Rigorous kinetic model considering positional specificity of lipase for enzymatic stepwise hydrolysis of triolein in biphasic oil-water system.

A rigorous kinetic model describing the stepwise triglyceride hydrolysis at the oil-water interface, based on the Ping Pong Bi Bi mechanism using suspended lipase having positional specificity, was constructed. The preference of the enzyme to cleave to the ester bonds at the edge and the center of the glycerol backbone of the substrates (tri-, di- or monoglyceride) was incorporated in the model. This model was applied to the experimental results for triolein hydrolysis using suspended Porcine pancreatic lipase (an sn-1,3 specific lipase) and Candida rugosa lipase (a non-specific lipase) in a biphasic oil-water system under various operating conditions. In order to discuss the model's advantages, other models that do not consider the positional specificity of the lipase were also applied to our experimental results. The model considering the positional specificity of the lipase gave results which fit better with the experimental data and described the effect of the initial enzyme concentration, the interfacial area, and the initial concentrations of triolein on the entire process of the stepwise triolein hydrolysis. This model also gives a good representation of the rate for cleaving the respective ester bonds of each substrate by each type of lipase.

Antiretroviral activity of Pterois volitans (red lionfish) venom in the early development of human immunodeficiency virus/acquired immunodeficiency syndrome antiretroviral alternative source.

AIM: This study aimed to investigate the antiviral activity of Pterois volitans phospholipase A2 (PV-PLA2) from Indonesia to human immunodeficiency virus (HIV). MATERIALS AND METHODS: Fresh venomous fin parts of wild PV specimens were collected from Java Sea waters. Then, it washed using phosphate buffer pH 7.0 and immersed in phosphate buffer pH 7.0 0.01 m containing CaCl2 0.001 m for 24 h. The immersed fin then allowed for extraction process by sonicating for 2×8 min with 80% pulse and 20 kHz output with temperature controlling to avoid denaturation. The crude venom (CV) extracted from the fin is allowed for purification by 80% ethanol (ET) precipitation and ammonium sulfate fractionation method. The purified PV-PLA2 then analyzed using Lowry's method, Marinette's method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide assay. After determining the purest and safest sample of six samples analyzed, the chosen sample then tested into simian retrovirus-2 (SRV2)-A549 culture (48×104 cells/mL at 1-4 ppm), and compared to the CV sample (1-4 ppm) and lamivudine (100 ppm). The culture then is analyzed using a quantitative real time-polymerase chain reaction to find out the copy number of SRV-2 virus in each culture. RESULTS: The protein's activity, concentration, and purity analysis revealed that the PV-PLA2 purified using ammonium sulfate fractionation has the highest activity (1.81 times higher than the CV at 80% fractionation) and has higher purity than the sample from ET fractionation. The testing of the sample purified using ammonium sulfate fractionation at 80% saturation level shown that it has a 97.78% inhibition level toward SRV2-A549 culture at 4 ppm. However, in comparison to lamivudine which has 99.55% inhibition level at 100 ppm, it needs much lower concentration to achieve the same result. CONCLUSION: The significant inhibition of SRV2-A549 culture shown that the PV-PLA2 extracted from PV venom has the potential to become anti-HIV substances. It would be worthwhile to further evaluate the antiretroviral activity of PV-PLA2 in the in vivo studies.

Antiviral activity of Acanthaster planci phospholipase A2 against human immunodeficiency virus.

AIM: Investigation of antiviral activity of Acanthaster planci phospholipase A2 (AP-PLA2) from moluccas to human immunodeficiency virus (HIV). MATERIALS AND METHODS: Crude venom (CV) and F20 (PLA2 with 20% fractioned by ammonium sulfate) as a sample of PLA 2 obtained from A. planci's extract were used. Enzymatic activity of PLA2 was determined using the degradation of phosphatidylcholine (PC). Activity test was performed using in vitro method using coculture of phytohemagglutinin-stimulated peripheral blood mononuclear cell (PBMC) from a blood donor and PBMC from HIV patient. Toxicity test of AP-PLA2 was done using lethal concentration required to kill 50% of the population (LC50). RESULTS: AP-PLA2 F20 had activity and purity by 15.66 times bigger than CV. The test showed that the LC50 of AP-PLA2 is 1.638 mg/ml. Antiviral analysis of AP-PLA2 in vitro showed the inhibition of HIV infection to PBMC. HIV culture with AP-PLA2 and without AP-PLA2 has shown the number of infected PBMC (0.299±0.212% and 9.718±0.802%). Subsequently, RNA amplification of HIV using reverse transcriptase-polymerase chain reaction resulted in the decrease of band intensity in gag gene of HIV. CONCLUSION: This research suggests that AP-PLA2 has the potential to develop as an antiviral agent because in vitro experiment showed its ability to decrease HIV infection in PBMC and the number of HIV ribonucleic acid in culture.