Molecular Cloning,Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from <Emphasis Type="Italic">Sargassum binderi</Emphasis> (Phaeophyta)

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C₆–C₁₄) to produce tri- and tetraketide pyrones. Mutations at H³³¹ and N³⁶⁴ caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His²²⁷ and Leu³⁶⁶ play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.     

Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen

Somatic embryogenesis in mangosteen (Garcinia mangstana L.) was investigated using seed and leaf segments cultured on Murashige and Skoog medium with treatments of 6-benzyladenine (BA) [2.0, 3.0, 4.0 µM] and 2,4-diclorophenoxyacetic acid (2,4-D) [4.5, 9.0, 13.5 µM]. There were four types of structures (globular, nodular compact, friable and spongy) formed. Two treatments resulted in embryogenic characteristics from seed cultures; the highest percentage 46.67?% of globular structure (resembling somatic embryos) grown on 3.0 µM BA and 80?% of nodular compact structures on 4.0 µM BA?+?13.5 µM 2,4-D. For the leaf culture, highest percentage, 93.33?% produced nodular compact structures on 2.0 µM BA?+?4.5 µM 2,4-D. Histological analysis showed that the globular structure has well-defined protoderm and separated from the original explant. Nodular compact structure also showed the presence of densely cytoplasmic meristematic cells with a high nucleoplasmic ratio. These characteristics observed in globular and nodular compact structure indicates somatic embryo formation. The globular structures which were converted into shoots and roots (60.00?%) showed atypical somatic embryogenesis in mangosteen. Metabolite fingerprinting was carried out using gas chromatography–mass spectrometry. Amino acids, carbohydrates, organic acids and fatty acids were found in both the embryogenic structures and non-embryogenic structures tested. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P?≤?0.05) for both types of structures. Principle component analysis suggested that amino acids and carbohydrates were the major compounds distinguishing embryogenic and non-embryogenic structures. Ornithine and mannose were present at significant level in embryogenic structures as compared to non-embryogenic ones while fructose was significantly higher in non-embryogenic structures.     

Reliability Measurement for Mixed Mode Failures of 33/11 Kilovolt Electric Power Distribution Stations

The reliability of the electrical distribution system is a contemporary research field due to diverse applications of electricity in everyday life and diverse industries. However a few research papers exist in literature. This paper proposes a methodology for assessing the reliability of 33/11 Kilovolt high-power stations based on average time between failures. The objective of this paper is to find the optimal fit for the failure data via time between failures. We determine the parameter estimation for all components of the station. We also estimate the reliability value of each component and the reliability value of the system as a whole. The best fitting distribution for the time between failures is a three parameter Dagum distribution with a scale parameter and shape parameters and . Our analysis reveals that the reliability value decreased by 38.2% in each 30 days. We believe that the current paper is the first to address this issue and its analysis. Thus, the results obtained in this research reflect its originality. We also suggest the practicality of using these results for power systems for both the maintenance of power systems models and preventive maintenance models.     

Transcriptome-wide effects of expansin gene manipulation in etiolated Arabidopsis seedling

Journal of Plant Research - Expansin is a non-enzymatic protein which plays a pivotal role in cell wall loosening by inducing stress relaxation and extension in the plant cell wall. Previous...     

S5 Lipase: an organic solvent tolerant enzyme

In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

GC-MS based metabolomics analysis reveals the effects of different agitation speeds on the level of proteinogenic amino acids in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> MG1363

Lactococcus lactis subsp. cremoris MG1363 is an opportunistic lactic acid bacterium (LAB) that has emerged as one of the most promising candidate cell factories. The availability of genome-level information and U.S. Federal Drug administration’s designation of ‘generally recognized as safe’ (GRAS) are two of the more important key factors for its wide-ranging applications in numerous biotechnological processes. Several studies have shown that various physiological conditions, such as temperature, salinity and pH, can influence the physiological growth of L. lactis; agitation, in particular, can increase the production of amino acids and fermentation by-products. However, the effect of different agitation speeds on the growth of L. lactis’ has rarely been examined. In the study reported here, we used a gas chromatography–mass spectrometry-based metabolomics approach to investigate the effects of different agitation speeds on the production of proteinogenic amino acids (PAAs) by L. lactis MG1363. Lactococcus lactis MG1363 was grown under four different agitation speeds (50, 100, 150 and 200 rpm) at a constant temperature of 30 °C, and the differences in the specific growth rate and levels of PAAs were determined. Approximately 15 PAAs with concentrations ranging from 0 to 50 mmol/L were detected under all conditions. Partial least squares discriminant analysis (PLS-DA) revealed a distinct difference when L. lactis was incubated at 100 and 150 rpm. Heatmap analysis showed that the levels of pyruvate-, glutamate- and aspartate-based amino acids were varied under the different agitation conditions. The time-series analysis showed an increment of lysine when L. lactis’ cells were cultured with shaking at 50, 100 and 200 rpm. Taken together, these results highlight the changes in the levels of PAAs in L. lactis cells in response to agitation. In addition, the collected dataset will be useful for optimization of ¹³C-labeling based experiments in L. lactis.     

Characterization of 10 novel microsatellite loci for the brown marbled grouper, Epinephelus fuscoguttatus (Serranidae)

Epinephelus fuscoguttatus is a commercially important marine fish species in southeast Asia. Due to overfishing and water pollution, this species has been declared as near-threatened. Thus, to provide information to help maintain and preserve the species, microsatellites were developed, using an enriched genomic library method. Thirty individuals were collected from the hatchery of the Fishery Research Institute, Terengganu, Malaysia. These individuals, from four to six years old, originated from Sabah and are maintained in captive culture as broodstock. Genomic DNA was extracted from the fins of selected individuals that weighed 3-8 kg. Ten microsatellite loci were found to be polymorphic in this population, with 5 to 21 alleles per locus. Observed and expected heterozygosities ranged from 0.53 to 0.97 and 0.59 to 0.95, respectively. Only one locus deviated significantly from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found among the pairs of loci. These polymorphic microsatellite loci will be used by the Malaysian Fishery Research Institute for investigating genetic diversity and for developing breeding strategies.     

Application of 16s rDNA and cytochrome <Emphasis Type="Italic">b</Emphasis> ribosomal markers in studies of lineage and fish populations structure of aquatic species

The most economically important form of aquaculture is fish farming, which is an industry that accounts for an ever increasing share of world fishery production. Molecular markers can be used to enhance the productivity of the aquaculture and fish industries to meet the increasing demand. Molecular markers can be identified via a DNA test regardless of the developmental stage, age or environmental challenges experienced by the organism. The application of 16s and cytochrome b markers has enabled rapid progress in investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and the construction of high resolution genetic linkage maps for aquaculture fisheries. In this review, the advantages of principles and potential power tools of 16s and cytochrome b markers are discussed. Main findings in term of trend, aspects and debates on the reviewed issue made from the model of aquatic species for the benefit of aquaculture genomics and aquaculture genetics research are discussed. The concepts in this review are illustrated with various research examples and results that relate theory to reality and provide a strong review of the current status of these biotechnology topics.     

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 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees 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.