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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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Zymographic analysis after SDS-PAGE gel electrophoresis of lipase from A. japonicus LAB01. Lane 1: lipase activity towards tributyrin; Lane 2: lipase activity towards oleic acid and dodecanol; Lane 3: molecular mass markers.
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fig2: Zymographic analysis after SDS-PAGE gel electrophoresis of lipase from A. japonicus LAB01. Lane 1: lipase activity towards tributyrin; Lane 2: lipase activity towards oleic acid and dodecanol; Lane 3: molecular mass markers.

Mentions: A. japonicus LAB01 lipases were purified using (NH4)2SO4 selective precipitation (60%) followed by gel filtration chromatography (Table 1). After two steps of purification, the lipase exhibited 3.5 × 104 U·mg−1 specific activity and 2.91-fold purification with an overall yield of 44.2%. The protein purification step profile showed a strong protein band with a molecular mass between 25 and 35 kDa estimated by SDS-PAGE (Figure 1). The zymographic analysis using tributyrin, oleic acid, and dodecanol as substrates revealed hydrolytic and synthetic activities based on bands that coincided with the migration of the principle protein (Figure 2). The partially purified lipase molecular mass was estimated to be approximately 25 kDa on SDS-PAGE. Most of the known Aspergillus sp. lipases reportedly have molecular masses in the range of 29−70 kDa [7, 10]. Another lipase from A. japonicus was purified by combining anion exchange (Q-Sepharose) and gel filtration (Sephadex G-100) chromatography by 3.44-fold with a 4.53% yield and a specific activity of 480 U/mg protein. The molecular weight of the enzyme was 43kDa according to SDS-PAGE [35]. Other lipase isoforms also differ in their molecular mass, such as those observed on Aspergillus niger. Two lipases from A. niger MZKI A116 were isolated by acetone precipitation followed by ion-exchange chromatography. The molecular weights were 43 kDa and 65 kDa, with isoelectric points of pH 4.1 and 4.2, respectively [36]. Recently, a novel lipase produced by the same fungus was found [37]. An extracellular lipase from the Aspergillus niger NCIM 1207 strain was purified by homogeneity using ammonium sulphate precipitation followed by phenyl-sepharose and Sephacryl-100 gel chromatography to yield a monomeric protein of 32.2 kDa.


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)

Zymographic analysis after SDS-PAGE gel electrophoresis of lipase from A. japonicus LAB01. Lane 1: lipase activity towards tributyrin; Lane 2: lipase activity towards oleic acid and dodecanol; Lane 3: molecular mass markers.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Zymographic analysis after SDS-PAGE gel electrophoresis of lipase from A. japonicus LAB01. Lane 1: lipase activity towards tributyrin; Lane 2: lipase activity towards oleic acid and dodecanol; Lane 3: molecular mass markers.
Mentions: A. japonicus LAB01 lipases were purified using (NH4)2SO4 selective precipitation (60%) followed by gel filtration chromatography (Table 1). After two steps of purification, the lipase exhibited 3.5 × 104 U·mg−1 specific activity and 2.91-fold purification with an overall yield of 44.2%. The protein purification step profile showed a strong protein band with a molecular mass between 25 and 35 kDa estimated by SDS-PAGE (Figure 1). The zymographic analysis using tributyrin, oleic acid, and dodecanol as substrates revealed hydrolytic and synthetic activities based on bands that coincided with the migration of the principle protein (Figure 2). The partially purified lipase molecular mass was estimated to be approximately 25 kDa on SDS-PAGE. Most of the known Aspergillus sp. lipases reportedly have molecular masses in the range of 29−70 kDa [7, 10]. Another lipase from A. japonicus was purified by combining anion exchange (Q-Sepharose) and gel filtration (Sephadex G-100) chromatography by 3.44-fold with a 4.53% yield and a specific activity of 480 U/mg protein. The molecular weight of the enzyme was 43kDa according to SDS-PAGE [35]. Other lipase isoforms also differ in their molecular mass, such as those observed on Aspergillus niger. Two lipases from A. niger MZKI A116 were isolated by acetone precipitation followed by ion-exchange chromatography. The molecular weights were 43 kDa and 65 kDa, with isoelectric points of pH 4.1 and 4.2, respectively [36]. Recently, a novel lipase produced by the same fungus was found [37]. An extracellular lipase from the Aspergillus niger NCIM 1207 strain was purified by homogeneity using ammonium sulphate precipitation followed by phenyl-sepharose and Sephacryl-100 gel chromatography to yield a monomeric protein of 32.2 kDa.

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