Purification and properties of heat-stable extracellular protease from Pseudomonads fluorescens BJ-10

Abstract

Pseudomonas fluorescens BJ-10, a kind of psychrotrophic bacteria, was isolated from raw milk. It produced an extracellular protease of 47 kDa by SDS-PAGE. The crude proteases were purified by ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. The specific activity of purified protease increased 61.38-fold. The optimum pH and temperature were pH 7.0 and 30°C, respectively. The purified protease was partially inhibited by DL-dithiothreitol, and the activity increased a little upon Fe 2+ addition. The protease showed typical heatstable behavior. After treatment at 100°C for 3 min, more than 94% activity remained. This work might lay the foundation for possible relationship between the heat stable protease and gelation of UHT milk.

Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk

“…The peptidases secreted by P. fluorescens have been under investigation for many decades and were characterised as thermo-resistant metallopeptidases (Kohlmann, Nielsen, & Ladisch, 1991a;Koka & Weimer, 2000;Kroll & Klostermeyer, 1984;Mayerhofer, Marshall, White & Lu, 1973;Zhang & Lv, 2014). These peptidases have been shown to be associated with the spoilage of milk (Baglini ere et al, 2013;Gaucher et al, 2011).…”

“…The combination of peptidase and lipase activity for Pseudomonas species was observed during agar diffusion assays by Hantsis-Zacharov and Halpern (2007). The secretion of extracellular lipases is described for several Pseudomonas species, such as Pseudomonas pseudoalcaligenes and P. fluorescens (Svendsen et al, 1995;Zhang & Lv, 2014). The lipase (lipA) and alkaline metallopeptidase (aprX) genes of P. fluorescens are located at the opposite ends of the same operon (14 kb), which contains eight genes, but they are regulated in different ways (McCarthy, Woods, & Beacham, 2004).…”

Multiplicação de Pseudomonas fluorescens em temperaturas de refrigeração e seu potencial proteolítico

“…A população de microrganismos psicro tróficos no leite cru pode alcançar altas concentrações quando ele é obtido em condições precárias de higiene, sendo as bactérias do gênero Pseudomonas encontradas com maior frequência em leite e derivados mantidos sob refrigeração (SAMARŽIJA et al, 2012;PERIN et al, 2012;ZHANG;LV, 2014;MAHROUS;MOUSA, 2012;RAAS et al, 2011, NÖRBERG et al, 2010. Esse gênero abrange espécies que se caracterizam por apresentar um curto tempo de geração a temperaturas de refrigeração (SAMARŽIJA et al, 2012;SØRHAUG;STEPANIAK, 1997).…”

“…Dentre as espécies de Pseudomonas, P. fluorescens constitui a de maior importância por produzir proteases termoestáveis que hidrolisam a caseína e diminuem o rendimento industrial e a qualidade sensorial dos produtos lácteos (MARTINS et al, 2015;BAGLINIÈRE et al, 2013;BAGLINIÈRE et al;ZHANG;LV, 2014;MAHROUS;MOUSA, 2012;NÖRBERG et al, 2010).…”

“…As enzimas proteolíticas produzidas por bactérias psicrotróficas podem resultar em vários problemas tecnológicos para a indústria laticinista, como a instabilidade do leite ao calor, a gelificação do leite UHT, a ocorrência de sabores e odores indesejáveis (PINTO et al, 2016;PINTO et al, 2014, ZENI et al, 2013BAGLINIÈRE et al, 2013;BAGLINIÈRE et al, 2012;SAMARŽIJA JA et al, 2012;ZHANG;LV, 2014;NÖRBERG et al, 2010;SØRHAUG;STEPANIAK, 1997) e a redução de rendimento na produção de queijo (ZHANG; LV, 2014).…”