de 5 L utilizando aeração de 1,5 vvm, a maior atividade da enzima foi 4,19 U/mL após 168 h de fermentação a 25ºC e 200 rpm; e com 3 vvm, foi obtido 4,38 U/mL após 144 h de fermentação. A quitinase (61 KDa) foi purificada cerca de 6,65 vezes em coluna de filtração em gel Sepharose CL 4B 200 com um rendimento de 46,61%. A enzima purificada foi capaz de lisar a parede celular de alguns fungos e formar protoplastos.

quitinase; Cellulosimicrobium cellulans; lise de fungos; protoplastos


FOOD MICROBIOLOGY

Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

Produção, purificação e aplicação da quitinase extracelular de Cellulosimicrobium cellulans 191

Luciana F. Fleuri* * Corresponding Author. Mailing address: Faculty of Food Engineering, Food Science Department, P.O Box 6121, CEP 13083862, UNICAMP - Campinas, SP, Brazil.; Tel.: (+55 19) 35212175.; Email: luciana@fea.unicamp.br ; Haroldo Y. Kawaguti; Hélia H. Sato

Laboratório de Bioquímica de Alimentos, Departamento de Ciências de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, Brasil

ABSTRACT

This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25ºC and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25ºC and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

Keywords: chitinase, Cellulosimicrobium cellulans, fungal lysis, protoplasts

RESUMO

O presente estudo visou a produção, purificação e aplicação da quitinase extracelular da linhagem Cellulosimicrobium cellulans 191. A maior produção de quitinase em frascos agitados foi 6,9 U/mL após 72 h de fermentação a 25ºC e 200 rpm. Em fermentador de 5 L utilizando aeração de 1,5 vvm, a maior atividade da enzima foi 4,19 U/mL após 168 h de fermentação a 25ºC e 200 rpm; e com 3 vvm, foi obtido 4,38 U/mL após 144 h de fermentação. A quitinase (61 KDa) foi purificada cerca de 6,65 vezes em coluna de filtração em gel Sepharose CL 4B 200 com um rendimento de 46,61%. A enzima purificada foi capaz de lisar a parede celular de alguns fungos e formar protoplastos.

Palavras-chave: quitinase, Cellulosimicrobium cellulans, lise de fungos, protoplastos

INTRODUCTION

Chitin is a polymer of N-acetylglucosamine with β-1,4 bonds (15). It has a highly ordered crystalline structure, as shown by X-ray diffraction studies, and is insoluble in water (24) and generally bound to other polysaccharides and proteins (15). Chitin chains present three forms of arrangement, denominated as α, β and γ. The α form is dominant and more stable and consists of alternating parallel and antiparallel chains; it occurs mainly in crustaceans, insects and fungi. The β form consists of parallel chains and occurs only in marine organisms. The γ form is still being elucidated (24).

Chitin is the main structural component of the cell wall of most fungi, but is susceptible to innumerable bacterial and fungal species acting as antagonists, due to their production of chitinolytic enzymes (25).

Chitinase (E.C. 3.2.1.14) {Poly [1,4-(N-acetyl-β-D-glucosamine)]glucanohydrolase} catalyses the hydrolysis of the β-1,4 bonds of the N-acetyl-β-D-glucosamine of chitin and chitin-dextrins. Chitinases can be used in controlling pathogenic fungi in plants and insects; in the production of biologically active chitin-oligosaccharides; in the production of single cell protein; in the preparation of mycolytic enzymes; and in the formation of fungal protoplasts (18).

In the agro-technological sector, emphasis is on the bio-fungicidal and bio-insecticidal effects of various chitinases produced by microrganisms. These effects relate to the hydrolysis of chitin in the fungal cell wall and insect carapace, respectively (6).

Chitinases are produced by various microrganisms, such as Cellulosimicrobium cellulans FXX (30), Cellulosimicrobium cellulans 191 (5), Trichoderma harzanium TUBF 781 (16), Penicillium aculeatum (2), Bacillus subtilis (29), Lecanicillium fungicola (21), Trichoderma harzanium (20), Paenibacillus sp. CHE-N1 (10, 11) and others.

This study aimed to produce, purify and apply the chitinase from Cellulosimicrobium cellulans strain 191 in the lysis of fungi and formation of protoplasts.

MATERIAL AND METHODS

Chitinase production in shaken flasks

The Cellulosimicrobium cellulans 191 was isolated from alcoholic fermentation residues by the Laboratory of Food Biochemistry and identified by the Korean Institute of Bioscience & Biotechnology. The microrganism was used to produce chitinase enzymes in shaken flasks in a culture medium composed of 4.0 g/L yeast extract; 2.0 g/L tryptone; 4.0 g/L MgSO4.7H2O; 1.2 g/L KH2PO4; 2.8 g/L K2HPO4; and 15 g/L neutralized chitin used as inducer (30). The fermentation was carried out in 500 mL Erlenmeyer flasks containing 100 mL of culture medium described above and incubated at 25ºC and 200 rpm for 72 h. 10 mL aliquots were aseptically transferred to 500 mL Erlenmeyer flasks containing 90 mL of the same culture medium. The flasks were incubated at 25ºC and 200 rpm for 72 h. After incubation, the culture media were centrifuged at 7,840 x g for 10 min at 5ºC and the supernatants used as crude enzyme preparation.

Chitinase production in a 5 L fermenter

The fermentation was carried out in a 5 L fermenter in the previously described culture medium, with 1.5 and 3 vvm of aeration. An experimental design was used to select the conditions of pH, temperature and aeration for chitinase production, the conditions selected being, respectively, 6.5; 25ºC and 200 rpm (7). Samples were collected at regular intervals after different time lapses. The pH was measured and the cell growth was estimated indirectly by measuring absorbance at 660 nm.

Chitinase assay

The assay procedure used in this research was originally described by Réissig et al. (23) and Sandhu et al. (27) and adapted by Fleuri & Sato (8). One unit of activity was defined as the formation of 1 µmoL N-acetylglucosamine under the assay conditions.

Chitinase purification

The crude chitinase preparation was applied to a CL4B200 Sepharose column (1.5 x 30 cm) equilibrated in 0.01 M sodium phosphate buffer, pH 7.0. The same buffer was applied to elute the adsorbed proteins and 1.5 mL fractions were collected every 3.5 minutes. The protein elution was followed by reading the absorbance at 280 nm. Fractions containing chitinase activity were pooled, dialysed against distilled water and freeze-dried. The protein concentration of the enzymatic solutions was determined by the method of Lowry et al. (14), using a standard of ovalbumin.

Electrophoresis of chitinase in SDS polyacrylamide gel

SDS-PAGE with 12% sodium dodecyl sulphate-polyacrylamide gel was performed to determine the molecular mass of the enzyme, as described by Laemmli (12). The standard protein mixture contained phosphorylase b (94.0 KDa), bovine albumin (67 KDa), ovalbumin (43.0 KDa), carbonic anhydrase (30.0 KDa), soybean trypsin inhibitor (20.1 KDa) and α-lactoalbumin (14.0 KDa).

Application of purified chitinase in the lysis of fungi and formation of protoplasts

Fungal cell production

The fungi Rhizopus oligosporus, Mucor miehei, Penicillium sp., Aspergillus oryzae, Streptomyces phaerochromogenes, Aspergillus niger, Paecylomyces sp. and Trichoderma viride were used in the study of lysis. The fungi were grown in potato dextrose agar plates for 10 days at 28ºC. The spore germination was carried out in 250 mL Erlenmeyer flasks containing 50 mL of a culture medium composed of 10 g/L soluble starch; 0.3 g/L vitamin-free casein; 2.0 g/L KNO3; 2.0 g/L NaCl; 0.05 g/L MgSO4 7 H2O; 0.01 g/L FeSO4 7 H2O; and 0.02 g/L CaCO3, at 28ºC and 150 rpm for 20 h. The material was centrifuged at 2,822 x g for 6 min at 5ºC. The mycelium was washed 3 times with distilled water and resuspended in 0.2M phosphate buffer, pH 5.8.

Application of purified chitinase in the lysis of fungi and formation of protoplasts

The reaction mixture containing 0.5 mL of fungal suspension in 0.2 M phosphate buffer, pH 5.8 and 0.5 U purified chitinase suspension/mL fungal suspension, was incubated at 30ºC for 2 h and stirred at regular intervals. Cell lysis was observed under an optical microscope with immersion. The reaction mixture containing the fungal suspension, but without the addition of the enzyme and incubated for the same time and temperature, was used as control.

RESULTS AND DISCUSSION

Chitinase production in shaken flasks

A yield of 6.9 U/mL chitinase was obtained by fermentation of C. cellulans strain 191 in shaken flasks in culture medium with an initial pH of 6.5 at 25ºC and 200 rpm, after 72 h of incubation.

Chitinase production in a 5 L fermenter

Figure 1 shows that, in the fermentation of C. cellulans strain 191 in a 5 L fermenter with culture medium containing 1.5% neutralised chitin at 25ºC, 200 rpm and aeration of 1.5 vvm, the maximum chitinase production (4.19 U/mL) was obtained after 168 h of fermentation, at the end of the stationary phase. The pH of the medium oscillated between 6.4 and 7.0 during fermentation.


Figure 2 shows that, in the fermentation of C. cellulans strain 191 in a 5 L fermenter with culture medium containing 1.5% neutralised chitin at 25ºC, 200 rpm and aeration of 3 vvm, the maximum chitinase production was obtained after 144 h of fermentation, coinciding with the range of microbial decline or death. A maximum chitinase yield of 4.38 U/mL was obtained. The pH of the culture medium oscillated between 6.6 and 7.3 during fermentation.


The increase in aeration from 1.5 vvm to 3 vvm in the cultivation of C. cellulans strain 191 in a 5 L fermenter resulted in a decrease in the time required to obtain the maximum chitinase yield. Using aeration values of 1.5 vvm and 3 vvm, chitinase yields of 4.19 U/mL and 4.38 U/mL were obtained after 168 h and 144 h of incubation, respectively. These results indicate that aeration rate directly influences the oxygen supply, which in turn might affect fermentation time.

As described by Liu et al. (13) in chitinase production by Verticillium lecanii in 5 and 30 L bioreactors, increased aeration affected the growth of the microrganism positively and, in this present study, it was observed that increased aeration resulted in chitinase production in a shorter time.

Kao et al. (11) verified that the highest chitinase production from Paenibacillus sp. CHE-N1 in a 5 L fermenter at 34.3ºC and 200 rpm was obtained at an aeration rate of 3 vvm, in comparision with yield using 1 and 2 vvm; while in this present study the change of aeration only affected the time taken to obtain highest enzyme production.

In this study, in shaken flasks, a chitinase yield of 6.9 U/mL was obtained after 72 h, while in the 5 L fermenter with aeration of 3 vvm, the yield was 4.38 U/mL after 144 h. Production in shaken flasks was thus 1.57 times greater than in a 5 L fermenter.

Chitinase purification

Preliminary studies of chitinase purification were conducted using DEAE-Sephadex A50 and DEAE-Sephacel DCL6B columns. Using a Sepharose CL4B200 gel filtration column equilibrated in 0.01 M phosphate buffer, pH 7.0 (Figure 3), the chitinase from C. cellulans strain 191 was purified 6.65 times with a recovery of 46.62%. One chitinase peak was obtained, denominated fraction Q1.


Figure 4 shows the SDS-PAGE electrophoresis of the purified chitinase, indicating the presence of a single protein band. The purified chitinase showed a molecular mass of 61 KDa in SDS-polyacrylamide gel.


The bacterial chitinases from Cellulomonas cellulans FXX (30), Bacillus sp. MH-1 (26), Bacillus sp. 13.26 (31), Bacillus subtilis W-118 (29) and the fungal chitinases from Metarhizium anisopliae (3), Trichoderma harzianum T198 (4), Streptomyces sp. NK1057 (17), Aeromonas schubertii (9), Penicillium aculeatum (2), and Aspergillus sp. S1-13 (22) were purified by many steps and presented different molecular masses.

In the present study, Cellulosimicrobium cellulans strain 191 was purified about 6.65 times with a yield of 46.61% using Sepharose CL4B200 gel filtration resin. In SDS-PAGE electrophoresis, the preparation presented a single band with a molecular mass of 61 KDa, similar to that of the chitinase M (62 KDa) from Bacillus sp. MH-1 (26) and the chitinase from Bacillus sp. 13.26 (60 KDa) (31).

Application of the purified chitinase in the lysis of fungi and formation of protoplasts

The purified chitinase from the C. cellulans strain 191 was used to study lysis of the fungi Rhizopus oligosporus, Mucor miehei, Penicillium sp., Aspergillus oryzae, Streptomyces phaerochromogenes, Aspergillus niger, Paecylomyces sp. and Trichoderma viride, by observation under an optical microscope with immersion.

Figure 5 illustrates the lysis of the fungi R. oligosporus, M. miehei, Penicillium sp., S. phaerochromogenes and T. viride by the purified chitinase as compared to their respective controls.


With the exception of Penicillium sp. and R. oligosporus, the fungi were highly hydrolysed, since only fragments were found on the slides when examined under the optical microscope with immersion. The purified chitinase degrades the cell wall chitin polymers and lyses various fungi, providing evidence of its potential as a bio-fungicide.

The fungi A. oryzae, A. niger and Paecylomyces sp. presented no alteration in cell structure after treatment with their respective controls. There are many differences in the composition and organization of fungal cell walls and this difference probably hindered the action of chitinase.

The digestion of mycelia and release of protoplasts were prominent in the case of Penicillium sp. and R. oligosporus. Protoplast production and fusion is an important tool in strain improvement to foster genetic recombination and develop hybrid strains in filamentous fungi. Pe'er and Chet (19), Tschen and Li (28) and Balasubramanian et al. (1) obtained protoplasts using the commercial lytic preparation Novozym 234. The same preparation is used in other studies to lyse yeast and fungal cell walls.

The present study showed that the purified chitinase from C. cellulans strain 191 presents potential for application in fungal control and protoplast formation.

CONCLUSIONS

The highest yield of chitinase (6.9 U/mL) was obtained in shaken flasks after 72 h of incubation of the microrganism C. cellulans strain 191 at 25ºC and 200 rpm, in a culture medium composed of 4.0 g/L yeast extract; 2.0 g/L tryptone; 4.0 g/L MgSO4.7H2O; 1.2 g/L KH2PO4; 2.8 g/L K2HPO4; and 15 g/L neutralised chitin. The chitinase (61 KDa), purified using Sepharose CL4B200 gel filtration resin, was capable of lysing the fungi Mucor miehei, Streptomyces phaerochromogenes and Trichoderma viride and capable of producing protoplasts of Rhizopus oligosporus and Penicillium sp.

ACKNOWLEDGEMENTS

The authors are grateful to FAPESP for conceding Masters and Doctoral scholarships.

Submitted: March 14, 2008; Returned to authors for corrections: August 26, 2008; Approved: May 08, 2009.

  • 1. Balasubramanian, N.; Juliet, A.A.; Srikalavani, P.; Lalithakumari, D. (2003). Release and regeneration of protoplasts from Trichothecium roseum. Can. J. Microbiol 49, 263-268.
  • 2. Binod, P.; Pusztahelyi, T.; Nagy, V.; Sandhya, C.; Szakács, G.; Pócsi, I.; Pandey, A. (2005). Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation. Enzyme Microbial Technol. 36, 880-887.
  • 3. De Siqueira Pinto, A.; Barreto, C.C.; Schrank, A.; Ulhoa, C.J.; Henning Vainstein, M. (1997). Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. Can. J. Microbiol. 43, 322-327.
  • 4. Deane, E.E.; Whipps, J.M.; Lynch, J.M.; Peberdy, J.F. (1998). The purification and characterization of Trichoderma harzianum exochitinase. Biochim. Biophys. Acta. 1383, 101110.
  • 5. Fleuri, L.F. (2003). Produção de β-1,3 glucanases, proteases líticas e quitinases por microrganismos e aplicação na lise de leveduras. Campinas, 141 p. (M. Sc. Dissertation. Faculdade de Engenharia de Alimentos. Unicamp).
  • 6. Fleuri, L.F; Sato, H.H. (2005). Produção, purificação, clonagem e aplicação de enzimas líticas. Quim. Nova. 28, 871-879.
  • 7. Fleuri, L.F; Sato, H.H. (2008). Estudo da influência de diferentes parâmetros na produção de enzimas líticas. Ciênc. Tecnol. Aliment. 28, 1-12.
  • 8. Fleuri, L.F; Sato, H.H. (2008). β-1,3 glucanases e quitinases: aplicação na lise de leveduras e inibição de fungos. Ciênc. Agrotec. 32, 1224-1231.
  • 9. Guo, S.H.; Chen, J.K.; Lee, W.C. (2004). Purification and characterization of extracellular chitinase from Aeromonas schubertii. Enzyme Microbial Technol. 35, 550-556.
  • 10. Kao, P.M.; Chen, C.I.; Huang, S.C.; Chang, Y.C.; Tsai, P.J.; Liu, Y.C. (2007). Effects of shear stress and mass transfer on chitinase production by Paenibacillus sp. CHE-N1. Biochem. Eng. J. 34, 172-178.
  • 11. Kao, P.M.; Chen, C.I.; Huang, S.C.; Chang, Y.C.; Tsai, P.J.; Liu, Y.C. (2007). Development of continuous chitinase production process in a membrane bioreactor by Paenibacillus sp. CHE-N1. Process Biochem. 42, 606-611.
  • 12. Laemmli, U.K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. 227, 680-685.
  • 13. Liu, B.L.; Kao, P.M.; Tzeng, Y.M.; Feng, K.C. (2003). Production of chitinase from Verticillium lecanii F091 using submerged fermentation. Enzyme Microbial Technol., 33, 410-415.
  • 14. Lowry, O.H.; Rosebrough, N.J.; Farr, L.A.; Randall, R.J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.
  • 15. Majeti, N.V.; Kumar, R. (2000). A review of chitin and chitosan applications. Reactive Func. Polymers. 46, 1-27.
  • 16. Nampoothiri, K.M.; Baiju, T.V.; Sandhya, C.; Sabu, A.; Szakacs, G.; Pandey, A. (2004). Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochem. 39, 1583-1590.
  • 17. Nawani, N.N.; Kapadnis, B.P. (2004). Production dynamics and characterization of chitinolytic system of Streptomyces sp NK1057, a well equipped chitin degrader. World J. Microbiol. Biotech. 20, 487-494.
  • 18. Patil, R.S.; Ghormade, V.; Desphande, M.V. (2000). Review: Chitinolytic enzymes: an exploration. Enzyme Microbial Technol. 26, 473-483.
  • 19. Pe'er, S.; Chet, I. (1990). Trichoderma protoplast fusion; a tool for improving biocontrol agents. Can. J. Microbiol. 36, 6-9.
  • 20. Prabavathy, V.R.; Mathivanan, N.; Sagadevan, E.; Murugesan, K.; Lalithakumari, D. (2006). Selffusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum. Bioresource Technol. 97, 2330-2334.
  • 21. Ramírez-Coutiño, L.; Marín-Cervantes, M.C.; Huerta, S.; Revah, S.; Shirai, K. (2006). Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases. Process Biochem. 41, 1106-1110.
  • 22. Rattanakit, N.; Yano, S.; Plikomol, A.; Wakayama, M.; Tachiki, T. (2007). Purification of Aspergillus sp. S113 chitinases and their role in saccharification of chitin in mash of solid-state culture with shellfish waste. J. Biosc. Bioeng. 103, 535-541.
  • 23. Réissig, J.L.; Strominger, J.L.; Leloir, L.F. (1955). A modified colorimetric method for the estimation of N-acetylamino sugar. J. Biol. Chem. 217, 959-699.
  • 24. Roberts, G.A.F. (1992). Chitin chemistry. Macmillan Press, London, pp. 55-58.
  • 25. Sahai, A.S.; Manocha, M.S. (1993). Chitinase of fungi and plants: their involvement in morphogenesis and hostparasite interactions. Microbiol. Rev., 11:317-338.
  • 26. Sakai, K.; Yokota, A.; Kurokawa, H.; Wakayama, M.; Moriguchi, M. (1998). Purification and characterization of three thermostable endochitinases of a novel Bacillus strain, MH-1, isolated from chitin-containing compost. Appl. Environ. Microbiol. 64, 33-97.
  • 27. Sandhu, D.K.; Wadhwa, V.; Bagga, P.S. (1989). Use of lytic enzymes for protoplast production in Trichoderma reesei QM9414. Enzyme Microbial Technol. 11, 21-25.
  • 28. Tschen, J.S.M.; Li, I.F. (1994). Optimization of formation and regeneration of protoplasts from biocontrol agents of Trichoderma sp. Mycoscience. 35, 257-263.
  • 29. Wang, S.L.; Lin, T.Y.; Yen, Y.H.; Liao, H.F.; Chen, Y.J. (2006). Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase. Carboh. Res. 341, 2507-2515.
  • 30. Yamaguchi, M.M. (2003). Seleção, produção e caracterização da enzima quitinase. Campinas, 83 p. (PhD. Thesis. Faculdade de Engenharia de Alimentos. Unicamp).
  • 31. Yuli, P.E.; Suhartono, M.T.; Rukayadi, Y.; Hwang, J.K.; Pyun, Y.R. (2004). Characteristics of thermostable chitinase enzymes from the Indonesian Bacillus sp 13.26. Enzyme Microbial Technol. 35, 147-153.
  • *
    Corresponding Author. Mailing address: Faculty of Food Engineering, Food Science Department, P.O Box 6121, CEP 13083862, UNICAMP - Campinas, SP, Brazil.; Tel.: (+55 19) 35212175.; Email:
  • Publication Dates

    • Publication in this collection
      18 Aug 2009
    • Date of issue
      Sept 2009

    History

    • Reviewed
      26 Aug 2008
    • Received
      14 Mar 2008
    • Accepted
      08 May 2009
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