Purification and biochemical characterization of an extracellular fructosyltransferase enzyme from Aspergillus niger sp. XOBP48: implication in fructooligosaccharide production.
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ABSTRACT:
SUBMITTER: Ojwach J
PROVIDER: S-EPMC7527383 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This study focused on the purification and characterization of an extracellular ?-d-fructofuranosidase or invertase from Aspergillus sojae JU12. The protein was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery. The apparent molecular mass of the enzyme was estimated to be ~ 35 kDa using SDS-PAGE and confirmed by deconvoluted mass spectrometry. The fungal ?-d-fructofuranosidase was suggested to be a monomer by native PAGE and zymography, and was found to be a glycoprotein possessing 68.92% carbohydrate content. The products of enzyme hydrolysis were detected by thin layer chromatography and revealed the monosaccharide units, d-glucose and d-fructose. ?-d-fructofuranosidase showed enhanced activity at broad pH 4.0-9.0 and activity at a temperature range from 30 to 70 °C, while the enzyme was stable at pH 8.0 and 40 °C, respectively. The ?-d-fructofuranosidase activity was lowered by metal ion inhibitors Ag2+ and Hg2+ whereas elevated by SDS and ?-ME. The fungal ?-d-fructofuranosidase was capable of hydrolyzing d-sucrose and the kinetics were determined by Lineweaver-Burk plot with Km of 10.17 mM and Vmax of 0.7801 µmol min-1. Additionally, the extracellular ?-d-fructofuranosidase demonstrated tolerance to high ethanol concentrations indicating its applicability in the production of alcoholic fermentation processes.
| S-EPMC5794676 | biostudies-literature
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.
| S-EPMC7350912 | biostudies-literature
Project description: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.
| S-EPMC7270473 | biostudies-literature
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.
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Project description:α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.
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Project description:Two new aspartic proteases, PepAb and PepAc (encoded by pepAb and pepAc), were heterologously expressed and biochemically characterized from Aspergillus niger F0215. They possessed a typical structure of pepsin-type aspartic protease with the conserved active residues D (84, 115), Y (131, 168) and D (281, 326), while their identity in amino acid sequences was only 19.0%. PepAb had maximum activity at pH 2.5 and 50 °C and PepAc at 3.0 and 50 °C. The specific activities of PepAb and PepAc toward casein were 1368.1 and 2081.4 U/mg, respectively. Their activities were significantly promoted by Cu2+ and Mn2+ and completely inhibited by pepstatin. PepAb exhibited higher catalytic efficiency (k cat/K m) toward soy protein isolates than casein, while PepAc showed higher catalytic efficiency toward casein. The hydrolysis capacities of PepAb and PepAc on soy protein isolates were slightly lower than that of previously identified A. niger aspartic protease, PepA (aspergillopepsin I), while the resultant peptide profiles were remarkably different for all three proteases.
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Project description:Fructosyltransferases have been used in the industrial production of fructooligosaccharides (FOS). However, it is still not possible to explain the difference in FOS production based on the variations observed in the FOS synthesizing enzymes of A. niger. In the present study, a novel fructosyltransferase (FT-A) from A. niger TCCC41686 with high FOS synthesis ability was characterized. The FT-A gene was obtained and expressed in Pichia pastoris. A homology model of FT-A showed that the changes of residues identified outside the conserved domains were found to have an effect on its characteristics and its FOS-synthesis capacity. The optimal activity of the recombinant FT-A was observed at 50 °C and pH 6.0, and it was stable below 50 °C and over the range of pH 3.0-11.0. The K m and V max values of FT-A were 151.13 g L-1 and 6.55 g L-1 min-1, respectively. The production of FOS using the recombinant FT-A remained above 60% during 50-80 min of synthesis based on sucrose as substrate. The novel fructosyltransferase (FT-A) investigated in this study can potentially be applied for the efficient industrial production of FOS. The results also provide more valuable information for explaining the relationship between the structure and function of the FT-A.
| S-EPMC9069702 | biostudies-literature
Project description:A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.
| S-EPMC1164865 | biostudies-other
Project description:Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).
| S-EPMC7767288 | biostudies-literature
Project description:In this study, a sn-1, 3 extracellular lipases from Aspergillus niger GZUF36 (PEXANL1) was expressed in Pichia pastoris, characterized, and the predicted structural model was analyzed. The optimized culture conditions of P. pastoris showed that the highest lipase activity of 66.5 ± 1.4 U/mL (P < 0.05) could be attained with 1% methanol and 96 h induction time. The purified PEXANL1 exhibited the highest activity at pH 4.0 and 40°C temperature, and its original activity remained unaltered in the majority of the organic solvents (20% v/v concentration). Triton X-100, Tween 20, Tween 80, and SDS at a concentration of 0.01% (w/v) enhanced, and all the metal ions tested inhibited activity of purified PEXANL. The results of ultrasound-assisted PEXANL1 catalyzed synthesis of 1,3-diaglycerides showed that the content of 1,3-diglycerides was rapidly increased to 36.90% with 25 min of ultrasound duration (P < 0.05) and later decreased to 19.93% with 35 min of ultrasound duration. The modeled structure of PEXANL1 by comparative modeling showed α/β hydrolase fold. Structural superposition and molecular docking results validated that Ser162, His274, and Asp217 residues of PEXANL1 were involved in the catalysis. Small-angle X-ray scattering analysis indicated the monomer properties of PEXANL1 in solution. The ab initio model of PEXANL1 overlapped with its modeling structure. This work presents a reliable structural model of A. niger lipase based on homology modeling and small-angle X-ray scattering. Besides, the data from this study will benefit the rational design of suitable crystalline lipase variants in the future.
| S-EPMC7994357 | biostudies-literature