Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk

Abstract

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli . The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca (+2) . The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca (+2) , on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.

Keywords: Pseudomonas fluorescens; extracellular lipase; extracellular protease; food deterioration; raw milk.

Figure 1. Production of extracellular hydrolytic enzymes by P. fluorescens . A: Proteolytic activity in the supernatant of TYEP medium; B: Lipolytic activity in the supernatant of TYEP medium; C: Samples of reconstituted skin milk powder (12%) inoculated with P. fluorescens 07A and 041 after 18 h of incubation at 25 °C. Data represent the average of duplicate experiments; D: Coomassie-stained SDS-PAGE and azocasein zymogram on 12% PAA-gels visualizing protease production by P. fluorescens grown in TYEP medium supplemented with 0.25% CaCl ₂ . Lanes S: molar mass standard (Biorad); lane 1: SDS-PAGE of ammonium sulfate precipitated proteins of P. fluorescens 041 supernatant; lane 2: azocasein zymogram of ammonium sulfate precipitated proteins of P. fluorescens 041 after proteins precipitation with ammonium sulfate.

Figure 2. Coomassie-stained SDS-PAGE and zymogram gel on 12% PAA-gels visualizing recombinant AprX and LipM. Lane S: molecular mass standard (Biorad); lane 1: SDS-PAGE of crude extract of E. coli XL1-Blue carrying pQE30-Xa-aprX-041; lane 2: SDS-PAGE of crude extract of E. coli XL1-Blue carrying pQE30-Xa-lipM-041; lane 3: SDS-PAGE of purified AprX; lane 4: azocasein zymogram of purified AprX; lane 5: SDS-PAGE of purified LipM; lane 6: MU-butyrate zymogram of purified LipM.

Figure 3. Biochemical characteristics of AprX. A: Temperature optimum of purified AprX on azocasein; B: pH-optimum of purified AprX on azocasein. C: Effect of heat treatment on proteolytic activity of purified AprX. Data represent the average of duplicate experiments.

Figure 4. Biochemical characteristics of LipM. A: Temperature optimum of purified LipM; B: pH-optimum of purified LipM. C: Effect of heat treatment for 60 min on lipolytic activity of purified LipM. Data represent the average of duplicate experiments.

Figure 5. Multiple sequence alignment of deduced protease AprX from P. fluorescens 041 (this study), P. fluorescens A506 (Genbank accession number AY298902 ), and P. fluorescens strain F (Genbank accession number DQ146945 ). The differences in amino acid residues are indicated by gray shading, and the catalytic domain of neutral zinc metalloprotease is underlined. Boxed residues are thought to participate in Calcium binding.

Figure 6. Multiple sequence alignment of deduced lipase LipM from P. fluorescens 041 (this study), Lip (Genbank accession number DQ305493 ), Lip68 (Genbank accession number AY694785 ), and LipA (Genbank accession number AF216702 ) from P. fluorescens . The differences in amino acid residues are indicated by gray shading and the catalytic domain of serine lipase is underlined.