Korea Research Institute of Bioscience and Biotechnology
KRIBB Open Access RepositoryNot a member yet
15834 research outputs found
Sort by
Purification of high purity docosahexaenoic acid from Schizochytrium sp. SH103 using preparative-scale HPLC
No full textHigh purity polyunsaturated fatty acids (>？95%) are essential for the synthesis of specialized pro-resolving lipid mediators (SPMs), such as protectins, resolvins, and maresins, which are used for clinical application. To date, high purity (>？95%) eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) have been produced through various manufacturing steps using fish oil. In this study, we optimized preparative high performance liquid chromatography (HPLC) process to purify high-purity DHA ethyl ester (DHAee; >？98%) from oleaginous microalgae Shizochytrium sp. SH103 containing at least 34% DHA content. The purity and yield of DHA were determined by reverse phase chromatography with changing the mobile phase velocity, loading amount, and mobile phase composition. On a semi-preparative scale, optimal DHA separation in isocratic elution was obtained with a mobile phase velocity of 0.5 mL/min, a loading amount of 10 mg/mL, and mobile phase composition of methanol/water (96:4, v/v), wherein the purity of DHA was 98.5%. This separation was scaled up to a preparative column, resulting in 99.0% DHA fraction with a yield of 79.8%. This result suggests that a large amount of high purity DHA can be produced from microalgae when scaling up a preparative column to an industrial column.
Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity
No full textVarious treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The results substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful method for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.
Design, synthesis, and evaluation of novel N'-substituted-1-(4-chlorobenzyl)-1 H-indol-3-carbohydrazides as antitumor agents
No full textIn continuity of our search for novel anticancer agents acting as procaspase activators, we have designed and synthesised two series of (E)-N′-benzylidene-carbohydrazides (4a？m) and (Z)-N'-(2-oxoindolin-3-ylidene)carbohydrazides (5a？g) incorporating 1-(4-chlorobenzyl)-1H-indole core. Bioevaluation showed that the compounds, especially compounds in series 4a？m, exhibited potent cytotoxicity against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI-H23, lung cancer). Within series 4a？m, compounds with 2-OH substituent (4g？i) exhibited very strong cytotoxicity in three human cancer cell lines assayed with IC50 values in the range of 0.56？0.83？μM. In particular, two compounds 4d and 4f bearing 4-Cl and 4-NO2 substituents, respectively, were the most potent in term of cytotoxicity with IC50 values of 0.011？0.001？μM. In caspase activation assay, compounds 4b and 4f were found to activate caspase activity by 314.3 and 270.7% relative to PAC-1. This investigation has demonstrated the potential of these simple acetohydrazides, especially compounds 4b, 4d, and 4f, as anticancer agents.
Molecular changes associated with spontaneous phenotypic variation of Paenibacillus polymyxa, a commonly used biocontrol agent, and temperature-dependent control of variation
No full textThere has been a growing interest in deploying plant growth-promoting rhizobacteria (PGPR) as a biological control agent (BCA) to reduce the use of agrochemicals. Spontaneous phenotypic variation of PGPR, which causes the loss of traits crucial for biocontrol, presents a large obstacle in producing commercial biocontrol products. Here, we report molecular changes associated with phenotypic variation in Paenibacillus polymyxa, a PGPR widely used for biocontrol worldwide, and a simple cultural change that can prevent the variation. Compared to B-type (non-variant) cells of P. polymyxa strain E681, its phenotypic variant, termed as F-type, fails to form spores, does not confer plant growth-promoting effect, and displays altered colony and cell morphology, motility, antagonism against other microbes, and biofilm formation. This variation was observed in all tested strains of P. polymyxa, but the frequency varied among them. RNA-seq analysis revealed differential regulation of many genes involved in sporulation, flagella synthesis, carbohydrate metabolism, and antimicrobial production in F-type cells, consistent with their pleiotropic phenotypic changes. F-type cells's sporulation was arrested at stage 0, and the key sporulation gene spo0A was upregulated only in B-type cells. The phenotypic variation could be prevented by altering the temperature for growth. When E681 was cultured at 20 °C or lower, it exhibited no variation for 7 days and still reached？~？108 cfu/mL, the level sufficient for commercial-scale production of biocontrol products.
Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption
No full textAcetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. Four genomic mutations were found in the evolved strain through whole-genome sequencing, and two major mutations (in cspC and patZ) mainly contributed to efficient utilization of acetate and tolerance to acetate. Transcriptomic reprogramming was examined by analyzing the genome-wide transcriptome with different carbon sources. The evolved strain showed high levels of intracellular ATP by upregulation of genes involved in NADH and ATP biosynthesis, which facilitated the production of enhanced green fluorescent protein, mevalonate, and n-butanol using acetate alone. This new strain, given its high acetate tolerance and high ATP levels, has potential as a starting host for cell factories targeting the production of acetyl-CoA-derived products from acetate or of products requiring high ATP levels.
Argyreia gyrobracteata Traiperm & Chitchak (Convolvulaceae): a new record to the flora of Vietnam
No full textArgyreia gyrobracteata Traiperm & Chitchak (Convolvulaceae) is newly recorded in Vietnam. It is similar to A. mekongensis Gagnep & Courch in having a white campanulate corolla but differs from that species by having whitish surface of the abaxial leaf, narrowly lanceolate or linear-oblong, curly, rolled up the bract with a longer length of the petiole and a shorter pedicel. It was previously known only in Thailand, but was recently found in Lam Dong Province, Vietnam, for the first time. The present study provides a detailed description, colorful photos and the distribution of the species. Furthermore, a comparison of the diagnostic characters between this species and a related species in Vietnam, A. mekongensis Gagnep & Courch, is provided.
Archaea, tiny helpers of land plants
No full textArchaea are members of most microbiomes. While archaea are highly abundant in extreme environments, they are less abundant and diverse in association with eukaryotic hosts. Nevertheless, archaea are a substantial constituent of plant-associated ecosystems in the aboveground and belowground phytobiome. Only a few studies have investigated the role of archaea in plant health and its potential symbiosis in ecosystems. This review discusses recent progress in identifying how archaea contribute to plant traits such as growth, adaptation to abiotic stresses, and immune activation. We synthesized the most recent functional and molecular data on archaea, including root colonization and the volatile emission to activate plant systemic immunity. These data represent a paradigm shift in our understanding of plant-microbiota interactions.
Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis
No full textPTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-Asp401Ala mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. The transient knockdown of PTPRT in dentate gyrus enhanced the depression-like behaviors of wild-type mice, while the rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. The chronic stress exposure reduced the expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expressions of GluR2 were attenuated through dysregulated tyrosine phosphorylation and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmissions of dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition the hippocampal expression of GABA transporter GAT3 was decreased and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced newborn granule cells in their dentate gyrus and the attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT on hippocampal neurogenesis as well as synaptic functions are involved in the pathogenesis of depressive disorder.
Synthesis of two new lipid mediators from docosahexaenoic acid by combinatorial catalysis involving enzymatic and chemical reaction
No full textOmega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.
Regioselective hydroxylation of phloretin, a bioactive compound from apples, by human cytochrome P450 enzymes
No full textPhloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6β-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.