Expression kinetics and plasmid stability of recombinant E. coli encoding urease-derived peptide with bioinsecticide activity

• Published in: Enzyme and microbial technology Vol. 41; no. 6; pp. 821 - 827
• Main Authors: Tomazetto, Geizecler, Mulinari, Fernanda, Stanisçuaski, Fernanda, Settembrini, Beatriz, Carlini, Célia Regina, Ayub, Marco Antônio Záchia
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-11-2007; Elsevier Science
• Subjects: Bioinsecticide; Biological and medical sciences; Bioreactor cultivation; Biotechnology; Canatoxin; Canavalia ensiformis; Dysdercus peruvianus; Escherichia coli; Fundamental and applied biological sciences. Psychology; Plasmid stability; Recombinant E. coli; Bioreactor cultivation; Recombinant E. coli; Canatoxin; Bioinsecticide; Plasmid stability; Stability; Peptides; Enzyme; Urease; Plasmid; Bioreactor; Hydrolases; Kinetics; Biopesticide; Culture; Microbial insecticide
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2007.07.006

The nucleotide sequence encoding for an insecticidal peptide derived from the
Canavalia ensiformis urease gene
jbureII (AF 468788), was cloned and expressed in the pET101/
Escherichia coli expression system. Bacterial cultivation in shaker with lactose as inducer produced 1.26
μg of recombinant peptide/mg protein, after 8
h of growth. The plasmid stability and the expression of the recombinant peptide were studied in bioreactor. Expression of the recombinant peptide was strongly affected by pH of cultures, with a decrease of more than 50% when acidification was freely allowed. Likewise, peptide production and plasmid stability were shown to be affected by aeration and agitation speed, both decreasing for higher values of oxygen mass transfer rates. Despite these difficulties, in bioreactor cultures carried out with controlled pH, low oxygen mass transfer rates and using lactose as inducer, we were able to achieve a total peptide production of 7.14
μg/mg protein, which represents approximately 2% of total cell protein. Bioassays were carried out using the purified peptide on insect models. The peptide fed to
Dysdercus peruvianus nymphs produced 100% mortality after 11 days, deaths starting with a lag phase of 3–4 days, confirming that the bioreactor-produced peptide retained its biological activity.