Synthesis activity-based zymography for detection of lipases and esterases

Kwon, Min-A; Kim, Hyun Suk; Hahm, Dae‐Hyun; Song, Jae Kwang

doi:10.1007/s10529-010-0485-4

Cited by 16 publications

(8 citation statements)

References 19 publications

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“…The gels were incubated for at least 4 hours with solution replacement every 30 minutes. The zymographic analysis was performed based on the lipase hydrolytic and synthetic activity using tributyrin [25], oleic acid, and dodecanol as substrates [26]. …”

Section: Methodsmentioning

confidence: 99%

Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

Souza

Oliveira

Santos

et al. 2014

BioMed Research International

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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 Cu2+, Ni2+, and Al3+ 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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Section: Methodsmentioning

confidence: 99%

Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

Souza

Oliveira

Santos

et al. 2014

BioMed Research International

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“…Electrophoresis in semi-denaturing conditions (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis- SDS-PAGE) was performed similarly to the methodology described by Laemmli (1970) [ 62 ]. The semi-denaturing character is conferred because the run buffer did not contain β-mercaptoethanol and the amount of SDS present was lower than the original methodology (0.01% SDS was used in this methodology), in addition, the samples were not boiled and after the run, buffer was added [ 63 ]. The gels were stained with a silver solution [ 64 ].…”

Section: Methodsmentioning

confidence: 99%

“…In order to visualize the lipolytic activity, the zymogram was developed as described by Kwon et al (2011) [ 63 ]. The assay led to the formation of complexes that were visualized as dark spots on the polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support

et al. 2018

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This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.

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“…Reakcioni pufer se uvek priprema svež. Gel je nakon završene elektroforeze i ispiranja/renaturacije potopljen u reakcioni pufer za zimogram, i inkubiran na 30 °C u trajanju od 30 min do 24 h, sve do pojave belih precipitata na transparentnom gelu [12].…”

Section: Eksperimentalni Deounclassified

Electrophoretic and zymographic techniques for production monitoring of two lipase forms from Candida antarctica DSM 70725

Dimitrijević

Veličković

Jankov

et al. 2012

Hem Ind

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Yeast Candida antarctica produces two lipase forms, which are widely used as catalysts in variety of organic reactions, many of which are applied on a large scale. In this work, production of two forms of lipase from C. antarctica DSM 70725 (CAL A and CAL B) was monitored during seven days of cultivation in the optimal medium using different electrophoretic and zymographic techniques. According to electrophoresis after silver staining, C. antarctica lipase A (molecular mass 45 kDa) was produced starting from the second day of cultivation. C. antarctica lipase B (CAL B) was also produced starting from the second day, but protein was present in the fermentation broth predominantly as dimer (molecular weight 66 kDa), while presence of monomeric form of CAL B (molecular weight of 33 kDa) was observed starting from the fourth day of cultivation. Both types of zymograms (based on hydrolysis and synthesis reactions) were used for detection of lipase activity in the fermentation broth. C. antarctica lipase A showed activity only in hydrolytic zymogram, when α-naphtyl butyrate was used as substrate. In the same zymogram, with α-naphtyl acetate as substrate no CAL A activity was detected. Similarly, CAL A showed no activity in synthesis based zymograms towards oleic acid and octanol as substrates, indicating that CAL A is not active towards very short or long-chain substrates. As opposite of CAL A, both monomeric and dimeric form of CAL B were detected in the all zymograms, suggesting that CAL B is active towards wide range of substrates, regardless to the chain length. Thus, zymogram based on hydrolysis of α-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics

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Synthesis activity-based zymography for detection of lipases and esterases

Cited by 16 publications

References 19 publications

Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support

Electrophoretic and zymographic techniques for production monitoring of two lipase forms from Candida antarctica DSM 70725

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