Proteinaze mlečno-kiselinskih bakterija: diverzitet u mezofilnim vrstama laktobacila i uticaj na aktivnost bakteriocina

Abstract

Mlečno-kiselinske bakterije su mikroorganizmi od izuzetnog značaja kako za industrijufermentisanih proizvoda, tako i za medicinu gde kao probiotici dobijaju sve veći značaj.Proteinaze i bakteriocini, molekuli koje ove bakterije sekretuju u ekstraćelijsku sredinu, imajuvažnu ulogu u oba ova aspekta.Proteinaze su veliki ekstraćelijski enzimi uključeni u prvi korak digestije proteina izvanćelijske sredine i neophodne su za efikasan rast bakterija u sredinama siromašnim malimpeptidima i slobodnim aminokiselinama. U ovom radu je analizirana proteolitička aktivnostrazličitih mezofilnih laktobacila, pripadnika Lb. casei i Lb. plantarum grupa prema pet različitihproteina mleka. Utvrđeno je da Lb. zeae LMG17315 poseduje najefikasniju proteolitičkuaktivnost, s obzirom da hidrolizuje sve ponuđene supstrate. Takođe pokazano je da se proteinazeovog soja odvajaju u medijum u aktivnom obliku. Lb. casei ATCC393, soj sa kontroverznimsistematičkim statusom, pokazao je sličnu, ali manje efikasnu proteolitičku aktivnost. Prisustvorazličitih proteinaznih gena u ispitivanim sojevima analizirano je DNK-DNK hibridizacijom iPCR metodom. Utvrđeno je da LMG17315 poseduje sekvence gena koje kodiraju katalitičkedomene za tri, a ATCC393 za jednu proteinazu. Kod soja LMG17315 je utvrđeno da posedujeprtP-sličan i prtR-sličan gen, dok je kod oba soja pronađen i novi proteinazni gen, označen kaoprtP1. S obzirom na dosadašnji problematičan sistematički status ovih sojeva urađena je injihova molekularna determinacija čime je stečen bolji uvid u njihove filogenetske odnose. Iakoje u relevantnoj literaturi predloženo da ATCC393 treba da bude reklasifikovan u vrstu Lb. zeae,dobijeni rezultati ukazuju na značajne razlike između soja ATCC393 i tipskog soja Lb. zeaeLMG17315.

Lactic acid bacteria are microorganisms of great importance for the industry of fermentedproducts as well as for medicine, where they are gaining great attentionas probiotics. Proteinasesand bacteriocins, molecules secreted by these bacteria, have important roles in both of theseaspects.Proteinases are large enzymes involved the in first steps of digestion of extracellularproteins and are necessary for efficient bacterial growth in environments with low concentrationsof small peptides and free amino acids. The proteolytic activity of different mesophiliclactobacilli, members of Lb. casei and Lb. plantarum groups, was tested against five main milkproteins, in this thesis. It was determined that Lb. zeae LMG17315 possessed the most efficientproteolytic activity, since it hydrolyzed all given substrates. In addition, it was shown thatproteinases of this strain could be released from cell wall in active form. Lb. casei ATCC393, astrain with controversial systematics, showed similar, but less efficient proteolytic activity.Presence of different proteinase genes in tested strains was analyzed with DNA-DNAhybridization and PCR. It was determined that strain LMG17315 possessed gene sequencescoding for catalytic domains of three proteinases, while ATCC393 possessed only one suchsequence. Two of these genes in strain LMG17315 were similar to previously characterized prtPand prtR genes, while the third one was a new proteinase gene, here named prtP1. The samegene was found in the genome of ATCC393. Considering the problematic taxonomic status ofthese strains, methods of molecular determination were applied for better understanding of theirphylogenetic relationship. Although many authors suggest that ATCC393 should be reclassifiedin taxon Lb. zeae, results obtained in this work highlighted some significant differences betweenthese strains.