Abstract
Two Bacillus cereus feather-degrading strains (23/1 and 6/2) were transformed using a recombinant plasmid p5.2 carrying the alkaline proteinase gene (aprE). A high level of the aprE gene expression was observed when the recombinant strains were grown on sporulation medium. The expression of the aprE gene proceeded during the early stationary phase and the p5.2 plasmid was segregationally and structurally stable in both strains. The two recombinant strains grown on a mineral medium with 1 % chicken feather as source of energy, carbon and nitrogen exhibited higher proteolytic activity (≈6-fold and 2.4-fold higher for strains 23/1 (p5.2) and 6/2 (p5.2), respectively. Keratinolytic activity increased ≈3.5-fold and 4.15-fold, respectively. The keratinolytic activity further increased when an optimized medium with yeast extract and corn oil was used. Considerable amounts of free amino acids were obtained after the biodegradation of feather which makes the new strains promising for application in feather-waste treatment to, e.g., transformation to animal feedstuff.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bernhardt K., Schrempf H., Goebel W.: Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J.Bacteriol.133, 897–903 (1978).
Bockle B., Muller R.: Reduction of disulfide bonds by Streptomyces pactum during growth on chicken feathers. Appl.Environ. Microbiol.63, 790–792 (1997).
Bron S., Luxen E.: Segregational instability of pUB 110-derived recombinant plasmids in Bacillus subtilis. Plasmid14, 235–244 (1989).
Cao L., Tan H., Liu Y., Xue X., Zhou S.: Characterization of a new keratinolytic Trichoderma atroviride strain F6 that completely degrades native chicken feather. Lett.Appl.Microbiol.46, 389–34 (2008).
Cliffe A.J., Law B.A.: A new method for the detection of microbial proteolytic enzyme in milk. J.Dairy Res.49, 209–219 (1982).
El-refai H.A., Abdel Naby M.A., Gaballa A., El-Araby M.H., Abdel Fattah A.F.: Improvement of the newly isolated Bacillus pumilus FH9 keratinolytic activity. Process Biochem.40, 2325–2332 (2004).
Gessesse A., Hatti-Kaul R., Gashe. B.A., Mattiasson B.: Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb.Technol.32, 519–524 (2003).
Leighton T.J., Doi R.H.: The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J.Biol.Chem.264, 3189–3195 (1971).
Leonhardt H., Alonso J.C.: Parameters affecting plasmid stability in Bacillus subtilis. Gene103, 107–111 (1991).
Lin X., Wang S.L., Miller E.S., Shih J.C.: Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. J.Ind. Microbiol.Biotechnol.19, 34–138 (1997).
Lin X., Inglis G.D., Yanke L.J., Cheng K.J.: Selection and characterization of feather-degrading bacteria from canola meal compost. J.Ind.Microbiol.Biotechnol.23, 49–153 (1999).
Lucas F.S., Broennimann O., Febbrano I., Heeb P.: High diversity among feather-degrading bacteria from a dry meadow soil. Microb.Ecol.45, 282–290 (2003).
Macaluso A., Mettus A.M.: Efficient transformation of Bacillus thuringiensis requires non-methylated plasmid DNA. J.Bacteriol.173, 1353–1356 (1991).
Maniatis T., Fritsch E., Sambrook J.: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor (USA) 1982.
Pearce K.N., Karahalios D., Friedman M.: Ninhydrin assay for proteolysis in ripening cheese. J.Food Sci.53, 432–435 (1988).
Ramnani P., Gupta R.: Optimization of medium composition for keratinase production from feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol.Appl.Biochem.40, 191–196 (2004).
Sangali S., Brandelli A.: Feather keratin hydrolysis by a Vibrio sp. strain kr2. J.Appl.Microbiol.89, 735–743 (2000).
Shoham Y., Demain A.L.: Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis. Enzyme Microb.Technol.12, 330–336 (1990).
Stewart M.R.J., Venables W.A., Manchee R.J.: Stability of single-stranded DNA plasmids during continuous culture of Bacillus subtilis, and the effect of host-chemostat experience. FEMS Microbiol.Lett.136, 317–323 (1996).
Suntornsuk W., Suntornsuk L.: Feather degradation by Bacillus sp. K46 in submerged cultivation. Bioresource Technol.86, 239–243 (2003).
Vehmaanpera J.O., Korhola M.P.: Stability of the recombinant plasmid carrying the Bacillus amyloliquefaciens α-amylase gene in B. subtilis. Appl.Microbiol.Biotechnol.23, 456–461 (1986).
Wang L.F., Doi R.H.: Promoter switch during development and termination site of the δ43 operon of Bacillus subtilis. Mol.Gen.Genet.207, 114–119 (1987).
Wang J.J., Rojanatovorn K., Shih J.C.: Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene. Biotechnol.Bioeng.87, 459–464 (2004).
Williams C.M., Richter C.S., Mackenzie J.M., Shih J.C.: Isolation, identification, and characterization of a feather-degrading bacterium. Appl.Environ.Microbiol.56, 1509–1515 (1990).
Zaghloul T.I., Kawamura F., Doi R.H.: Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion. J.Bacteriol.164, 550–555 (1985).
Zaghloul T.I., Abdelaziz A., Mostafa M.H.: High level of expression and stability of the cloned alkaline protease (aprA) gene in Bacillus subtilis. Enzyme Microb.Technol.16, 534–537 (1994).
Zaghloul T.I., Al-Bahra M., Al-Zzmeh H.: Isolation, identification and keratinolytic activity of some feather-degrading bacterial isolates. Appl.Biochem.Biotechnol.70–72, 207–213 (1998).
Zaghloul T.I., Haroun M.A., El-Gayar K., Abdelal A.: Recycling of keratin-containing materials (chicken feather) through genetically engineered bacteria. Polymer-Plastics Technol.Eng.43, 1589–1599 (2004).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ouled-Haddar, H., Zaghloul, T.I. & Saeed, H.M. Expression of alkaline proteinase gene in two recombinant Bacillus cereus feather-degrading strains. Folia Microbiol 55, 23–27 (2010). https://doi.org/10.1007/s12223-010-0004-y
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12223-010-0004-y