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

Abstract

Sn-1,3 extracellular GZUF36 lipase (EXANL1) has wide application potential in the food industry. However, the 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 BL21 (DE3) cells using pET-28a as the expression vector. The temperature-induced conditions were optimized, and we successfully achieved its active expression in . These conditions significantly influenced the active expression of EXANL1 ( < 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 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.