Isolation, Characterization and Amino Acid Composition of a Bacteriocin Produced by Bacillus methylotrophicus Strain BM47

Poznato je da bakterije roda Bacillus proizvode široki spektar proteinskih antibiotičkih spojeva što inhibiraju različite saprofitne i patogene mikroorganizme. U ovom je radu opisan i identificiran kao bakteriocin peptid iz soja bakterije Bacillus methylotrophicus BM47, prethodno izolirane iz prirodnog termalnog izvora u Bugarskoj. Ispitivanje je antimikrobnog djelovanja in vitro nepročišćene suspenzije bakteriocina iz B. methylotrophicus BM47 pokazalo učinak na fitopatogene plijesni Fusarium moniliforme, Aspergillus awamori, Penicillium sp., Aspergillus niger te na Gram-negativnu bakteriju Pseudomonas aeruginosa. Antimikrobno djelovanje nepročišćene suspenzije bakteriocina djelomično su inhibirali enzimi tripsin, Alcalase®, Savinase®, proteinaza K, papain i Esperase®, dok katalaza nije imala učinkovito djelovanje. Nepročišćena je suspenzija bakteriocina bila otporna na promjene u pH-vrijednostima, ali je bila osjetljiva na djelovanje topline i većine ispitanih organskih otapala i detergenata. Radi dobivanja udjela aktivnog proteina, sirovi je ekstrakt bakteriocina pročišćen pomoću brze tekućinske kromatografije proteina (engl. FPLC) i jake anionske kolone za ionsku izmjenu. Analiza tricin-natrij dodecil sulfat – poliakrilamid gel elektroforezom pokazala je da pročišćeni bakteriocin ima molekulsku masu od 19 578 Da, dok je visokodjelotvornom tekućinskom kromatografijom utvrđeno da sadržava 17 vrsta aminokiselina s najvećim molarnim udjelom serina (29,3 %), valina (10,3 %), alanina (9,8 %) i tirozina (7,1 %).Members of the bacterial genus Bacillus are known as producers of a broad spectrum of antibiotic compounds of proteinaceous nature that possess inhibitory activity against different saprophytic and pathogenic microorganisms. In the current research, a peptide synthesized by Bacillus methylotrophicus strain BM47, previously isolated from a natural thermal spring in Bulgaria, was identified and characterized as a bacteriocin. In vitro antimicrobial screening of the crude bacteriocin substance of B. methylotrophicus BM47 showedn activity against the plant pathogenic fungi Fusarium moniliforme, Aspergillus awamori, Penicillium sp., Aspergillus niger and Gram-negative bacterium Pseudomonas aeruginosa. The antimicrobial activity of the crude bacteriocin substance was partially inhibited by the enzymes trypsin, Alcalase®, Savinase®, proteinase K, papain and Esperase®, while catalase was not effective. The crude bacteriocin substance was relatively pH resistant, but sensitive to the action of heat and most organic solvents and detergents tested. To obtain the active protein fractions, crude bacteriocin substance was purified by fast protein liquid chromatography (FPLC) using a strong anion exchange column. Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purified bacteriocin had molecular mass of 19 578 Da. The amino acid analysis performed by high-performance liquid chromatography (HPLC) revealed that the isolated bacteriocin consisted of 17 types of amino acids, with the highest mol fraction expressed as percent of serine (29.3), valine (10.3), alanine (9.8) and tyrosine (7.1)