Screening, Identification, and optimization of extracellular lipase production of yeast (Cryptococcus flavescens) isolated from a tree canopy fern in Mount Makiling Forest Reserve, Philippines

Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes vastly used in industrial applications. The current study aims to screen lipase-producing yeasts isolated from a tree canopy fern from the Makiling Forest Reserve (MFR), Philippines and to optimize conditions that can maximize the mass production and activity of the enzyme. From the 144 isolates, B1-7 showed the highest lipase activity in both solid (EIA 7.6) and liquid selection media (0.082 U/mL- min). Molecular identification using Internally Transcribed Spacer (ITS) primers and microscopic observation revealed that the isolate was Cryptococcus flavescens, a generally regarded as safe (GRAS) microorganism. Response Surface Method (Box-Behnken Design) showed that the maximum lipase activity (0.66 U/mL-min) and a biomass of 4 g/L were achieved at 5.0 Carbon:Nitrogen ratio, pH 6.0 and 0.5% inducer (Tween 20). Also, C:N-% inducer interaction and inducer concentration significantly affected lipase activity. After a 72h fed-batch fermentation experiment, lipase activity was ten- fold lower than the optimization results and a negative correlation (r=-0.405) between lipase activity and biomass suggested the non-dependence of lipase activity to biomass availability. Lastly, total sugar concentration remained constant implying that the organism used the degradative products of lipase as its carbon source. In conclusion, C. flavescens from MFR can be utilized for mass lipase production, but it was recommended that other parameters be examined and optimized

Screening, identification and optimization of lipase production by a yeast isolated from a tree canopy fern in Mt. Makiling Forest Reserve, Philippines

Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes generally used in several industrial applications. This study aimed to screen lipase-producing yeast isolates obtained from Makiling Forest Reserve (MFR), Philippines and to optimize conditions required for the mass production of the enzyme. From the 144 isolates, B1-7 showed the highest lipase activity in both solid and liquid selection media (7.6 EAI and 0.082 U/mL · min activity, respectively). Molecular identification through ITS amplification and phenotypic characterization revealed the putative identity of isolate as Cryptococcus flavescens. Response Surface Method (Box- Behnken Design), suggested that the optimum conditions for maximum lipase production in liquid medium (0.66 U/mL · min) were 5.0 Carbon to Nitrogen ratio (C:N), pH 6.0 and 0.5% inducer. Lipase activity was shown to be significantly affected by the interaction between C: N and inducer (p = 0.010) and inducer alone (p = 0.040). After a 72h fed-batch fermentation experiment, the obtained lipase activity was 0.10 U/mL · min, which was 6-fold lower than expected yield indicating other factors (e.g. oxygen saturation) affecting lipase production. Lipase activity and biomass were also found to be negatively correlated (r = -0.405) suggesting that lipase production did not depend on the available cell population. Lastly, total sugar concentration remained constant implying that the degradative products of lipase were used as the carbon source for cell growth. In conclusion, C. flavescens from MFR with further production optimization can be utilized for mass lipase production