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Gene Expression, Biochemical Characterization of a sn-1, 3 Extracellular Lipase From Aspergillus niger GZUF36 and Its Model-Structure Analysis.

ABSTRACT: 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.

SUBMITTER: Xing S

PROVIDER: S-EPMC7994357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Gene Expression, Biochemical Characterization of a sn-1, 3 Extracellular Lipase From Aspergillus niger GZUF36 and Its Model-Structure Analysis.

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