PARTIAL PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR METALLOPEPTIDASE PRODUCED BY Bacillus amyloliquefaciens FE-K1

Abstract

The aim of this study was to purify and characterize the peptidase of Bacillus amyloliquefaciens (Fukumoto) (strain FE-K1) isolated from ropey bread. Peptidases were purified from crude enzyme solution by affinity chromatography with an efficiency of 25 % and a purification coefficient of 1.53. The optimum pH of partially purified peptidase (PPPase) solution was determined as 7.5 and the peptidases retained approximately 90 % of their initial activity in the pH range 7.0-8.5 following incubation at 37°C for 2 h. The optimum temperature for the PPPase was 60°C. The approximate molecular weight of the PPPase was determined as 36 kDa. Inactivation of the PPPase in the presence of O-FEN and EDTA showed them to be metallopeptidases and 5 mM of K +1 and 5 mM of Mn +2 ions increased the enzyme activity by 4 % and 6.15 %, respectively. The presence of Hg +2 , Fe +3 and SDS (0.1-1.0 % w/v) caused inactivation whereas the enzyme retained most of its activity in the presence of 0.1-1.0 % (v/v) Triton X-100, Tween 20 and Tween 80 and 1-20 % (v/v) xylene, ethanol, acetone and acetonitrile. Characterization of the PPPase revealed the enzyme as a neutral serine metallopeptidase compatible with some organic solvents and surfactants.Özet: Bu çalışmanın amacı, sünmüş ekmeklerden izole edilen Bacillus amyloliquefaciens (Fukumoto) (suş FE-K1) ile elde edilen peptidazı saflaştırmak ve karakterize etmektir. Peptidazlar ham enzim çözeltisinden afinite kromatografisi ile % 25 verim ve 1,53 saflaştırma katsayısı ile saflaştırılmıştır. Kısmi olarak saflaştırılmış peptidaz (PPPaz) çözeltisinin optimum pH değeri 7,5 olarak tespit edilmiş olup, pH 7,5-8,0 aralığında peptidaz, 37°C'de 2 saat inkübasyonun ardından başlangıç aktivitesini yaklaşık % 90 oranında korumuştur. PPPaz'ın optimum sıcaklığı 60°C'dir. PPPaz'ın yaklaşık molekül ağırlığı 36 kDa olarak belirlenmiştir. PPPaz'ın O-FEN ve EDTA varlığında inaktive olması, enzimin metallopeptidaz olduğunu göstermiştir. Ayrıca 5 mM K +1 ve 5 mM Mn +2 , enzimin aktivitesini sırasıyla % 4 ve % 6,15 oranında artırmıştır. Hg +2 , Fe +3 ve SDS (% 0,1-1,0 w/v) varlığı enzimin inaktivasyonuna neden olurken, % 0,1-1,0 (v/v) Triton X-100, Tween 20 ve Tween 80; ve % 1-20 (v/v) ksilen, etanol, aseton ve asetonitril varlığında enzim aktivitesini büyük ölçüde korumuştur. PPPaz'ın karakterizasyonu, enzimin bazı organik çözücü ve yüzey aktif maddelerle ile uyumlu nötr bir serin metalopeptidaz olduğunu ortaya çıkarmıştır.

Essential oil of Cymbopogon martini, source of geraniol, as a potential antibacterial agent against Bacillus subtilis, a pathogen of the bakery industry

“…Due to the action of proteolytic and amylolytic enzymes released by Bacillus, the texture of the bread is modified and becomes viscous. 22 One of the species considered to be the most common causative agent of rope in bakery products is B. subtilis, 23 however, as it takes a relatively long time for the stiffness to develop, the deterioration is often only detected…”

Essential oil of Cymbopogon martini, source of geraniol, as a potential antibacterial agent against Bacillus subtilis, a pathogen of the bakery industry

“…Due to the action of proteolytic and amylolytic enzymes released by Bacillus, the texture of the bread is modified and becomes viscous. 22 One of the species considered to be the most common causative agent of rope in bakery products is B. subtilis, 23 however, as it takes a relatively long time for the stiffness to develop, the deterioration is often only detected once the consumer has purchased the product. There is increasing interest in developing new strategies to inactivate spores.…”