Quantification of Bacillus thuringiensis delta endotoxins by protein elution from polyacrylamide

MENTEL, MARÍA ISABEL; BAIGORÍ, MARIO DOMINGO; PERA, LICIA MARÍA

Córdoba

Congreso; XI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL; 2015

SAMIGE

Bacillus thuringiensis(Bt) is a gram-positive and spore forming bacterium. It is characterized by the production of insecticidal crystal proteins (Cry) named delta-endotoxins, which exhibit larvicidal toxicity upon ingestion by susceptible insect larvae. Biopesticides derived from Bt are the most prominent biological agents for selective control of pest insects. They are composed of a mixture of spores, crystals and minor cell debris harvested from the culture media at the end of the fermentation process. The production of Bt as biological control currently focuses on the use of different supplements and substrates to find the best combinations to obtain an efficient product, using operational conditions that facilitate the reduction of the production costs at an industrial scale. This can be achieved through the use of low cost culture media such as residues from agro-industry so that production can become economically viable. Furthermore, several studies have reported the utilization of several media based on complex substrates, which are efficient for Bt bioinsecticides production. Our group already designed a production medium based on the use of agro-industrial residues. The aim of this work is to find a method for delta endotoxin quantification compatible with complex culture medium. The native isolate Bt RT was used throughout this study. The Cry concentration was determined by the method of dye elution in SDS-isopropanol using bovine serum albumin as a standard. Electrophoresis was conducted essentially as described by Laemmli. Gels were stained with Coomassie Brilliant Blue R250. The amount of protein in the Cry1Ac bands was determined measuring the optical density at 594nm of the elution solution. Thus, a very simple method for delta endotoxin quantification has been developed. This method allows us to quantify the Cry1Ac proteins produced in a complex medium without the interference of any undesired proteins. In addition, the highest Cry1Ac concentration was observed after three days of fermentation.