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Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

ABSTRACT: There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125 mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075 M NaCl, 10% n-hexanol and 30 min, respectively. Under these conditions, the forward extraction rate reached 90.3%?±?3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5 M KCl, 6.5, 60 min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7 kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC-MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.

SUBMITTER: Chen C

PROVIDER: S-EPMC6542905 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from <i>Aspergillus niger</i> GZUF36.

Chen Cuicui C Tian Hua H Xing Shuqi S Li Cuiqin C Zeng Xuefeng X He Laping L

Journal of food science and technology 20190506 6

There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel <i>Aspergillus niger</i> GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, ...[more]

PMID: 31205345

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Project description:BackgroundExtracellular production of fungal lipases especially the lipases obtained from the Aspergilli has gained immense interest in recent years due to its diverse biotechnological applications. In this study, we focused on determining the fermentation parameters required for the optimal lipase production.MethodsA total of 256 fungal isolates were obtained from oil seeds. From each genus, one isolate was selected to evaluate lipase production using phenol red and tributyrin plate assays. Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. The highest lipase producer isolates were identified using 18S ribosomal RNA gene sequencing. The genetic variability was determined by random amplified polymorphic DNA (RAPD) analysis and the dendrogram was constructed using the unweighted pair group method with arithmetic averages method. The isolates were examined in a submerged fermentation culture (Smf) to measure the effect of temperature, pH, incubation time, carbon source, nitrogen source, inoculum volume, and lipid source on lipase production.ResultsEleven isolates belonging to the genus Aspergillus were analyzed for lipase production where they were found to be the highest lipase producers among various fungal genera. All the tested isolates were identified as A. niger using 18s rRNA sequencing. Genetic diversity was evaluated among all of the studied A. niger isolates using RAPD primers. The RAPD primers were used to amplify 285 loci, of which five were polymorphic (1.75%) and seven were monomorphic (2.45%). Thus, a high level of genetic diversity was observed among all isolates. The tributyrin test and the lipase activity assay identified five strains of A. niger as high lipase producers, and their optimal enzyme activities were 709.74, 532.54, 735.64, 794.62, and 787.69 U/ml. The optimal conditions for lipase production were as follows: 40 °C, pH 7.5, 1% fructose as the carbon source, 1% yeast extract as the nitrogen source, 2% palm oil, 2.5 × 107 spores/ml suspension, and 3 days of incubation.ConclusionsThe current study provides a comprehensive characterization of the optimal conditions, which are essential to enhance lipase production in five A. niger isolates.

Dataset Information

Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

Publications

Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from <i>Aspergillus niger</i> GZUF36.

Similar Datasets

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