Production, Characterization And Expression Of An Organic Solvent Tolerant Lipase From Pseudomonas Aeruginosa S5s5

Lipolytic bacterium was screened from five pure bacteria cultures available in Enzyme and Microbial Technology laboratory in UPM. The stock cultures were tested for lipase production. Two isolates (S5 and 205W) showed the highest activity in tripticase soy broth and brain heart infusions. These isolates were further incubated in different basal media. Isolate S5 was shown to give higher activity (0.327 Ulml) than isolate 205W in media MI and stable in various organic solvents tested. Therefore isolate S5 was chosen for further studies. Based on its morphological, biochemical characteristics and 16s rDNA sequence, strain S5 was identified as Pseudomonas aeruginosa. P. aeruginosa lipase exhibited the highest relative activity with n-hexane (410%) for 20 min reaction. Optimum lipase production was obtained at pH 7.0 and 37°C at static condition with peptone as the best nitrogen source and olive oil as the best carbon source. The best inoculum size was 6%. The surfactants, Tween 60 and Tween 80 were found to enhance for bacterial growth and lipase production by S5. The lipase was purified to homogeneity by affinity column chromatography and anion exchange column chromatography. The purified lipase was highly homogeneous as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and the molecular mass was estimated to be 60 kDa by SDS-PAGE and G-100 gel filtration column chromatography. The optimum temperature and pH of the purified enzyme was 45°C and pH 9.0, respectively. S5 lipase was stable at pH 6-9 for 30 min. The half-life of the S5 lipase at 45°C and 50°C was 2 h and 1 h, respectively. The lipase exhibited high stability in the presence of n-dodecane, 1-pentanol and toluene. As for metal ions, it was found that ca2+ stimulated lipase activity in 15 min incubation time, while EDTA had no effect on lipase activity. However, the S5 lipase was strongly inhibited by the addition of 1 mM phenyl methyl sulfonyl fluoride (PMSF) (87% inhibition) and 1 rnM of Pepstatin (76% inhibition) after 30 min incubation. The S5 lipase exhibited the highest activity in the presence of palm oil as a substrate and followed by coconut oil. S5 lipase was found to have the highest activity against triolein which possess longer carbon chain length. S5 lipase is a non-specific lipase as shown by triolein hydrolysis. The gene encoding for the intracellular lipase of P. aeruginosa strain S5 was isolated via genomic DNA library and cloned into pRSET. The cloned sequence included two open reading frames (OW) consisting of 1575 bp for the first ORF (ORF1) and 582 bp for the second ORF (ORF2). The OW2 was located at the downstream and function as the act gene for O w l . The conserved pentapeptide Gly- X- Ser- X-Gly was located in the ORF1. Catalytic triad resembling of that serine protease, consisting of serine, histidine, aspartic acid or glutarnic acid residues was present in this lipase gene. Expression in E.coli resulted a 100-fold increase in enzyme activity after 9 h induction with 0.75 mM IPTG. The recombinant plasmid revealed a size of 60 kDa on SDS-PAGE. The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h incubation at 37OC. Predicted 3D structure of S5 lipase revealed topological organization of a / @-hydrolase fold consisting of 10 a-Helices and 5 @-strands. The Ramachandran plot of S5 lipase showed that 85.8% (229) of residues lie in the most-favored region and only 2.2% (6) of residue lie in generously allowed regions and 1 residue lie in disallowed region

THE RED BUTTERFLY WING (CHRISTIA VESPERTILIONIS): A PROMISING CANCER CURE IN MALAYSIA

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Organic solvent tolerant lipases

The nature of the enzymes will be denatured in the presence of the organic solvents. Solvents not only affect enzyme stability but also change enzyme specificity. Most of the reports have been published concerning solvent stable enzymes. However, these enzymes are not naturally solvent stable enzymes that direct attempts from screening of organic solvent tolerant bacteria. In this chapter, we review the production and characterization of organic solvent tolerant lipases on the first attemps to screen organic solvent tolerant lipases from Bacillus sphaericus 205y, Bacillus sp. stain 42 and Pseudomonas sp. S5. All of these bacteria were proven to be organic solvent tolerant lipase producers. These lipases were stable in the organic solvents with log P value between 2.0 to 3.6. Organic solvent tolerant lipases from Pseudomonas sp. S5, B. sphaericus 205y, Bacillus sp. strain 42 were successfully purified to homogeneity. The optimum temperatures of these lipases were 45◦C, 55◦C and 70◦C, respectively. These purified lipases exhibited great stability and high activity in most of the organic solvents tested, therefore, these valuable criteria should not be neglected as important products in different fields could be developed from these lipases

Evaluation of potential molecular interaction between quorum sensing receptor, LuxP and grouper fatty acids: in-silico screening and simulation

Pathologically relevant behaviors of Vibrio, such as the expression of virulence factors, biofilm production, and swarming motility, have been shown to be controlled by quorum sensing. The autoinducer-2 quorum sensing receptor protein LuxP is one of the target proteins for drug development to suppress the virulence of Vibrio. Here, we reported the potential molecular interaction of fatty acids identified in vibriosis-resistant grouper with LuxP. Fatty acid, 4-oxodocosahexaenoic acid (4R8) showed significant binding affinity toward LuxP (−6.0 kcal/mol) based on molecular docking analysis. The dynamic behavior of the protein–ligand complex was illustrated by molecular dynamic simulations. The fluctuation of the protein backbone, the stability of ligand binding, and hydrogen bond interactions were assessed, suggesting 4R8 possesses potential interaction with LuxP, which was supported by the low binding free energy (−29.144 kJ/mol) calculated using the molecular mechanics Poisson–Boltzmann surface area

Effects of Derivatization on the Metabolite Profiling of the Cadmium-Tolerant Mangrove Fungus Trichoderma atroviride Using GC-MS Analysis

The mangrove fungus Trichoderma atroviride was found to be tolerant to the heavy metal cadmium and it is of high interest to profile its metabolites to gain insight into its response to cadmium toxicity. This study aimed to investigate the effect of derivatization agents on the number and types of metabolites present in the cadmium-tolerant T. atroviride, detected using GC-MS analysis. The intracellular and extracellular metabolites of T. atroviride treated with cadmium for ten days were derivatized using silylation and alkylation reactions. The results showed that a higher number of metabolites were identified when the three different derivatization agents were used: BSTFA, TBDMSTFA, and MCF. More types of metabolites were identified by silylation, making it suitable for non-targeted metabolites profiling study.  Silylation is efficient for the analysis of sugars and their derivatives while alkylation is suitable for a targeted study involving amino acids and organic acids. Statistical analysis for the data set of identified metabolites was performed using Metaboanalyst 3.0 followed by visualization using Partial Least Square-Discriminant Analysis. The plots showed clear separations of metabolites between the different types of derivatization agents and between control and cadmium-treated samples. A more comprehensive metabolite profile of T. atroviride obtained using different derivatization agents in this study, followed by distinct metabolites detected between control and treated samples, will provide good baseline information for future investigations including the pathways and biomarkers responsible for the fungal tolerance to cadmium toxicity

Uncaria gambir (W. Hunter) Roxb: From phytochemical composition to pharmacological importance

Purpose: To present an overview of the ethnopharmacology, phytochemistry, and pharmacological effects of the ‘wonder’ plant, Uncaria gambir (W. Hunter) Roxb.Methods: The literature search for information on phytochemical composition and pharmacological importance of U. gambir was undertaken using diverse electronic search engines, including Google, Scopus, Web of Science, scientific literature, and databases (Pubmed, Springer and Science Direct). Other relevant literature sources include books, book chapters, conference papers, theses, and other scientific publications.Results: Uncaria gambir Roxb possesses significant medicinal potentials as an antioxidant, anthelmintic, antibacterial, anti-diabetic, and for the management of osteoarthritis. Interest has increased among researchers for the utilization of this plant in complementary medicine, for example, to relieve sore throat, spongy gum, and dysentery, to treat atherosclerosis and obesity, and to prolong sexual intercourse.Conclusion: Uncaria gambir demonstrates significant pharmacological properties. This review will be useful for prospective research and development of this ethnomedicinal plant into potentially valuable health products. Keywords: Anthelmintic, Antibacterial,Anti-Diabetic, Osteoarthritis, Uncaria gambi

Homogeneous nature of Malaysian Marine fish Epinephelus fuscoguttatus (Perciformes; Serranidae): Evidence based on molecular markers, morphology and fourier transform infrared analysis

Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance–Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish

High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5

An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 °C and 9.0, respectively. The purified lipase was stable at 45 °C and pH 6–9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography