Thermostable lipase from Pichia sp. strain RT: Identification, production and characterization

Lipases are hydrolytic enzymes that have been widely used in industries. In order to meet the industrial demands, thermostable lipases which can withstand high temperatures are required. We sought to optimize the lipase production and to characterize the crude lipase from a locally isolated yeast. Yeast from rotten tomato (from Selangor, Malaysia) which has been identified Pichia sp. strain RT and was found to have thermostable lipase. The morphological features of the isolate were analyzed using SEM and TEM. The microscopy examination confirmed that strain RT was a yeast species. Amplification of lipase gene from cDNA library of the Pichia sp. strain RT showed the gene possessed 100% similarity to the hypothetical protein of Meyerozyma guilliermondii ATCC6260. In addition, the yeast achieved the maximum lipase activity at 30°C with 200 rpm after 72 hours of cultivation. The lipase has the optimum temperature at 75°C and retained nearly 50% of residual activity after 30 minutes of pre-incubation at 75°C. The optimal pH for RT lipase was at pH 9 and it highly stable within pH range 7–10. Physical parameters and the characteristics of crude lipase in strain RT have been successfully investigated. This finding has given an insight for the use of newly isolated thermostable lipase for industrial applications.

Heterologous Expression and Characterization of Thermostable Lipases from Geobacillus thermoleovorans PPD2 through Escherichia coli

ABSTRACT: Heterologous expression and purification of thermostable lipase from Geobacillus thermoleovorans PPD2 had been carried out through Escherichia coli BL21 as host. Two bands obtained showed lipolytic activity with the size at around 51 (LipA) and 43 (LipB) kDa, respectively. The activities were identified by zymogram analysis, while the control protein from Escherichia coli BL21(DE3) do not show any lipolytic activity. Purification of crude extract using chromatography affinity Ni-NTA resulted one dominant band of LipA, meanwhile LipB did not appeared on the gel. Another purification for LipB was carried out by acetone fractionation. Both of LipA and LipB showed high activity toward medium chain length substrates, with optimum activity at 50oC and pH 8.5. The activities of LipA and LipB showed tolerance toward short chain alcohols, such as methanol, ethanol, n-propanol, and isopropanol.

Geometric Simulation Approach for Grading and Assessing the Thermostability of CALBs

Candida antarcticalipase B (CALB) is a known stable and highly active enzyme used widely in biodiesel synthesis. In this work, the stability of native (4K6G) and mutant (4K5Q) CALB was studied through various structural parameters using conformational sampling approach. The contours of polar surface area and surface area of mutant CALB were 11357.67 Å2and 30007.4 Å2, respectively, showing an enhanced stability compared to native CALB with a statistically significantPvalue of < 0.0001. Moreover, simulated thermal denaturation of CALB, a process involving dilution of hydrogen bond, significantly shielded against different intervals of energy application in mutant CALB revealing its augmentation of structural rigidity against native CALB. Finally, computational docking analysis showed an increase in the binding affinity of CALB and its substrate (triglyceride) in mutant CALB with Atomic Contact Energy (ACE) of −91.23 kcal/mol compared to native CALB (ACE of −70.3 kcal/mol). The computational observations proposed that the use of mutant CALB (4K5Q) could serve as a best template for production of biodiesel in the future. Additionally, it can also be used as a template to identify efficient thermostable lipases through further mutations.