Most of anti-retrogradation amylases could not be used in rice cooking due to their low optimal temperature. To overcome this disadvantage, an extremely thermostable maltogenic amylase from ...Thermofilum pendens at 3.5, 35 or 80U/g substrate was incubated at 90, 95, or 100°C with rice starch at 0.5% (w/v) or 5% (w/v), or polished rice grains at 50% (w/w), respectively. Its hydrolytic patterns and anti-retrogradation roles were evaluated. The amylose content of rice starch decreased from 16.6 to 8.6% after incubation of 120min, while the amylopectin content remained almost constant with a somewhat increase in ratio of short-to-long branch-chains. Substrate specificity analysis of the enzyme showed that its kcat/Km value on amylose was 0.98s−1mLmg−1, whereas that for amylopectin was 0.30s−1mLmg−1, indicating an exceptionally high preference toward amylose. Retrogradation rates of gelatinized rice starch and rice meal decreased significantly during storage of 4°C.
•Rice starch or meal treated by TfMA had low retrogradation rate at 4°C.•TfMA with optimal temperature of 95°C could be directly added during the cooking.•TfMA reduced amylose and increased ratio of short-to-long chain of amylopectin.
The gene encoding a β-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and ...released glucose or mannose from p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal β-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (kcat/Km = 3.05) with strong substrate affinity for such natural substrates as cellobiose (Km = 0.149) and mannobiose (Km = 0.147). Genistin solubilized in 10–40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds.
In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria
Thermoanaerobacter pseudoethanolicus
39E ...and expressed in
Escherichia coli
. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (
K
d
) and the maximum specific binding (
B
max
) of Tp39E to corn starch granules were 0.537 μM and 5.79 μM/g, respectively, at pH 5.5 and 20 °C.
99
APU
1357
with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.
•B. licheniformis α-amylase produces mainly maltohexaose from maltooctaose and maltononaose.•Maltononaose gave the smallest Km value and the highest kcat.•The enzyme has nine subsites: six in the ...non-reducing and three at the reducing end-binding site.
The action pattern of Bacillus licheniformis thermostable α-amylase (BLA) was analyzed using a series of 14C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest Km=0.36mM, the highest kcat=12.86s−1, and a kcat/Km value of 35.72s−1mM−1, producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.
► Ethanol reduces autophagy activity by suppressing AMPK activity in hepatocytes. ► Puerarin recovers autophagy activity by stimulating AMPK and suppressing mTOR. ► Puerarin plays hepatoprotective ...role in ethanol-treated hepatocytes. ► Puerarin might be used for treating alcoholic liver damage.
We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100mM) for 24h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5′AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.
TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities ...for α-1,4-transferase and α-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the “flexible loop” (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in α-1,4-transferase activity and a reduced level of α-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the α-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.
► The presence of 4αGTase-treated starch in the inner water phase improved EE. ► 4αGTase-treated starch solution was liquid in the preparation stage at 20wt.%. ► 4αGTase-treated starch solution ...became a solid gel during storage at 20wt.%. ► 20wt.% 4αGTase-treated starch was the most effective for improving EE. ► The amount of PGPR required was reduced by adding 4αGTase-treated starch.
The present study was performed to investigate the possibility of using 4-α-glucanotransferase (4αGTase)-treated starch in W/O/W emulsions to increase their encapsulation efficiency (EE) and stability. Emulsions were prepared using soybean oil, polyglycerol polyricinoleate (PGPR), 4αGTase-treated starch and Tween 20. The mean diameter of W/O/W droplets ranged from 4 to 10μm depending on the sonication time. When the dye was loaded in the internal water phase, the emulsion prepared by sonication for 1 and 2min showed a high EE of the dye (>90%). The W/O/W emulsion prepared by sonication for 3min showed an EE of<90%, but this EE was improved by adding 4αGTase-treated starch to the internal water phase. 4αGTase-treated starch was added to the internal water phase of W/O/W emulsions prepared with a low concentration of PGPR, and the PGPR concentration required to maintain an EE>90% was reduced. W/O/W emulsions containing 4αGTase-treated starch also showed better stability against heating and shearing stresses. These results indicated that 4αGTase-treated starch could be used in the preparation of W/O/W emulsions, which would allow the formulation of W/O/W emulsions with a reduced surfactant concentration.
Glycogen is the predominant polysaccharide in living cells. Many microorganisms accumulate glycogen, which serves as an energy reserve to cope with harsh environmental conditions. Therefore, the ...functions of enzymes involved in glycogen synthesis and degradation must be deciphered to understand the survival mechanisms of microbes. However, these enzymes in bacteria, most of which are glycosyl transferases or glycosidases, have not been fully characterized. Although there are similarities, the processes of glycogen synthesis and degradation in bacteria are quite distinct from the same processes in eukaryotes. Considerable progress has been made in understanding the mode of glycogen metabolism in Escherichia coli. In addition to the common core pathway, the virulence factors of infecting enteropathogenic bacteria appear to be involved in glycogen degradation. This review will focus on the following: (i) enzymes involved in glycogen degradation in E. coli, (ii) comparisons of the glycogen enzymes within enterobacteria, and (iii) glycogen as a carbon source for infectious microbes.
•Replacement of His235 with Glu in BLA induces transglycosylation activity.•A mutant enzyme, H235E, exhibits high substrate transglycosylation activity.•Glu235 near subsite +1 recognizes the ...non-reducing end glucose of the acceptor molecule.•The subsite +1 in a wide-open geometry results in an α-1,4-transfer mode.
To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM−1s−1 for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM−1s−1 for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235—located on a wide open groove near subsite +1—is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.
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