• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1636-1643
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• 2014

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA, in which acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. To understand the molecular mechanisms of n-butanol biosynthesis, we determined the crystal structure of 3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD). The monomer structure of CbHBD exhibits a two-domain topology, with N- and C-terminal domains, and the dimerization of the enzyme was mostly constituted at the C-terminal domain. The mode of cofactor binding to CbHBD was elucidated by determining the crystal structure of the enzyme in complex with $NAD^+$. We also determined the enzyme's structure in complex with its acetoacetyl-CoA substrate, revealing that the adenosine diphosphate moiety was not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule. Using this structural information, we performed a series of site-directed mutagenesis experiments on the enzyme, such as changing residues located near the substrate-binding site, and finally developed a highly efficient CbHBD K50A/K54A/L232Y triple mutant enzyme that exhibited approximately 5-fold higher enzyme activity than did the wild type. The increased enzyme activity of the mutant was confirmed by enzyme kinetic measurements. The highly efficient mutant enzyme should be useful for increasing the production rate of n-butanol.

• Journal of Life Science
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• v.26 no.3
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• pp.353-358
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• 2016

Thiolase is an enzyme that catalyzes condensation reactions between two acetyl-CoA molecules to produce acetoacetyl-CoA. As thiolase catalyzes is the first reaction in the production of n-butanol, knowledge of the molecular and regulatory mechanism of the enzyme is crucial for synthesizing high-value biofuel. Thiolase from Clostridium butyricum (CbTHL) was expressed, purified, and crystallized. X-ray diffraction data were collected from the crystals, and the 3-dimentional structure of the enzyme was determined at 2.0 Å. The overall structure of thiolase was similar to that of type II biosynthetic thiolases, such as thiolase from C. acetobutylicum (CaTHL). The superposition of this structure with that of CaTHL complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of CbTHL. The catalytic site of CbTHL contains three conserved residues, Cys88, His349, and Cys379, which may function as a covalent nucleophile, general base, and second nucleophile, respectively. For substrate binding, the way in which CbTHL stabilized the ADP moiety of CoA was unlike that of other thiolases, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar to that of other enzymes. The most interesting observation in the CbTHL structure was that the enzyme was regulated through redox-switch modulation, using a reversible disulfide bond.

• Journal of the Korean Applied Science and Technology
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• v.38 no.2
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• pp.476-487
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• 2021

To prepare acrylic resin coatings containing 70% of solids, we used n-butyl methacrylate(BMA), methyl methacrylate(MMA), 2-hydroxyethyl methacrylate(2-HEMA), and acetoacetoxyethyl acrylate(AAEA), caprolactone acrylate(CLA) as raw materials, the glass transition temperature(T_g) of acrylic copolymer was adjusted around 50 ℃. The viscosity and molecular weight of the acrylic resins was increased with increasing OH values. Di-tert-amyl peroxide was found to be the suitable initiator to get high-solids acrylic resins. The optimum reaction conditions found in the study are 5 wt% of initiator, 4 wt% of chain transfer agent, 4 hrs of dropping time, and 140 ℃ of reaction temperature. The structure of the synthesized resins were characterized by FT-IR and ¹H-NMR spectroscopy. Number average molecular weight of 1900~2600 and molecular wight distribution of 1.4~2.1 were obtained. Crosslinked acrylic urethane clear coatings were obtained by curing reaction between the synthesized acrylic resins and hexamethylene diisocyanate trimer(Desmodur N-3300), the equivalent ratio of NCO/OH was 1.2/1.0. The physical properties from the following studies were carried out: viscosity(Zahn cup #2), adhesion, drying time, pot-life, pensil hardness, and 60° specular gloss. Various properties of the acrylic urethane clear coatings were also evaluated on the coating specimens. Adhesion property to a substrate, drying time, pot-life, pencil hardness, and 60° specular gloss of prepared paint showed quite good properties. Futhermore, prepared paint containing 10% of CLA showed quite good properties for adhesion, low viscosity and high hardness.

• Analytical Science and Technology
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• v.29 no.1
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• pp.49-55
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• 2016

A ketone body (acetoacetic acid, β-hydroxybutyric acid, and acetone) increases from blood or urine when bio-energy dependence pays more fatty acid than glucose. However, in case oxidation of fat is greater than the capacity of the citric acid cycle the fatty acid oxidation is made from acetoacetyl CoA to acetoacetate then, again form β-hydroxyburytic acid to acetone, the diffusion take place into the blood. Enzymes that oxidize ketone body in the brain and nerve tissue blood ketone dody is increased during prolonged fasting, brain used it as energy. In this study, we developed the rapid two step derivatization method for sensitive detection of the ketone body by GC-MS/SIM. The plasma was deproteinized and then the hydroxy and carboxyl groups of ketone body are subjected to extraction and drying then, keto-group were derivatized with hydoxylamine at 60℃ for 30 min for oximation. Then it was trimetyl-silylated with BSTFA at 80℃ for 30 min and analyzed using a GC-MS. The linear ranges were in between 0.001 μg/mL and 250 μg/mL for β-hydroxy butyrate, and acetoacetate. The method detection limits were below 0.1 pg over each target compound determined. The mean recoveries (%) of target compounds were ranged from 88.2 % to 92.3 % at 1 µg/mL, from 89.5 % to 94.8 % at 10 μg/mL, with RSD of 6.3-9.4 %. This method could be applied to quantification of ketone bodies which are seen in the keto-acidosis in children and adults from a variety of diseases that cause ketones in the blood and urine.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.26-35
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• 2014

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at $20^{\circ}C$. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were $40^{\circ}C$ and 7, with a $K_m$ value of $7.9{\mu}M$ and a $V_{max}$ of $0.63{\mu}M/min$ when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at $55^{\circ}C$and 9, a $K_m$ value of $14.7{\mu}M$, and a $V_{max}$ of $0.67{\mu}M/min$ when benzoyl-CoA was used as limited substrate.