Home >Archive

Volume: 4, Issue: 4, July-August, 2016
DOI: 10.7324/JABB.2016.40401

Research Article

Production and Characterization of Collagenase by Penicillium sp. UCP 1286 Isolated From Caatinga Soil

Maria Carolina de Albuquerque Wanderley1 2, Jose Manoel Wanderley Duarte Neto2, Carolina de Albuquerque Lima3, Sara Isabel da Cruz Silverio4, Jose Luiz de Lima Filho2, Jose Antonio Couto Teixeira4, Ana Lucia Figueiredo Porto5

  Author Affiliations


A new Penicillium sp. strain isolated from the soil of Caatinga, a Brazilian Biome (UCP 1286) was selected for collagenase production. Fermentation system allowing obtention of collagenolytic activity about 2.7 times higher than existing data, with the highest values of collagenolytic and specific activity (379.80 U/mL, 1460.77 U/mg, respectively), after 126 hours. Applying a factorial design, enzyme production was increased by about 65% compared to the preliminary results. The factorial design demonstrated the existence of two factors with statistical significance on the production of the enzyme: pH and temperature, both with negative effects. Enzyme was found to be more active at pH 9.0 and 37 °C, and also to be very stable in comparison with the collagenase produced by other microorganisms. The enzyme seems to belong to collagenolytic serine proteases family. Concerning the substrate specificity, it was observed that the highest enzyme activity corresponds to azocoll, there was no relevant activity on azocasein and the enzyme showed to be more specific to type V collagen and gelatin than the commercial colagenase produced by Clostridium histolyticum. Major band observed at electrophoresis was approximately 37 kDa. Zymogram analysis confirmed the collagenolytic activity. All data indicates this enzyme as promising biotechnology product.


Collagenolytic; Enzymes; Factorial Design; Fermentation; Filamentous Fungi; Specificity.

Citation: Wanderley MCA, Neto JMWD, Lima CA, Silverio SIC, Filho JLL, Teixeira JAC, Porto ALF. Production and Characterization of Collagenase by Penicillium sp. UCP 1286 Isolated From Caatinga Soil. J App Biol Biotech. 2016; 4 (04): 001-010. DOI: 10.7324/JABB.2016.40401

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.


1. Jain R, Jain PC. Production and partial characterization of collagenase of Streptomyces exfoliatus CFS 1068 using poultry feather. Indian J Exp Biol 2010;48:174-8.

2. Suphatharaprateep W, Cheirsilp B, Jongjareonrak A. Production and properties of two collagenases from bacteria and their application for collagen extraction. N Biotechnol 2011;28:649-55.

3. Di Lullo G a., Sweeney SM, Körkkö J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem 2002;277:4223-31.

4. M\üller WEG. The origin of metazoan complexity: porifera as integrated animals. Integr Comp Biol 2003;43:3-10.

5. Tran LH, Nagano H. Isolation and Characteristics of Bacillus subtilis CN2 and its Collagenase Production. Food Microbiol Saf 2002;67:3-6.

6. Wu Q, Li C, Li C, Chen H, Shuliang L. Purification and characterization of a novel collagenase from Bacillus pumilus Col-J. Appl Biochem Biotechnol 2010;160:129-39.

7. Dresden MH. Evidence for the role of collagenase in collagen metabolism. Nature 1971.

8. Lima CA, Viana Marques DA, Neto BB, Lima Filho JL, Carneiro-da-Cunha MG, Porto ALF. Fermentation medium for collagenase production by Penicillium aurantiogriseum URM4622. Biotechnol Prog 2011;27:1470-7.

9. Lima CA, Filho JLL, Neto BB, Converti A, Carneiro da Cunha MG, Porto ALF. Production and characterization of a collagenolytic serine proteinase by Penicillium aurantiogriseum URM 4622: A factorial study. Biotechnol Bioprocess Eng 2011;16:549-60.

10. Lima CA, J\únior ACVF, Filho JLL, Converti A, Marques D a. V, Carneiro-da-Cunha MG, et al. Two-phase partitioning and partial characterization of a collagenase from Penicillium aurantiogriseum URM4622: Application to collagen hydrolysis. Biochem Eng J 2013;75:64-71.

11. Ravanti L, Kahari VM. Matrix metalloproteases in wound repair. Int J Mol Med 2000;6:391-407.

12. Shanmughapriya S, Kiran GS, Natarajaseenivasan K. Optimization of extracellular thermotolerant alkaline protease produced by marine Roseobacter sp. (MMD040). Bioproc Biosys Eng 2008;31:427-33.

13. Barret AJ, Rawlings ND, Woessner JF. Handbook of Proteolytic Enzymes. 2nd. ed. Oxford, U. K.: 2004.

14. Hamdy HS. Extracellular collagenase from Rhizoctonia solani : Production , purification and characterization. Indian J Biotechnol 2008;7:333-40.

15. Rao MB, Tanksale a M, Ghatge MS, Deshpande V V. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597-635.

16. Daboor SM, Budge SM, Ghaly AE, Brooks S, Dave D. Extraction and Purification of Collagenase Enzymes : A Critical Review. Am J Biochem Biotechnol 2010;6:239-63.

17. Mookthiar K, Steinbrink SD, Van Wart HE. Mode of hydrolysis of collagen-like peptidase by class I and class II Clostridium hystolyticum collagenases: Evidence for both endopeptidase and tripeptidyl-carboxypeptidase activities. Biochemistry1 985;24:6527-33.

18. Peterkofsky B. Bacterial collagenase. Methods Enzym 1982;82:453-71.

19. Sandhya C, Sumantha A, Szakacs G, Pandey A. Comparative evaluation of neural protease production by Aspergillus oryzae in submerged and solid state fermentation. Process Biochem 2005;40:2689-94.

20. Yakovleva MB, Khoang TL, Nikitina ZK. Collagenolytic activity in several species of deuteromycetes under various storage conditions. Appl Biochem Microbiol 2006;42:431-4.

21. de Siqueira ACR, da Rosa NG, Motta CMS, Cabral H. Peptidase with keratinolytic activity secreted by Aspergillus terreus during solid-state fermentation. Brazilian Arch Biol Technol 2014;57:514-22.

22. Rosso BU, Lima CDA, Porto TS, de Oliveira Nascimento C, Pessoa A, Converti A, et al. Partitioning and extraction of collagenase from Penicillium aurantiogriseum in poly(ethylene glycol)/phosphate aqueous two-phase system. Fluid Phase Equilib 2012;335:20-5.

23. Lima CA, Rodrigues PMB, Porto TS, Viana D a., Lima Filho JL, Porto ALF, et al. Production of a collagenase from Candida albicans URM3622. Biochem Eng J 2009;43:315-20.

24. Viani FC, Cazares Viani PR, Gutierrez Rivera IN, da Silva ÉG, Paula CR, Gambale W. Actividad proteolítica extracelular y análisis molecular de cepas de Microsporum canis aisladas de gatos con y sin sintomatología. Rev Iberoam Micol 2007;24:19-23.

25. Ikram-Ul-Haq H, Mukhtar. Biosynthesis of acid proteases by Penicillium griseoroseum IH-02 in solid-state fermentation. Pakist J Bot 2007;39:2717-24.

26. Menezes R, Sampaio E, Giongo V, P\érez-Marin A. Biogeochemical cycling in terrestrial ecosystems of the Caatinga Biome. Brazilian J Biol 2012;72:643-53.

27. Ferreira ACC, Leite LFC, de Ara\újo ASF, Eisenhauer N. Land-Use Type Effects on Soil Organic Carbon and Microbial Properties in a Semi-Arid Region of Northeast Brazil. L Degrad Dev 2014;178:n/a - n/a.

28. Pacchioni RG, Carvalho FM, Thompson CE, Faustino ALF, Nicolini F, Pereira TS, et al. Taxonomic and functional profiles of soil samples from Atlantic forest and Caatinga biomes in northeastern Brazil. Microbiologyopen 2014;3:299-315.

29. Chavira RJ, Burnett TJ, Hageman JH. Assaying Proteinases with Azocoll. Anal Biochem 1984;136:446-50.

30. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.

31. Endo A, Murakawa S, Shimizu H, Shiraishi Y. Purification and properties of collagenase from Streptomyces species. J Biochem 1987;102:163-70.

32. Rosen H. A modified ninhydrin colorimetric analysis for amino acids. Arch Biochem Biophys 1975;67:10-5.

33. Moore S, Stein WH. Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem 1948;176:367-88.

34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.

35. Baehaki A, Sukamo, Syah D, Setyahadi S, Suhartono MT. Production and characterization of collagenolytic protease from Bacillus licheniformis F11.4 originated from Indonesia. Asian J Chem 2014;26:2861-4.

36. Mahmoud Y a.-G, Abu El-Souod SM, El-Shourbagy SM, El-Badry ASM. Characterisation and inhibition effect of cetrimide on collagenase produced by Aspergillus flavus, isolated from mycotic ulcers. Ann Microbiol 2007;57:109-13.

37. Ok T, Hashinaga F. Detection and production of extracellular collagenolytic enzyme from Zygosaccharomyces rouxii. J Gen Appl Microbiol 1996;42:517-23.

38. Chellapan S, Jasmin C, Basheer SM, Elyas K, Bhat SG, Chandrasekaran M. Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid station fermentation. Process Biochem 2006;41:956-61.

39. Sakurai Y, Inoue H, Nishii W, Takahashi T, Iino Y, Yamamoto M, et al. Purification and characterization of a major collagenase from Streptomyces parvulus. Biosci Biotechnol Biochem 2009;73:21-8.

40. Adachi E, Hayashi T. “In vitro” formation of fine fibrils with a D-periodic banding pattern type V collagen. Relat Res 1985;5:225-32.

41. Kiriviko KI, Mlyllyla R. Biosynthesis of the collagens. In: Piez KA, Reddi AH, organizadores. Extracell. Matrix Biochem., New York: Elsevier; 1984, p. 83-118.

42. Moreland LW, Gay RE, Gay S. Collagen autoantibodies in patients with vasculitis and systemic lupus erythematosus. Clin Immunol Immunopathol 1991;60:412-218.

43. James GT. Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 1978;86:574-9.

44. Baehaki A, Suhartono MT, Syah D, Sitanggang AB, Setyahadi S, Meinhardt F. Purification and characterization of collagenase from Bacillus licheniformis F11.4. African J Microbiol Res 2012;6:2373-9.

45. Roy P, Colas B, Durand P. Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crab (Carcinus maenas) digestive gland. Comp Biochem Physiol 1994;36:247-301.

46. Matsushita K, Toyama H, Adachi O. Respiratory chains and bioenergetics of acetic acid bacteria. Adv Microb Physiol 1994;36:247-301.

47. Kristjánsson MM, Gudmundsd\óttir S, Fox JW, Bjamason JB. Characterization of collagenolytic serine proreinase from the Atlantic cod (Gadus morhua). Comp Biochem Physiol 1995;110:707-17.

48. Sivakumar P, Sampath P, Chandrakasan G. Collagenolytic metalloprotease (gelatinase) from the hepatopancreas of the marine crab, Scylla serrata. Comp Biochem Physiol 1999;123:273-9.

Article Metrics

Similar Articles