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ABSTRACT: We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h 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.
Biochemical and Biophysical Research Communications 09/2011; 414(2):361-6. · 2.48 Impact Factor
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Jong-Tae Park,
Jae-Hoon Shim,
Phuong Lan Tran,
In-Hee Hong,
Hwan-Ung Yong,
Ershita Fitria Oktavina,
Hai Dang Nguyen, Jung-Wan Kim,
Tae Soo Lee,
Sung-Hoon Park,
Winfried Boos,
Kwan-Hwa Park
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ABSTRACT: Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.
Journal of bacteriology 03/2011; 193(10):2517-26. · 3.94 Impact Factor
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ABSTRACT: Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five α-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the α-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except α-amylase production, and fermented alcohol better than commercial wine yeasts.
Mycobiology. 03/2011; 39(1):33-9.
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ABSTRACT: Multi-substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed β-cyclodextrin (β-CD) to maltose and glucose. It exhibited substrate preference for β-CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an α-amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 62-O-α-maltosyl-maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer–dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association–dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards β-CD decreased to 80%. Therefore, multi-substrate specificity of neopullulanase was likely to be modulated by the shift of monomer–dimer association equilibrium.
Biotechnology and Applied Biochemistry 12/2010; 35(1):27 - 34. · 1.53 Impact Factor
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Ok-Hee Kim,
Young-Ok Kim,
Jae-Hoon Shim,
Yun-Shin Jung,
Woo-Jin Jung,
Won-Chan Choi,
Heeseob Lee,
Sang-Jun Lee,
Kyung-Kil Kim,
Joong-Huck Auh,
Hyeonjin Kim, Jung-Wan Kim,
Tae-Kwang Oh,
Byung-Chul Oh
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ABSTRACT: Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate’s binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca2+-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0−9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca2+-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca2+-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca2+-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.
10/2010;
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Ok-Hee Kim,
Young-Ok Kim,
Jae-Hoon Shim,
Yun-Shin Jung,
Woo-Jin Jung,
Won-Chan Choi,
Heeseob Lee,
Sang-Jun Lee,
Kyung-Kil Kim,
Joong-Huck Auh,
Hyeonjin Kim, Jung-Wan Kim,
Tae-Kwang Oh,
Byung-Chul Oh
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ABSTRACT: Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.
Biochemistry 10/2010; 49(47):10216-27. · 3.42 Impact Factor
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Chong-Hyo Choi, Jung-Wan Kim,
Cheon-Seok Park,
Kwan-Hwa Park,
Myung-Ok Kyung,
Jong-Tae Park,
Chang-Kyu Lee,
Young-Wan Kim,
Jong-Hyuck Lee,
Sung-Hoon Park,
Jae-Hoon Shim,
Xue-Hong Li
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ABSTRACT: Abstract To increase the water solubility of puerarin, an isoflavonoid derived from Radix puerariae, a puerarin inclusion complex with cycloamylose was enzymatically synthesized by combining maltogenic amylase reactions from Bacillus stearothermophilus (BSMA) and 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The puerarin transfer products, including maltosyl-α-(1→6)-puerarin as a major product generated by BSMA, were reacted with TSαGT in the presence of amylose. The molecular weights and chemical structures of the reaction products were determined using TLC, HPLC and MALDI-TOF/MS. An analysis of the reaction products revealed that the maltosyl-α-(1→6)-puerarin–cycloamylose complex was formed by an elongation reaction and cyclization of TSαGT. The results indicate that TSαGT does not have substrate affinity towards puerarin or glucosyl α-(1→6)-puerarin; however, it did have an affinity towards maltosyl-α-(1→6)-puerarin in which the first glucose of the maltosyl residue is linked through an O-glucosidic bond.
06/2010; 28(3):209-214.
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Dan Li,
Jong-Tae Park,
Xiaolei Li,
Sukyung Kim,
Seungjae Lee,
Jae-Hoon Shim,
Sung-Hoon Park,
Jaeho Cha,
Byong-Hoon Lee, Jung-Wan Kim,
Kwan-Hwa Park
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ABSTRACT: A gene encoding a hyperthermostable maltogenic amylase of Staphylothermus marinus (SMMA) was cloned and overexpressed in Escherichia coli. SMMA consisted of 696 amino acids with a predicted molecular mass of 82.5 kDa. The enzyme was active in acidic conditions (pH 3.5-5.0), with an optimal pH of 5.0, and was extremely thermostable, with a temperature optimum of 100 degrees C and a melting temperature of 109 degrees C, both of which extremely favored the starch conversion process. SMMA hydrolyzed linear malto-oligosaccharides, starch, cyclodextrins, and cycloamylose, primarily to maltose and glucose, and showed highest activity toward acarbose and pullulan, hydrolyzed to acarviosine-glucose and panose, respectively. Investigation of the cleavage mode using (14)C-maltoheptaose revealed that SMMA preferentially hydrolyzed the first and second glycosidic bonds from the reducing end. To our knowledge, this enzyme is the most thermostable maltogenic amylase yet reported, and might be of potential value in the food and starch industries.
New Biotechnology 04/2010; 27(4):300-7. · 2.76 Impact Factor
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ABSTRACT: A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol−1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The Km and kcat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s−1 mM, respectively. The overall catalytic efficiency (kcat/Km) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.
07/2009; 23(2):79-87.
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Van Dao Nguyen,
Byoung-Cheol Min,
Myung-Ok Kyung,
Jong-Tae Park,
Byong Hoon Lee,
Chung-Hyo Choi,
Nam-Seok Seo,
Yong-Ro Kim,
Dong Uk Ahn,
Sung-Joon Lee,
Cheon-Seok Park, Jung-Wan Kim,
Kwan-Hwa Park
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ABSTRACT: Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one-year-old kudzu root cultivated in Vietnam.
To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI(+) TOF MS-MS, (1)H-, (13)C-NMR and enzymatic hydrolysis.
Samples were prepared by extraction of one-year-old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV-light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the (13)C and (1)H NMR spectra recorded at 100.40 and 400.0 MHz, respectively.
ESI(+) TOF MS-MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by (1)H-, (13)C-NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an alpha-1,6-glycosidic bond. This compound is the first naturally-occurring 8-[alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl-alpha-1,6-puerarin in kudzu root was 2.3 mg/g as determined by HPLC.
The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water-soluble glycosylated puerarin.
Phytochemical Analysis 07/2009; 20(6):450-5. · 2.63 Impact Factor
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Jae-Hoon Shim,
Jong-Tae Park,
Jung-Sun Hong,
Ki Woo Kim,
Myo-Jeong Kim,
Jung-Hyuk Auh,
Young-Wan Kim,
Cheon-Seok Park,
Winfried Boos, Jung-Wan Kim,
Kwan-Hwa Park
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ABSTRACT: The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.
Journal of bacteriology 06/2009; 191(15):4835-44. · 3.94 Impact Factor
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Hee-Kwon Kang,
Hyunju Cha,
Tae-Joo Yang,
Jong-Tae Park,
Seungjae Lee,
Young-Wan Kim,
Joong-Hyuck Auh,
Yasuyo Okada, Jung-Wan Kim,
Jaeho Cha,
Chung Ho Kim,
Kwan-Hwa Park
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ABSTRACT: Di-O-alpha-maltosyl-beta-cyclodextrin ((G2)(2)-beta-CD) was synthesized from 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-beta-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-beta-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-beta-CD, suggesting that TreX transferred the maltosyl residue of a G2-beta-CD to another molecule of G2-beta-CD by forming an alpha-1,6-glucosidic linkage. When [(14)C]-maltose and maltosyl-beta-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-beta-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-beta-CD occurred exclusively via transglycosylation of an alpha-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6(1),6(3)- and 6(1),6(4)-dimaltosyl-beta-CD. Inhibition studies with beta-CD on the transglycosylation activity revealed that beta-CD was a mixed-type inhibitor, with a K(i) value of 55.6 micromol/mL. Thus, dimaltosyl-beta-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of beta-CD. Our findings suggest that the high yield of (G2)(2)-beta-CD from G2-beta-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of beta-CD.
Biochemical and Biophysical Research Communications 03/2008; 366(1):98-103. · 2.48 Impact Factor
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ABSTRACT: In this study, the effects of phosphate concentration and carbon source on the patterns of alkaline phosphatase (APase) and phospholipase (PLase) expression in Vibrio vulnificus ATCC 29307 were assessed under various conditions. The activities of these enzymes were repressed by excess phosphate (4 mM) in the culture medium, but this repression was reversed upon the onset of phosphate starvation in low phosphate defined medium (LPDM) containing 0.2 mM of phosphate at approximately the end of the exponential growth phase. The expressions of the two enzymes were also influenced by different carbon sources, including glucose, fructose, maltose, glycerol, and sodium acetate at different levels. The APase activity was derepressed most profoundly in LPDM containing fructose as a sole carbon source. However, the repression/derepression of the enzyme by phosphate was not observed in media containing glycerol or sodium acetate. In LPDM-glycerol or sodium acetate, the growth rate was quite low. The highest levels of PLase activity were detected in LPDMsodium acetate, followed by LPDM-fructose. PLase was not fully repressed by high phosphate concentrations when sodium acetate was utilized as the sole carbon source. These results showed that multiple regulatory systems, including the phosphate regulon, may perform a function in the expression of both or either APase and PLC, in the broader context of the survival of V. vulnificus.
The Journal of Microbiology 09/2007; 45(4):311-7. · 1.10 Impact Factor
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ABSTRACT: To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k(cat)/K(m) value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.
Bioscience Biotechnology and Biochemistry 07/2007; 71(6):1564-7. · 1.28 Impact Factor
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ABSTRACT: A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.
Journal of Agricultural and Food Chemistry 07/2007; 55(12):4735-40. · 2.82 Impact Factor
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ABSTRACT: A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal temperature and pH of 95 degrees C and 5.0, respectively, retaining 97% of its activity at 100 degrees C. Incubation at 60 degrees C for 20 min during the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with starch indicated that cyclic alpha-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed by an increase in beta-CD. The major final product of PFCGT cyclization was beta-CD, and thus the enzyme is a beta-CGTase.
Extremophiles 06/2007; 11(3):537-41. · 2.94 Impact Factor
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ABSTRACT: Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.
Biochimica et Biophysica Acta 06/2007; 1774(5):661-9. · 4.66 Impact Factor
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ABSTRACT: A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.
Bioscience Biotechnology and Biochemistry 06/2007; 71(5):1348-52. · 1.28 Impact Factor
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ABSTRACT: ABSTRACTA branched oligosaccharides (BOS) mixture was prepared from liquefied corn starch using Bacillus lichenifrmis maltogenic amylase (BLMA). A highly concentrated BOS was prepared by removing glucose and maltose through yeast fermentation, which increased the BOS content. Water activity (aw) of bread went from 0.87 to 0.82 when 10% (w/w) or to 0.78 when 20% (w/w) BOS was added. BOS lowered aw more effectively as concentration of BOS increased. The water sorption isotherm of BOS was much higher than that of sucrose at aw, 0.1–0.8. BOS also prevented starch retrogradation in bread, probably due to steric hinderance and to the state of water retained by BOS. Relative sweetness of BOS was ± 17.5% of sucrose. The BOS mixture produced by BLMA might provide a new type humectant for foods that is low in sweetness and retards retrogradation.
Journal of Food Science 08/2006; 60(3):516 - 521. · 1.66 Impact Factor
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Hee-Seob Lee,
Jin-Soo Kim,
Kyuho Shim,
Jung-Woo Kim,
Kuniyo Inouye,
Hiroshi Oneda,
Young-Wan Kim,
Kyung-Ah Cheong,
Hyunju Cha,
Eui-Jeon Woo,
Joong Hyuck Auh,
Sung-Joon Lee, Jung-Wan Kim,
Kwan-Hwa Park
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ABSTRACT: As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.
FEBS Journal 02/2006; 273(1):109-21. · 3.79 Impact Factor
