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Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

ABSTRACT: Sn-1,3 extracellular Aspergillus niger GZUF36 lipase (EXANL1) has wide application potential in the food industry. However, the A. niger strain has defects such as easy degradation and instability in the expression of sn-1,3 lipase. To obtain a stable expression of this lipase and its subsequent enzymatic properties, the gene encoding EXANL1 was cloned and expressed in Escherichia coli BL21 (DE3) cells using pET-28a as the expression vector. The temperature-induced conditions were optimized, and we successfully achieved its active expression in E. coli. These conditions significantly influenced the active expression of EXANL1 (P < 0.05), and the highest enzyme activity of the supernatant of lysis cells expressed at 20 °C was at 7.02 ± 0.05 U/mL. The expressed recombinant EXANL1 was purified using Ni-NTA, showing an estimated relative molecular mass of 35 kDa. The recombinant EXANL1 exhibited maximum activity at 35 °C and pH 4.0, with a wide acid pH range. Thin-layer chromatography analysis showed that the enzyme displayed sn-1,3 positional selectivity toward triolein. The recombinant EXANL1 could maintain its relative activities (> 80%) after 24 h of incubation at pH 3-10, suggesting its suitability for a wide range of industrial applications. After comparing these properties with those of the other A. niger lipases, we found that some key amino acids may play a decisive role in enzymology. This work laid a foundation for the stable expression of the EXANL1 gene and its potential industrial application.

SUBMITTER: Xing S

PROVIDER: S-EPMC7270473 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn???) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp???-Val??? peptide was shortened to Asp???-Arg??? in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (?86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

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.

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Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

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