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.

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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.