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Lipase from Thermomyces lanuginosus: From production by solid-state fermentation to isolation

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) catalyze a broad range of reactions based on eco friendly techniques in various industrial fields such as biodiesel production, surfactants and ester synthesis, racemic resolution, wastewater bio-treatment, etc. Due to their properties, lipase presents a great alternative to the harsh conditions linked with an application of the chemicals. Lipase from Thermomyces lanuginosus (TLL) represents the basophilic single-chain protein with potential application in different areas due to temperature tolerance, great pH range stability, and different substrates specificity. Production of the TLL based on the fermentation process in solid-state conditions due to industrial-waste utilization presents a great potential in bio-economy. Solid-state fermentation (SSF) was conducted in laboratory jars with pumpkin seed pomace as the substrate. Inoculation of the substrate was performed with a defined number of mycelium discs (Ø = 1 cm) of T. lanuginosus resuspended in the distilled and sterile water. The effect of substrate content (ms = 30 – 70 g), initial concentration of biomass (nmycelium discs = 3 – 7), moisture content (wH2O = 50 – 70 %), duration (t = 2 – 4 days), and temperature (T = 35 – 45 °C) on TLL production were performed according to the Box-Behnken three- level experimental plan with six replications for the central points. Optimization of the lipase production was achieved using response surface methodology (RSM). Numerical optimization was applied to determinate optimal conditions for lipase production which were as follow: ms = 50 g ; nmycelium discs = 7 ; wH2O = 60 % ; t = 2 days ; T = 45 °C. Validation of the model was performed according to the optimal conditions for SSF and experimental result (15.769 U mL-3) was in good correlation with model prediction (15.862 U mL-3). After SSF optimization, next step in process development was lipase purification. In our previous research, partial purification of lipase produced from T. lanuginosus by SSF lead to a 17.03 fold increase in a specific activity, with a recovery of 1% achieved after gel chromatography [1]. Obtained results clearly indicate necessity of additional optimization of TLL isolation processes in order to achieve higher lipase activity. Therefore, all isolation processes, namely precipitation, dialysis and gel chromatography were optimized which resulted in an increase of TLL specific activity. After isolation, effects of various metal ions, carbon and nitrogen sources on activity of purified TLL were determined. As a final step, temperature and pH optimum as well as storage stability of TLL were determined.

lipase ; Thermomyces lanuginosus ; solid-state fermentation ; lipase purification

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