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Lipolytic potential of Aspergillus japonicus LAB01: production, partial purification, and characterisation of an extracellular lipase.

Souza LT, Oliveira JS, dos Santos VL, Regis WC, Santoro MM, Resende RR - Biomed Res Int (2014)

Bottom Line: The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature.Among the tested ions, only Cu(2+), Ni(2+), and Al(3+) showed inhibitory effects.The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling, Nanobiotechnology and Enzymology Laboratory, Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil.

ABSTRACT
Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu(2+), Ni(2+), and Al(3+) showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

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Effects of pH on the stability (a) and activity (b) of A. japonicus LAB01 lipase. (a) Partially purified lipase samples were incubated for 90 min at 30°C in the presence of different buffers (100 mM): citrate phosphate (pH 3.0–6.0), sodium phosphate (pH 7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 10.0), and the residual activity was measured using pNPP at pH 8.0 and 37°C. (b) Partially purified lipase samples were assayed in buffers B from pH 7.0 to 9.5 using pNPP as substrate at 37°C.
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fig4: Effects of pH on the stability (a) and activity (b) of A. japonicus LAB01 lipase. (a) Partially purified lipase samples were incubated for 90 min at 30°C in the presence of different buffers (100 mM): citrate phosphate (pH 3.0–6.0), sodium phosphate (pH 7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 10.0), and the residual activity was measured using pNPP at pH 8.0 and 37°C. (b) Partially purified lipase samples were assayed in buffers B from pH 7.0 to 9.5 using pNPP as substrate at 37°C.

Mentions: The lipase stability was evaluated in the pH range of 3.0 to 10.0 using different buffers at the same concentration (100 mM) (Figure 4). The enzyme remained stable from pH 5.0 to 9.0 during preincubation for 90 min at 30°C, where the enzyme retained at least 80% of its initial activity. Furthermore, increasing or decreasing the pH values dramatically reduced the enzyme stability. The A. japonicus LAB01 lipase was more active at slightly alkaline pH values, and the optimal pH was 8.5, which is similar to the values reported for lipases from A. carneus [13]. The purified enzyme from our strain did not show differences in the optimum pH and pH stability; consequently, the lipase could be considered a resistant alkaline lipase. Alkaline lipases are required for applications in the detergent industries, suggesting a possible use in this area. The majority of the lipases of Aspergillus origin showed maximum activities in neutral or near neutral pH values (6.0–7.5), such as those obtained from A. japonicus [35], A. niger MTCC 2594 [39], A. nidulans [40], A. niger F044 [41], A. awamori [42], A. niger MYA135 [27], and A. terreus [43]. A. niger NCIM 1207 lipase, which exhibited an optimum acidic pH of 2.5, was an exception to this rule [37].


Lipolytic potential of Aspergillus japonicus LAB01: production, partial purification, and characterisation of an extracellular lipase.

Souza LT, Oliveira JS, dos Santos VL, Regis WC, Santoro MM, Resende RR - Biomed Res Int (2014)

Effects of pH on the stability (a) and activity (b) of A. japonicus LAB01 lipase. (a) Partially purified lipase samples were incubated for 90 min at 30°C in the presence of different buffers (100 mM): citrate phosphate (pH 3.0–6.0), sodium phosphate (pH 7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 10.0), and the residual activity was measured using pNPP at pH 8.0 and 37°C. (b) Partially purified lipase samples were assayed in buffers B from pH 7.0 to 9.5 using pNPP as substrate at 37°C.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230215&req=5

fig4: Effects of pH on the stability (a) and activity (b) of A. japonicus LAB01 lipase. (a) Partially purified lipase samples were incubated for 90 min at 30°C in the presence of different buffers (100 mM): citrate phosphate (pH 3.0–6.0), sodium phosphate (pH 7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 10.0), and the residual activity was measured using pNPP at pH 8.0 and 37°C. (b) Partially purified lipase samples were assayed in buffers B from pH 7.0 to 9.5 using pNPP as substrate at 37°C.
Mentions: The lipase stability was evaluated in the pH range of 3.0 to 10.0 using different buffers at the same concentration (100 mM) (Figure 4). The enzyme remained stable from pH 5.0 to 9.0 during preincubation for 90 min at 30°C, where the enzyme retained at least 80% of its initial activity. Furthermore, increasing or decreasing the pH values dramatically reduced the enzyme stability. The A. japonicus LAB01 lipase was more active at slightly alkaline pH values, and the optimal pH was 8.5, which is similar to the values reported for lipases from A. carneus [13]. The purified enzyme from our strain did not show differences in the optimum pH and pH stability; consequently, the lipase could be considered a resistant alkaline lipase. Alkaline lipases are required for applications in the detergent industries, suggesting a possible use in this area. The majority of the lipases of Aspergillus origin showed maximum activities in neutral or near neutral pH values (6.0–7.5), such as those obtained from A. japonicus [35], A. niger MTCC 2594 [39], A. nidulans [40], A. niger F044 [41], A. awamori [42], A. niger MYA135 [27], and A. terreus [43]. A. niger NCIM 1207 lipase, which exhibited an optimum acidic pH of 2.5, was an exception to this rule [37].

Bottom Line: The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature.Among the tested ions, only Cu(2+), Ni(2+), and Al(3+) showed inhibitory effects.The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling, Nanobiotechnology and Enzymology Laboratory, Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil.

ABSTRACT
Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu(2+), Ni(2+), and Al(3+) showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

Show MeSH
Related in: MedlinePlus