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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 temperature on stability (a) and activity (b) of the partially purified A. japonicus LAB01 lipase. (a) Temperature stability profile was determined by measuring the residual activity at pH 8.0 and 37°C after incubating the lipase at 45°C for 720 min. (b) Optimal temperature was determined by measuring the enzyme activity at different temperatures at pH 8.0.
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fig5: Effects of temperature on stability (a) and activity (b) of the partially purified A. japonicus LAB01 lipase. (a) Temperature stability profile was determined by measuring the residual activity at pH 8.0 and 37°C after incubating the lipase at 45°C for 720 min. (b) Optimal temperature was determined by measuring the enzyme activity at different temperatures at pH 8.0.

Mentions: The purified lipase was active in the temperature range of 20–45°C, with maximal activity at 45°C. Its activity sharply decreased above the optimum temperature (Figure 5). The characteristics of A. japonicus LAB01 lipase were not consistent with those of others found in Aspergillus genus, including A. niger MTCC 2594 [39], A. carneus [13], and A. niger MYA135 [44]. In all of these cases, the optimum temperature was 37°C, and the enzymes were not very active at temperatures above 40°C. The recombinant A. fumigatus lipase, whose optimal temperature was 65°C, constitutes an exception. However, this enzyme was not stable at this temperature, losing almost 100% of its activity after 1 h of incubation [29]. The thermal stability profile of A. japonicus LAB01 lipase indicated that this enzyme was stable at 45°C for at least 3 h, retaining 70% of its original activity. The high thermal stability of the enzyme is similar to that found for the thermostable lipases obtained from R. homothallicus, which had a half-life equal to 0.72 hours at 50°C [45]. The thermal stability of lipase from our strain indicates its potential application in the modification of vegetable oils, mainly oils that have high melting points, which are solid at room temperature. In general, fungal lipases are not stable at temperatures above 40°C. Some studies have reported lipases with moderate thermostability, as observed for P. wortmanii lipase, which retained 90 and 55% of its initial enzymatic activity after 60 min of incubation at 45 and 50°C, respectively [46]. P. cyclopium lipase was stable for 1 hour when incubated at 30°C and only lost 10% of its activity after 1 hour of incubation at 35°C [47]. P. crustosum lipase lost 75% of its enzymatic activity after incubation for 1 hour at 45°C [48].


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 temperature on stability (a) and activity (b) of the partially purified A. japonicus LAB01 lipase. (a) Temperature stability profile was determined by measuring the residual activity at pH 8.0 and 37°C after incubating the lipase at 45°C for 720 min. (b) Optimal temperature was determined by measuring the enzyme activity at different temperatures at pH 8.0.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effects of temperature on stability (a) and activity (b) of the partially purified A. japonicus LAB01 lipase. (a) Temperature stability profile was determined by measuring the residual activity at pH 8.0 and 37°C after incubating the lipase at 45°C for 720 min. (b) Optimal temperature was determined by measuring the enzyme activity at different temperatures at pH 8.0.
Mentions: The purified lipase was active in the temperature range of 20–45°C, with maximal activity at 45°C. Its activity sharply decreased above the optimum temperature (Figure 5). The characteristics of A. japonicus LAB01 lipase were not consistent with those of others found in Aspergillus genus, including A. niger MTCC 2594 [39], A. carneus [13], and A. niger MYA135 [44]. In all of these cases, the optimum temperature was 37°C, and the enzymes were not very active at temperatures above 40°C. The recombinant A. fumigatus lipase, whose optimal temperature was 65°C, constitutes an exception. However, this enzyme was not stable at this temperature, losing almost 100% of its activity after 1 h of incubation [29]. The thermal stability profile of A. japonicus LAB01 lipase indicated that this enzyme was stable at 45°C for at least 3 h, retaining 70% of its original activity. The high thermal stability of the enzyme is similar to that found for the thermostable lipases obtained from R. homothallicus, which had a half-life equal to 0.72 hours at 50°C [45]. The thermal stability of lipase from our strain indicates its potential application in the modification of vegetable oils, mainly oils that have high melting points, which are solid at room temperature. In general, fungal lipases are not stable at temperatures above 40°C. Some studies have reported lipases with moderate thermostability, as observed for P. wortmanii lipase, which retained 90 and 55% of its initial enzymatic activity after 60 min of incubation at 45 and 50°C, respectively [46]. P. cyclopium lipase was stable for 1 hour when incubated at 30°C and only lost 10% of its activity after 1 hour of incubation at 35°C [47]. P. crustosum lipase lost 75% of its enzymatic activity after incubation for 1 hour at 45°C [48].

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