ABSTRACT
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor-hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes.
Subject(s)
Phospholipase D/ultrastructure , Amino Acid Sequence , Catalytic Domain , Drug Design , Humans , Isoenzymes/metabolism , Phospholipase D/metabolism , Phospholipase D/physiology , Phosphoric Diester Hydrolases/metabolism , Structure-Activity RelationshipABSTRACT
BTK is a tyrosine kinase playing an important role in B cell and myeloid cell functions through B cell receptor (BCR) signaling and Fc receptor (FcR) signaling. Selective inhibition of BTK has the potential to provide therapeutical benefits to patients suffering from autoimmune diseases. Here we report the design, optimization, and characterization of novel potent and highly selective covalent BTK inhibitors. Starting from a piperazinone hit derived from a selective reversible inhibitor, we solved the whole blood cellular potency issue by introducing an electrophilic warhead to reach Cys481. This design led to a covalent irreversible BTK inhibitor series with excellent kinase selectivity as well as good whole blood CD69 cellular potency. Optimization of metabolic stability led to representative compounds like 42, which demonstrated strong cellular target occupancy and inhibition of B-cell proliferation measured by proximal and distal functional activity.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Agammaglobulinaemia Tyrosine Kinase/metabolism , B-Lymphocytes/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
This study aimed at purification of phycocyanin (PC) from Phormidium tergestinum using an aqueous two-phase system (ATPS) comprised of polyethylene glycol (PEG) and salts. The partitioning efficiency of PC in ATPS and the effect of phase composition, pH, crude loading, and neutral salts on purification factor and yield were investigated. Results showed that PC was selectively partitioned toward bottom phase of the system containing potassium phosphate. Under optimum conditions of 20% (w/w) PEG 4000, 10% (w/w) potassium phosphate, 20% (v/v) crude load at pH 7, with addition of 0.5% (w/w) NaCl, PC from P. tergestinum was partially purified up to 5.34-fold with a yield of 87.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of PC was â¼19 kDa. Results from this study demonstrated ATPS could be used as a potential approach for the purification of PC from P. tergestinum.
Subject(s)
Phycocyanin/chemistry , Biomass , Biotechnology/methods , Electrophoresis, Polyacrylamide Gel , Food Technology/methods , Hydrogen-Ion Concentration , Molecular Weight , Phormidium/metabolism , Phosphates/chemistry , Polyethylene Glycols/chemistry , Potassium Compounds/chemistry , Static Electricity , Temperature , Water/chemistryABSTRACT
The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70-80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 109 to 1010 CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.
Subject(s)
Cryopreservation/methods , Freeze Drying , Lactobacillus acidophilus/drug effects , Lactobacillus acidophilus/physiology , Microbial Viability/drug effects , Capsules , Cell Survival/drug effects , Lactobacillus acidophilus/cytology , Polysaccharides/pharmacologyABSTRACT
Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.
Subject(s)
Protein Kinase C-theta/metabolism , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Binding Sites , Cell Line , Humans , Inhibitory Concentration 50 , Interleukin-2/metabolism , Mice , Mice, Knockout , Molecular Docking Simulation , Phosphorylation/drug effects , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Structure, Tertiary , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Structure-Activity Relationship , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase , Brain , Multiple Sclerosis , Protein Kinase Inhibitors , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Agammaglobulinaemia Tyrosine Kinase/metabolism , Multiple Sclerosis/drug therapy , Humans , Animals , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/chemistry , Brain/metabolism , Mice , Drug Discovery , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Rats , Structure-Activity Relationship , Cell Proliferation/drug effects , FemaleABSTRACT
Glycogen synthase kinase 3 (GSK3) remains a therapeutic target of interest for diverse clinical indications. However, one hurdle in the development of small molecule GSK3 inhibitors has been safety concerns related to pan-inhibition of both GSK3 paralogs, leading to activation of the Wnt/ß-catenin pathway and potential for aberrant cell proliferation. Development of GSK3α or GSK3ß paralog-selective inhibitors that could offer an improved safety profile has been reported but further advancement has been hampered by the lack of structural information for GSK3α. Here we report for the first time the crystal structure for GSK3α, both in apo form and bound to a paralog-selective inhibitor. Taking advantage of this new structural information, we describe the design and in vitro testing of novel compounds with up to â¼37-fold selectivity for GSK3α over GSK3ß with favorable drug-like properties. Furthermore, using chemoproteomics, we confirm that acute inhibition of GSK3α can lower tau phosphorylation at disease-relevant sites in vivo, with a high degree of selectivity over GSK3ß and other kinases. Altogether, our studies advance prior efforts to develop GSK3 inhibitors by describing GSK3α structure and novel GSK3α inhibitors with improved selectivity, potency, and activity in disease-relevant systems.
Subject(s)
Glycogen Synthase Kinase 3 , Protein Serine-Threonine Kinases , Glycogen Synthase Kinase 3 beta , Phosphorylation , Cell Proliferation/physiologyABSTRACT
Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.
ABSTRACT
Multiple Sclerosis is a chronic autoimmune neurodegenerative disorder of the central nervous system (CNS) that is characterized by inflammation, demyelination, and axonal injury leading to permeant disability. In the early stage of MS, inflammation is the primary driver of the disease progression. There remains an unmet need to develop high efficacy therapies with superior safety profiles to prevent the inflammation processes leading to disability. Herein, we describe the discovery of BIIB091, a structurally distinct orthosteric ATP competitive, reversible inhibitor that binds the BTK protein in a DFG-in confirmation designed to sequester Tyr-551, an important phosphorylation site on BTK, into an inactive conformation with excellent affinity. Preclinical studies demonstrated BIB091 to be a high potency molecule with good drug-like properties and a safety/tolerability profile suitable for clinical development as a highly selective, reversible BTKi for treating autoimmune diseases such as MS.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase , Drug Discovery , Multiple Sclerosis , Protein Kinase Inhibitors , Animals , Male , Rats , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Macaca fascicularis , Multiple Sclerosis/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Rats, Sprague-Dawley , Tissue DistributionABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) is one of the key mediators of the cellular stress response that regulates inflammation and apoptosis. To probe the therapeutic value of modulating this pathway in preclinical models of neurological disease, we further optimized the profile of our previously reported inhibitor 3. This effort led to the discovery of 32, a potent (cell IC50 = 25 nM) and selective ASK1 inhibitor with suitable pharmacokinetic and brain penetration (rat Cl/Clu = 1.6/56 L/h/kg and Kp,uu = 0.46) for proof-of-pharmacology studies. Specifically, the ability of 32 to inhibit ASK1 in the central nervous system (CNS) was evaluated in a human tau transgenic (Tg4510) mouse model exhibiting elevated brain inflammation. In this study, transgenic animals treated with 32 (at 3, 10, and 30 mg/kg, BID/PO for 4 days) showed a robust reduction of inflammatory markers (e.g., IL-1ß) in the cortex, thus confirming inhibition of ASK1 in the CNS.
Subject(s)
Brain/drug effects , Drug Discovery , Inflammation/drug therapy , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Animals , Brain/metabolism , Dose-Response Relationship, Drug , Humans , Inflammation/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Mice , Mice, Transgenic , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer's disease and other tauopathies.
Subject(s)
Brain/metabolism , Drug Design , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , tau Proteins/metabolism , Animals , Humans , Indazoles/chemistry , Indazoles/metabolism , Indazoles/pharmacology , Mice , Molecular Targeted Therapy , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , RatsABSTRACT
BACKGROUND: The banana or scientifically referred to as Musa sp., is one of the most popular fruits all over the world. Almost all parts of a banana tree, including the fruits, stem juice, and flowers are commonly used as traditional medicine for treating diarrhoea (unripe), menorrhagia, diabetes, dysentery, and antiulcerogenic, hypoglycemic, antilithic, hypolipidemic conditions, plus antioxidant actions, inflammation, pains and even snakebites. The study carried out was to evaluate in vitro anti-urolithiatic activity from different types of Musa pseudo-stems. METHODS: Observing anti-urolithiathic activity via in vitro nucleation and aggregation assay using a spectrophotometer followed by microscopic observation. A total of 12 methanolic extracts were tested to determine the potential extracts in anti-urolithiasis activities. Cystone was used as a positive control. RESULTS: The results manifested an inhibition of nucleation activity (0.11 ± 2.32% to 55.39 ± 1.01%) and an aggregation activity (4.34 ± 0.68% to 58.78 ± 1.81%) at 360 min of incubation time. The highest inhibition percentage in nucleation assay was obtained by the Musa acuminate x balbiciana Colla cv "Awak Legor" methanolic pseudo-stem extract (2D) which was 55.39 ± 1.01%at 60 min of incubation time compared to the cystone at 30.87 ± 0.74%. On the other hand,the Musa acuminate x balbiciana Colla cv "Awak Legor" methanolic bagasse extract (3D) had the highest inhibition percentage in the aggregation assay incubated at 360 min which was obtained at 58.78 ± 1.8%; 5.53% higher than the cystone (53.25%).The microscopic image showed a great reduction in the calcium oxalate (CaOx) crystals formation and the size of crystals in 2D and 3D extracts, respectively, as compared to negative control. CONCLUSIONS: The results obtained from this study suggest that the extracts are potential sources of alternative medicine for kidney stones disease.
Subject(s)
Musa/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , Urolithiasis/drug therapy , Calcium Oxalate/metabolism , Humans , Malaysia , Plant StemsABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress response. To investigate the therapeutic value of modulating this pathway in neurological disease, we have completed medicinal chemistry studies to identify novel CNS-penetrant ASK1 inhibitors starting from peripherally restricted compounds reported in the literature. This effort led to the discovery of 21, a novel ASK1 inhibitor with good potency (cell IC50 = 138 nM), low clearance (rat Cl/Clu = 0.36/6.7 L h-1 kg-1) and good CNS penetration (rat K p,uu = 0.38).
ABSTRACT
Structural analysis of a known apoptosis signal-regulating kinase 1 (ASK1) inhibitor bound to its kinase domain led to the design and synthesis of the novel macrocyclic inhibitor 8 (cell IC50 = 1.2 µM). The profile of this compound was optimized for CNS penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC50 = 95 nM; 26, cell IC50 = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER = 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (Kpuu = 0.17) and analog 26 had high CNS penetration (Kpuu = 1.0).
Subject(s)
MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacology , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Animals , Brain/metabolism , Drug Design , Humans , MAP Kinase Kinase Kinase 5/metabolism , Macrocyclic Compounds/chemistry , Molecular Structure , RatsABSTRACT
Carbon dioxide assisted particle formation combined with electrospraying using supercritical CO2 (scCO2) as an aid (Carbon Dioxide Assisted Nebulization-Electrodeposition, CAN-ED) was used to produce Bortezomib loaded poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) nanoparticles for sustained release. The morphology and structure of the prepared nanoparticles were investigated by SEM, TEM and FT-IR spectroscopy. Average diameter of particles obtained was 155nm and the average core sizes of P(3HB-co-3HHx) nanoparticles were between 6 and 13nm. The drug loading capacity, drug release and stability of Bortezomib loaded P(3HB-co-3HHx) nanoparticles were analyzed. The maximum loading capacity was achieved at pH=6.0 in phosphate buffer (K2HPO4/KH2PO4). It was found that temperature did not affect the stability of Bortezomib loaded nanoparticles and it was good both at 37°C and 4°C. This study pointed out that CAN-ED is a green method to produce P(3HB-co-3HHx) nanoparticles for pH responsive targeting of Bortezomib especially to parts of the body where size exclusion is not crucial.
Subject(s)
3-Hydroxybutyric Acid/chemical synthesis , Bortezomib/chemistry , Caproates/chemical synthesis , Drug Liberation , Nanoparticles/chemistry , 3-Hydroxybutyric Acid/chemistry , Bortezomib/therapeutic use , Caproates/chemistry , Carbon Dioxide/chemistry , Electroplating , Humans , Hydrogen-Ion Concentration , Microscopy, Electrochemical, Scanning , Nanoparticles/therapeutic use , Nanotechnology/methods , Spectroscopy, Fourier Transform Infrared , TemperatureABSTRACT
Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation, Neoplastic , NF-kappa B/metabolism , Osteosarcoma/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclins/genetics , Cyclins/metabolism , Gene Expression Profiling , High-Throughput Screening Assays , Humans , Indoles/pharmacology , Lymphotoxin beta Receptor/antagonists & inhibitors , Lymphotoxin beta Receptor/genetics , Lymphotoxin beta Receptor/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B p52 Subunit/genetics , NF-kappa B p52 Subunit/metabolism , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Propionates/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteome , Signal Transduction , TWEAK Receptor/antagonists & inhibitors , TWEAK Receptor/genetics , TWEAK Receptor/metabolism , Tumor Cells, Cultured , NF-kappaB-Inducing KinaseABSTRACT
A combination of palm olein (POl) and fructose was used as carbon source for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by recombinant Cupriavidus necator Re2058/pCB113. Cultures grown using 5g/L PO alone as carbon source produced cell dry weight (CDW) of 5.13g/L, 67% PHA/CDW and accumulated a copolymer containing 27mol% 3HHx in shake flask cultures. When cultures were grown in 5g/L fructose alone as the carbon source they produced CDW of 2.32g/L, 11% PHA/CDW and accumulated only poly(3-hydroxybutyrate) [P(3HB)] homopolymer. When the cells were cultured in 5g/L POl in combination with 7g/L fructose, CDW of 7.41g/L and 80% PHA/CDW was obtained with 17mol% 3HHx monomer fraction. Biosynthesis was carried out using a 13L fermenter to study the accumulation of 3HHx monomer fraction in the bacterial cells at different time point. The molecular weights of P(3HB-co-3HHx) with 4-15mol% 3HHx monomer were in the range between 5.47-6.85×105Da, which were at least two fold higher than previously reported values. Interestingly, the increase in Mw of the copolymer along with the increase in 3HHx molar fraction was observed. The viscoelastic property of the copolymer further confirmed the increase in Mw.
Subject(s)
3-Hydroxybutyric Acid/biosynthesis , 3-Hydroxybutyric Acid/chemistry , Caproates/chemistry , Cupriavidus necator/metabolism , Fructose/metabolism , Palm Oil/metabolism , Acyltransferases/metabolism , Biotechnology , Cupriavidus necator/enzymology , Molecular WeightABSTRACT
Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.
Subject(s)
Cupriavidus necator/metabolism , Larva/physiology , Polyhydroxyalkanoates/isolation & purification , Polyhydroxyalkanoates/metabolism , Tenebrio/physiology , Animals , Electrophoresis, Polyacrylamide Gel , Feces/chemistry , Polyhydroxyalkanoates/analysis , Polyhydroxyalkanoates/chemistryABSTRACT
Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of an organic alkali was validated for the compositional analysis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] accumulated in whole bacterial cells. Recombinant Cupriavidus necator Re2058/pCB113 was grown in a batch fermentation with different concentration of palm oil and fructose in order to control the molar fraction of 3HHx in P(3HB-co-3HHx) produced in the cells. Trace amounts (30µg) of freeze-dried cells were directly subjected to THM-GC in the presence of tetramethylammonium hydroxide (TMAH) at 400°C. The obtained chromatograms clearly showed nine characteristic peaks, attributed to the THM products from 3HB and 3HHx units in the polymer chains, without any appreciable interference by the bacterial matrix components. Based on these peak intensities, the copolymer compositions were determined rapidly without using any cumbersome and lengthy sample pretreatment as in conventional GC method. Moreover, the compositions thus obtained were strongly correlated with those by NMR and conventional GC involving solvent extraction.