Production and Biochemical Characterization of Insecticidal Enzymes from Aspergillus Fumigatus Toward Callosobruchus Maculatus

Overview

Journal Curr Microbiol

Specialty Microbiology

Date 2006 May 30

PMID 16732450

Authors

Jackeline L Pereira

Octavio L Franco

Eliane F Noronha

Affiliations

Soon will be listed here.

Abstract

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM: phenylmethylsulfonyl fluoride (100%) and 50 microM leupeptin (67.94%).

References

Punja Z, Utkhede R . Using fungi and yeasts to manage vegetable crop diseases. Trends Biotechnol. 2003; 21(9):400-7. DOI: 10.1016/S0167-7799(03)00193-8. View

Chakrabarti S, Matsumura N, Ranu R . Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2). Curr Microbiol. 2000; 40(4):239-44. DOI: 10.1007/s002849910048. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Xia G, Zhou J, Yang S, Zhang S, Jin C . A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem. 2001; 268(14):4079-85. DOI: 10.1046/j.1432-1327.2001.02323.x. View

Kogan T, Jadoun J, Mittelman L, Hirschberg K, Osherov N . Involvement of secreted Aspergillus fumigatus proteases in disruption of the actin fiber cytoskeleton and loss of focal adhesion sites in infected A549 lung pneumocytes. J Infect Dis. 2004; 189(11):1965-73. DOI: 10.1086/420850. View

Palmieri G, Bianco C, Cennamo G, Giardina P, Marino G, Monti M . Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus. Appl Environ Microbiol. 2001; 67(6):2754-9. PMC: 92935. DOI: 10.1128/AEM.67.6.2754-2759.2001. View

Petinate S, Branquinha M, Coelho R, Vermelho A, Giovanni-De-Simone S . Purification and partial characterization of an extracellular serine-proteinase of Streptomyces cyaneus isolated from Brazilian cerrado soil. J Appl Microbiol. 1999; 87(4):557-63. DOI: 10.1046/j.1365-2672.1999.00852.x. View

Khan A, Williams K, Molloy M, Nevalainen H . Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels. Protein Expr Purif. 2004; 32(2):210-20. DOI: 10.1016/j.pep.2003.07.007. View

Tomschy A, Brugger R, Lehmann M, Svendsen A, Vogel K, Kostrewa D . Engineering of phytase for improved activity at low pH. Appl Environ Microbiol. 2002; 68(4):1907-13. PMC: 123903. DOI: 10.1128/AEM.68.4.1907-1913.2002. View

10.

Kramer K, Muthukrishnan S . Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol. 1998; 27(11):887-900. DOI: 10.1016/s0965-1748(97)00078-7. View

11.

Maurer K . Detergent proteases. Curr Opin Biotechnol. 2004; 15(4):330-4. DOI: 10.1016/j.copbio.2004.06.005. View

12.

Krikstaponis A, Lugauskas A, Prazmo Z, Dutkiewicz J . Enzymatic activities of Aspergillus fumigatus strains isolated from the air at waste landfills. Ann Agric Environ Med. 2001; 8(2):227-34. View

13.

Franco O, Rigden D, Melo F, Bloch Jr C, Silva C, Grossi de Sa M . Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities. Eur J Biochem. 2000; 267(8):2166-73. DOI: 10.1046/j.1432-1327.2000.01199.x. View

14.

Rao M, Tanksale A, Ghatge M, Deshpande V . Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev. 1998; 62(3):597-635. PMC: 98927. DOI: 10.1128/MMBR.62.3.597-635.1998. View

15.

Bouchara J, Tronchin G, Larcher G, Chabasse D . The search for virulence determinants in Aspergillus fumigatus. Trends Microbiol. 1995; 3(8):327-30. DOI: 10.1016/s0966-842x(00)88965-9. View

16.

Viterbo A, Harel M, Chet I . Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots. FEMS Microbiol Lett. 2004; 238(1):151-8. DOI: 10.1016/j.femsle.2004.07.028. View

17.

Campos R, Arruda W, Boldo J, Silva M, de Barros N, de Azevedo J . Boophilus microplus infection by Beauveria amorpha and Beauveria bassiana: SEM analysis and regulation of subtilisin-like proteases and chitinases. Curr Microbiol. 2005; 50(5):257-61. DOI: 10.1007/s00284-004-4460-y. View

18.

Santos RMDB , Firmino A, De Sa C, Felix C . Keratinolytic activity of Aspergillus fumigatus fresenius. Curr Microbiol. 1996; 33(6):364-70. DOI: 10.1007/s002849900129. View

19.

Escott G, Hearn V, Adams D . Inducible chitinolytic system of Aspergillus fumigatus. Microbiology (Reading). 1998; 144 ( Pt 6):1575-1581. DOI: 10.1099/00221287-144-6-1575. View

20.

Bradford M . 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. DOI: 10.1016/0003-2697(76)90527-3. View