Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

Abstract

An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH₂-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0-12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry.

Keywords: Characterization; Detergent-stable; HPLC, high-performance liquid chromatography; NaDC, sodium deoxycholic acid; NaTDC, sodium taurodeoxy cholic acid; OD, optical density; PCR, polymerase chain reaction; Purification; S. aureus, Staphylococcus aureus; SAL, Staphylococcus aureus lipase; SDS, sodium dodecyl sulfate; SEL, Staphylococcus epidermidis lipase; SHyL, Staphylococcus hyicus lipase; SL1, Staphylococcus sp. lipase; SSL, Staphylococcus simulans lipase; SXL, Staphylococcus xylosus lipase; Staphylococcus aureus lipase; TC18, triolein; TC3, tripropionin; TC4, tributryin; TC8, trioctanoin; TFA, tri fluoroacetic acid; Thermo-alkaline; rDNA, ribosomal deoxy ribo nucleic acid; rpm, revolutions per minute.

Figure 1

Chromatography on RP-HPLC column and SDS–PAGE (15%) of SAL4. (A) RP-HPLC on a C-18 column, elution was performed at room temperature within 40 min using a gradient from 0 to 80% acetonitrile at a flow rate of 1 mL/min. The gradient is indicated by the dotted line. The absorbance was measured at 280 nm. AU: Arbitrary Units. (B) 15%-SDS–PAGE of pure SAL4. Lane 1, molecular mass markers (Pharmacia); lane 2, 7 μg of purified SAL4 eluted from RP-HPLC. (C) Alignment of the amino acid sequences of the mature forms of SAL4 (Present study, KF67886), SAL3 (Horchani et al., 2009), SSL (Sayari et al., 2001), SEL3 (Rosenstein et al., 2000) and SHL (Tiesinga et al., 2007).

Figure 2

(A) Hydrolysis rate of TC3 by SAL4 as function of substrate concentration. The release of propionic acid was recorded continuously at pH 7 and 37 °C using a pH-stat. The CMC of TC3 (12 mM) is indicated by vertical dotted lines. (B) Kinetics of hydrolysis of TC4, TC8, or TC18 emulsions by SAL4 (11U). Lipase activity was followed at pH 12 and 60 °C. Each data point represents an average of at least two independent experiments, each in triplicate.

Figure 3

Temperature effect of on SAL4 stability (A) and activity (B). pH effect on enzyme activity (C) and stability (D) of SAL4. All experiments were repeated at least three times.

Figure 4

(A) Influence of Ca²⁺ concentration on SAL4 activity. The star symbol indicates the absence of lipase activity checked in the absence of CaCl₂ and in the presence of 10 mM EDTA. (B) Influence of metal ions concentrations (2 mM each) on SAL4 activity was studied using olive oil emulsion as substrate under optimal conditions of pH and temperature. The control represents 100% of lipase activity in the absence of metal ions under the same condition. Values represent the mean of three replicates.