Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. ...vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings.
Background information
The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, ...therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.
Results
Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt‐interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab‐interactome using putative parasite Rab‐effectors identified by homology to Ypt‐effectors. We demonstrate its potential by wet‐bench testing three predictions; that casein kinase‐1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP‐dependent protein kinase A (PfPKA‐C) is a PfRab5A and PfRab7 effector.
Conclusions
The establishment of a shared set of physical Ypt/PfRab‐effector proteins sheds light on a core set Plasmodium Rab‐interactants shared with yeast. The PfRab‐interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA‐C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab‐recruited kinases potentially play a role in early and late endosome function in malaria parasites.
Rabs are important regulators of vesicular traffic via their recruitment of specific effectors. To identify Rab effectors for the human malaria parasite Plasmodium falciparum we made a yeast Ypt‐interactome and exploited this to build a parasite Rab‐interactome. The kinase PfCK1 was shown to be a specific PfRab5B effector, whereas the catalytic subunit of PfPKA‐C an effector of both PfRab5A and PfRab7. The recruitment of PfPKA to PfRab5A+ and PfRab7+ vesicles suggests that the cAMP‐dependent kinase potentially plays a role in early and late endosome function in malaria parasites.
Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477
U/mL). This lipase exhibited maximum stability in
n-hexane retaining about ...97.8% activity for 24
h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14
U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The
K
m and
V
max values for partially purified BMV2 lipase were found to be 1.56
mM and 5.62
μmoles/mg
min. The metal ions Ca
2+, Mg
2+ and Mn
2+ had stimulatory effect on lipase activity, whereas Cu
2+, Fe
2+ and Zn
2+ strongly inhibited the lipase activity. EDTA and PMSF at 10
mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.
In our study
Candida rugosa lipase coated with various surfactants viz. AOT, CTAB, and Tween 80 was immobilized in microemulsion based organogels (MBGs) and investigated for the production of ethyl ...isovalerate, a fruity flavor ester. Combinations of different surfactant coated lipase and the surfactant used for preparation of MBG were tried for esterification activity. Amongst various combinations used, CTAB coated lipase immobilized in AOT based MBGs exhibited maximum ethyl isovalerate synthesis (0.082
mol/L) in 9 days. The CTAB coated lipase immobilized in AOT based organogel showed eight-fold increase in the esterification activity as compared to the native lipase. We also found that, increase in the concentration of the CTAB led to the decrease in the catalytic activity of lipase immobilized in AOT based organogels. The AOT based organogels containing CTAB coated lipase could be reused five times, without significant loss in esterification activity. However, after fifth cycle the esterification activity decreased to 40% of the initial activity. The activation energy for catalytic activity of the untreated and surfactant coated lipase was found to be 29.8 and 9.15
kJ, respectively, which depicts some structural change in surfactant coated lipase. Thus, the surfactant coated lipase immobilized in MBGs exhibited significantly higher stability and activity in comparison to uncoated lipase immobilized in MBGs. This strategy seems to hold a good promise for the synthetic flavor industry.
In our previous study, a surfactant-coated Candida rugosa lipase immobilized in microemulsion-based organogels was exploited for the synthesis of ethyl isovalerate. In the present study, we are ...focusing on the effective reuse of lipase immobilized in microemulsion-based organogels (MBGs) in terms of retainment of the catalytic activity. As water is one of the co-products in esterification reactions, the removal of water becomes a priority to allow the reaction to work in the forward direction and to prevent back hydrolysis. Taking this fact into consideration, the lipase-containing microemulsion-based organogels were given pretreatment and/or several intermittent treatments with dry reverse micellar solution of AOT in organic solvent during repeated cycles of ester synthesis. The pretreated MBGs with dry reverse micellar solution exhibited lower water content and higher initial rates of esterification in comparison with untreated freshly prepared MBGs. The esterification efficiency of untreated MBGs started decreasing after 5 cycles of reuse and was almost completely lost by the end of the 8∨th cycle. In contrast, pretreated MBGs exhibited a gradual decrease in esterification efficiency after 5 cycles and retained about 80% of the initial activity at the end of the 8∨th cycle. The intermittent treatment of MBGs after every 3 cycles resulted in enhanced reusability of immobilized lipase for up to 9 cycles without significant loss in esterification activity, after which it resulted in a slow decrease in activity with about 27% lower activity at the end of the 12∨th cycle. Furthermore, the treatment conditions such as concentration of AOT in liquid dessicant and time of treatment were optimized with respect to our system. The granulated MBGs proved to be better in terms of initial esterification rates (1.2-fold) as compared with the pelleted MBGs.
Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100
±
20
nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent ...for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40
°C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The
V
max and
K
m of immobilized lipase at 25
°C for pNPP hydrolysis were found to be 212
μmol/min/mg and 0.3
mM, whereas those for free lipase were 26.17
μmol/min and 1.427
mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.
Abstract
Magnetic nanoparticles were synthesized by co-precipitation under hydrothermal conditions. The average diameter of the magnetic nanoparticles was found to be in the range of 15 ± 5 nm with ...an average surface area of 112.15 m2 g−1. Immobilization of lipase on magnetite nanoparticles was confirmed by FTIR, differential scanning calorimetry and thermal gravimetric analysis. The activation energy of the free enzyme was 1.9-fold higher than that of the immobilized lipase for hydrolytic reactions. Additionally, the lower KM and higher Vmax values of the immobilized enzyme for hydrolysis of 4-nitrophenyl palmitate indicated an increased efficiency of the immobilized lipase. The immobilized lipase exhibited higher esterification efficiency compared with free lipase for synthesis of ethyl isovalerate. It also exhibited fairly good reusability, with about 8.5% reduction in esterification efficiency for ethyl isovalerate synthesis over ten cycles of reuse.
Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent ...for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25-40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The Vmax and Km of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 is a subset of mol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 is a subset of mol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.
In our previous study, a surfactant-coated Candida rugosa lipase immobilized in microemulsion-based organogels was exploited for the synthesis of ethyl isovalerate. In the present study, we are ...focusing on the effective reuse of lipase immobilized in microemulsion-based organogels (MBGs) in terms of retainment of the catalytic activity. As water is one of the co-products in esterification reactions, the removal of water becomes a priority to allow the reaction to work in the forward direction and to prevent back hydrolysis. Taking this fact into consideration, the lipase-containing microemulsion-based organogels were given pretreatment and/or several intermittent treatments with dry reverse micellar solution of AOT in organic solvent during repeated cycles of ester synthesis. The pretreated MBGs with dry reverse micellar solution exhibited lower water content and higher initial rates of esterification in comparison with untreated freshly prepared MBGs. The esterification efficiency of untreated MBGs started decreasing after 5 cycles of reuse and was almost completely lost by the end of the $8^{th}$ cycle. In contrast, pretreated MBGs exhibited a gradual decrease in esterification efficiency after 5 cycles and retained about 80% of the initial activity at the end of the $8^{th}$ cycle. The intermittent treatment of MBGs after every 3 cycles resulted in enhanced reusability of immobilized lipase for up to 9 cycles without significant loss in esterification activity, after which it resulted in a slow decrease in activity with about 27% lower activity at the end of the $12^{th}$ cycle. Furthermore, the treatment conditions such as concentration of AOT in liquid dessicant and time of treatment were optimized with respect to our system. The granulated MBGs proved to be better in terms of initial esterification rates (1.2-fold) as compared with the pelleted MBGs.
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