In industrial application, immobilized lipase are typically not reused and served as industrial waste after a certain process is completed. The capacity on the reusability of the spent lipase is not well studied. This current study embarks on reusing the remaining lipase from the spent immobilized enzyme. Active lipases were recovered using a simple reverse micellar extraction (RME). RME is the extraction process of targeted biomolecules using an organic solvent and a surfactant. This method was the first attempt reported on the recovery of the lipase from the used immobilized lipase. RME of the spent lipase was done using the nonionic Triton X-100 surfactant and toluene. Various parameters were optimized to maximize the lipase recovery from the used immobilized lipase. The optimum forward extraction condition was 0.075 M KCl, and backward conditions were at 0.15 M Triton X-100/toluene (pH 6, 2 M KCl) with recovery of 66%. The extracted lipase was immobilized via simple adsorption into the ethanol pretreated carrier. The optimum conditions of immobilization resulted in 96% of the extracted lipase was reimmobilized. The reimmobilized lipase was incubated for 20 h in pH 6 buffer at 50 °C of water bath shaker. The reimmobilized lipase still had 27% residual activity after 18 h of incubation, which higher thermal stability compared to the free lipase. In conclusion, the free lipase was successfully extracted from the spent immobilized lipase and reimmobilized into the new support. It exhibited high thermal stability, and the reusability of the spent lipase will promote continued use of industrial lipase and reduce the cost of the manufacturing process.
The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for non-scavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease.
Microbial arsenite oxidation is an essential biogeochemical process whereby more toxic arsenite is oxidized to the less toxic arsenate. Thiomonas strains represent an important arsenite oxidizer found ubiquitous in acid mine drainage. In the present study, the arsenite oxidase gene (aioBA) was cloned from Thiomonas delicata DSM 16361, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant Aio consisted of two subunits with the respective molecular weights of 91 and 21 kDa according to SDS-PAGE. Aio catalysis was optimum at pH 5.5 and 50-55 °C. Aio exhibited stability under acidic conditions (pH 2.5-6). The V max and K m values of the enzyme were found to be 4 µmol min(-1) mg(-1) and 14.2 µM, respectively. SDS and Triton X-100 were found to inhibit the enzyme activity. The homology model of Aio showed correlation with the acidophilic adaptation of the enzyme. This is the first characterization studies of Aio from a species belonging to the Thiomonas genus. The arsenite oxidase was found to be among the acid-tolerant Aio reported to date and has the potential to be used for biosensor and bioremediation applications in acidic environments.
Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30⁻60 °C and pH range of 6⁻11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na⁺, Mg2+, K⁺ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.
In this study, a Lactic acid bacteria (LAB) strain was isolated on MRS medium from gastro-intestinal tissues of Broadhead catfish (Clarias macrocephalus). Out of 50 isolates, 25 isolates were found to be positive on lactose utilisation test and were identified to be gram positive cocci. Using disc diffusion methods, one out of 22 isolates, i.e., a strain A5 demonstrated inhibitions against three indicator organisms; Bacillus cereus, Staphylococcus aureus and Salmonella thyphimurium. Partial 16S rDNA sequencing identified isolate A5 as a member of Lactococcus lactis, with 100% DNA homology. Cell free supernatant fluid from Lactococcus lactis A5 showed inhibitory activities against both gram positive pathogens (Bacillus cereus and Staphylococcus aureus) and gram negative pathogens (Salmonella thyphimurium). Chloroform precipitated bacteriocin retained antagonistic activities in the presence of catalase and lysozyme; and was completely inactivated by Proteinase K treatment. The bacteriocin has a molecular weight of 3.4 kDa, based on SDS-PAGE analysis and the extract was heat stable at 37°C and 65°C, for 15 minutes. The antibacterial activity was suppressed with the addition of EDTA but was significantly increased with the addition of SDS, Triton X-100, Tween 20 and Tween 80. This bacteriocin belongs to class 1 bacteriocin, which was shown to have a nisin-like properties. This strain can be used as potential probiotics in animal or aquaculture feeding; and the bacteriocin it produces will be useful in food preservative.
Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2) < pH < DTT < Triton X-100.