EXPRESION OPTIMIZATION OF A PUTATIVE AMYLO- PULLULANASE FROM LACTOCOCCUS LACTIS, USING NICE SYSTEM

MAGNI, C; TUBIO, G; PEREZ, C; BLANCATO, VÍCTOR S.

CABA

Congreso; LIII Reunión Anual de SAIB; 2017

SAIB

L. lactis is one of the most commonly used lactic acid bacteria in fermented food production. Because it is considered Generally Recognized As Safe (GRAS), the implementation of this strain in biotechnological processes aims to simplify the downstream processing and to diminish contamination risks with toxins in a wide range of commercial products. The NICE system can be used to produce large amount of recombinant proteins using nisin as inductor enabling its application for I+D and industrial enzymes production. Novel amylo-pullulanase, neo-pullulanase, pullulanase and other enzymes that debranch sugar polymers into simple di- or tri-saccharides are of industrial and agricultural relevance e.g. as a food supplement. The aim of this study was to over-express and characterize a putative amylo-pullulanase (apu) induced when L. lactis IL1403 was grown in the presence of starch or in L. lactis KF147 when growing on plant tissues. To simplify protein detection, purification and further application in several food matrixes, the L. lactis IL1403 apu gene was fused to usp secretion sequence in the N-terminus and/or Histidine-tag in the C-terminus for Ni2+-NTA affinity purification. These sequences were provided by a corresponding expression plasmid derived from pNZ8048 where apu gene was cloned under the control of Pnis promoter. Expression was optimized in L. lactis NZ9000 and NZ9000 Clp- HtrA- strains, the best conditions for protein overexpression were 5ng/ml nisin and 3 hs of induction at 30ºC. Intracellular over-expression could be observed by coomasie staining of SDS-PAGE gels, but no secreted protein was detected in medium supernatant by coomassie staining or western blot using anti-his antibodies. Enzyme activity was measured by the DNS method with 1% pullulan solution as substrate, giving 0.282 U/ml of L. lactis crude extract. Suggesting that the overexpressed protein has the predicted function, but further optimization will be required to increase its activity.Keywords: GRAS, protein expression, lactic acid bacteria