CONICET | Buscador de Institutos y Recursos Humanos

fermented food production. Because it is considered Generally RecognizedAs Safe (GRAS), the implementation of this strain in biotechnologicalprocesses aims to simplify the downstream processingand to diminish contamination risks with toxins in a wide rangeof commercial products. The NICE system can be used to producelarge amount of recombinant proteins using nisin as inductor enablingits application for I+D and industrial enzymes production.Novel amylo-pullulanase, neo-pullulanase, pullulanase and otherenzymes that debranch sugar polymers into simple di- or tri-saccharidesare of industrial and agricultural relevance e.g. as a food supplement.The aim of this study was to over-express and characterizea putative amylo-pullulanase (apu) induced when L. lactis IL1403was grown in the presence of starch or in L. lactis KF147 whengrowing on plant tissues. To simplify protein detection, purificationand further application in several food matrixes, the L. lactis IL1403apu gene was fused to usp secretion sequence in the N-terminusand/or Histidine-tag in the C-terminus for Ni2+-NTA affinity purification.These sequences were provided by a corresponding expressionplasmid derived from pNZ8048 where apu gene was clonedunder the control of Pnis promoter. Expression was optimized in L.lactis NZ9000 and NZ9000 Clp- HtrA- strains, the best conditions forprotein overexpression were 5ng/ml nisin and 3 hs of induction at30ºC. Intracellular over-expression could be observed by coomasiestaining of SDS-PAGE gels, but no secreted protein was detectedin medium supernatant by coomassie staining or western blot usinganti-his antibodies. Enzyme activity was measured by the DNSmethod with 1% pullulan solution as substrate, giving 0.282 U/ml ofL. lactis crude extract. Suggesting that the overexpressed proteinhas the predicted function, but further optimization will be requiredto increase its activity.