Bioprocesso de produção de L-asparaginase e desenvolvimento de biocatalisador enzimático para evitar formação de acrilamida em alimentos

Abstract

The heating of some plant-based foods in industry induces the production of acrylamide through the Maillard reaction, that occurs between reducing sugars and the amino acid L-asparagine (Asn). The acrylamide is a neurotoxic and genotoxic compound, and probable human carcinogen. Acrylamide’s harmful effects on human health are sufficient to evoke the recommendation of its mitigation by the World Health Organization, in products like French fries, rich and free sugars and Asn. Treating these foods with L-asparaginase (L-ASNase), before heating, can reduce Asn concentration, the precursor of acrylamide. This work aims to produce L-ASNase in high concentration in the fermentation broth through fermentative process, and to develop a biocatalyst. The biocatalyst implies in L-ASNase immobilization on a mesoporous silica, SBA-15. The L-ASNase II genes, from Escherichia coli (ansB, encoding EcAII) and from Bacillus subtilis (ansZ, encoding BsAII), were cloned into vectors for periplasmatic constitutive expression, being inserted into E. coli cells. The expression products were obtained from the fermented broth through precipitation. It was obtained high yield of recombinant enzymes on the extracts from fermented broth of agitated

tubes, reaching 14810 U∙L -1 of L-ASNase activity. Inferior results were obtained on agitated flasks. The EcAII extract was used on immobilization experiments on SBA-15. Different immobilization and coating conditions were evaluated. EcAII adsorption on SBA-15 was ineffective, with no recovered activity, compared to 8% from covalent bonding on activated SBA-15. The addition of amino acids in the immobilization increased the global yield, reaching 39% and 97% utilizing L-glutamine (Gln) (SBA- E(Gln)) and Asn (SBA-E(Asn)), respectively. In general, SBA-E(Asn) and its derivative coated with polyethyleneimine, SBA-E-PEI, showed better performance than the free enzyme in higher temperatures, increasing thermal stability and optimal activity temperature and improving denaturation profiles. SBA-E(Asn) and SBA-E-PEI kept above 50% of its initial activity after 10 cycles of reuse on operational stability at 55 °C, proving to be adequate biocatalysts to industrial application. Applying the immobilization condition to BsAII and new experiments to improve the thermal stability should be made in future works, as well as applying the biocatalysts on potatoes to mitigate acrylamide formation.