• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.457-464
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• 2008

An extracellular enzyme (RMEBE) possessing ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferring activity was purified to homogeneity from Rhodothermus marin us by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex-200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at $80^{\circ}C$. Its half-life was determined to be 73.7 and 16.7 min at 80 and $85^{\circ}C$, respectively. The enzyme was also halophilic and highly halotolerant up to about 2M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial ${\alpha}-(1{\rightarrow}4)$-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferase activity for small maltooligosaccharides, producing disproportionated ${\alpha}-(1{\rightarrow}6)$-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.

• Korean Journal of Food Science and Technology
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• v.26 no.6
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• pp.819-823
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• 1994

For the development of rice-derived fat replacing ingredient, low dextrose equivalent (D.E.) malto dextrin was prepared by enzyme hydrolysis, and its physical and rheological properties were studied. The molecular sizes of rice maltodextrin were measured by gel permeation chromatography on Sephadex G-50. Gel permeation column chromatograms showed a large single peak, suggesting a limited hydrolysis, and the average degree of polymerization decreased from 72.8 for 3 D.E. maltodextrin to 48.7 for 6 D.E. maltodextrin. Cold water solubility of maltodextrin was increased with increasing D.E. value and its values ranged from 47.3% to 71.3%. 8% solution of rice maltodextrin showed pseudoplastic behavior. Flow behavior index was decreased as D.E. value was increased.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.969-975
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• 2003

Two conserved amino acid residues in the extra sugar-binding space near the catalytic site of Thermus maltogenic amylase (ThMA) were analyzed for their role in the hydrolysis and transglycosylation activity of the enzyme. Site-directed mutagenesis was carried out by replacing N33l with a lysine (N331K), E332 with a histidine (E332H), or by replacing both residues at the same time (N331K/E332H). The measured $K_m$ values indicated that affinities toward all substrates tested, including starch, pullulan, ${\beta}-cyclomaltodextrin$, and acarbose, were lower in all the mutants compared to that of wild-type ThMA, leading to reduced hydrolysis activity. In addition, the lower ratio of transglycosylation to hydrolysis in the mutants compared to that in the wild-type ThMA indicated that these mutants preferred hydrolysis to the transglycosylation reaction. These results demonstrated that the conserved dipeptide at 331 and 332 of ThMA is directly involved in the formation and accumulation of transfer products by accommodating acceptor sugar molecules.

• Microbiology and Biotechnology Letters
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• v.10 no.2
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• pp.123-132
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• 1982

These experiments were conducted to investigate the enzymatic characteristics of the purified $\alpha$-amylase (F-2A) of Bacillus circulans F-2 and the digestion rate of various starches. 1. The molecular weight was estimated to be 93000 by SDS-polyacrylamide disc gel electrophoresis. The isoelectric point was about pH 5.0. The optimum pH for the enzyme action was 6.0-6.5 and the stable pH ranged pH 5.5-12.0. The optimum temperature was 6$0^{\circ}C$, and the purified $\alpha$-amylase was stable below 4$0^{\circ}C$. 2. The purified $\alpha$-amylase was activated by Mn$^{＋＋}$ and Co$^{＋＋}$, whereas it was inhibited by Ag$^{＋}$, HT$^{＋＋}$, Cu$^{＋＋}$ and Pb$^{＋＋}$. 3. The purified $\alpha$-amylase is considered to have no sulfhydryl residue essential for its catalytic activity. 4. Michaelis constant (Km) was 1.704 mg/$m\ell$. Activation energy between 25-4$0^{\circ}C$ was 12.297 Kcal/mole, and between 40-6$0^{\circ}C$, it was 7.831 Kcal/mole. 5. The hydrolysis product from soluble starch, amylose and amylopectin in the early stage of hydrolysis was G$_{6}$, and as hydrolysis proceeds, G$_4$and G$_2$appeared. 6. Products from each oligosaccarides are as follows: G$_4$longrightarrow G$_2$＋ G$_2$,G$_3$ ＋G$_1$,G$_{5}$longrightarrow G$_4$＋G$_1$,G$_{6}$longrightarrowG$_4$＋ G$_2$,G$_{7}$ G$_4$,G$_{8}$longrightarrow G$_4$＋G$_4$, 7. On raw potato starch, raw sago starch and raw yam starch, the purified enzyme exhibited a remarkably high digestion rate than Porcine pancreatic amylase and Streptococcus bovis amylase.

• YAKHAK HOEJI
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• v.6 no.2
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• pp.18-22
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• 1962

Purified preparation of .betha.-amylase is obtained from radish root by the means of fractional precipitation with ammonium sulfate. Purified preparation saccharifies the starch, .betha.-maltose being formed. Dextrinization in the true sense does not take place. Hydrolysis ceases when approximately 50% of the theoretical yield of maltose is obtained and there remains a substance (to be .betha.-limit dextrin) which gives a blue-violet with iodine, no glucose being formed. Stability of preparation is optimal at pH 4-9 and more completely inactivated at 65.deg. in fifteen minutes. .betha.-Amylase of radish exhibits optimal activity at and near pH 5.0, which varied depending upon the buffer. Calcium and chloride ions do not effect the activities of enzyme. The results of experiments with oxidizing, alkylating and mercaptide-forming reagents which have been reported to be specific for sulfhydryl groups confirm that free sulfhydryl groups are essential to the activity of .betha.-amylase from radish.

• Mycobiology
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• v.29 no.4
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• pp.183-189
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• 2001

Extracellular amylase properties were examined with the mycelium of Tricholoma matsutake isolated from ectomycorrhizal roots of Pinus densiflora. The molecular weights of $\alpha$-amylase and glucoamylase were estimated as 34.2 kD and 11.5 kD, respectively, after eluted through Superdex 75 column. The optimum pH of the purified enzyme was found in a range of pH $5.0{\sim}6.0$, with a peak at pH 5.0. The activities of these enzymes were stable from $4^{\circ}C\;to\;30^{\circ}C$. The $\alpha$-amylase of T. matsutake readily hydrolyzed soluble starch and amylose-B, while it weakly hydrolyzed glycogen, dextrin, amylose and amylose-A. The main products of hydrolysis were confirmed to be glucose, maltose and maltotriose on the basis of the similarities in the thin layer chromatographic mobility.

• Archives of Pharmacal Research
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• v.8 no.2
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• pp.67-75
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• 1985

To find emylase inhibitors produced by microorganisms from soil, a strain which had a strong inhibitory activity against bacteria .alpha.-amylase was isolated from the soil smaple collected in Seoul. The morphological and physiological characteristics of this strain on several media and its utilization of carbon sources showed that it was one of Streptomyces specties according to the international Streptomyces Project method. The amylase inhibitor of this strain was purified by means of acetone precipitation, adsorption on Amberlite XAD-2, and column chromatography on Amberlite CG-50 and SP-Sephadex C-25. The inhibitor was stable at the pH range of 1-10 and at 100.deg.C for half an hour, and had inhibitory activities against other amylases such as salivary .alpha.-amylase, pancreatic .alpha.-amylase, fungal .alpha.-amylase and glucoamylase. The kinetic studies of the inhibitor showed that its inhibitory effect on starch hydrolysis by .alpha.-amylase was non-competitive.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.363-369
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• 2016

A putative cyclomaltodextrinase gene (licd) was found from the genome of Listeria innocua ATCC 33090. The licd gene is located in the gene cluster involved in maltose/maltodextrin utilization, which consists of various genes encoding maltose phosphorylase and sugar ABC transporters. The structural gene encodes 591 amino acids with a predicted molecular mass of 68.6 kDa, which shares less than 58% of amino acid sequence identity with other known CDase family enzymes. The licd gene was cloned, and the dimeric enzyme with C-terminal six-histidines was successfully produced and purified from recombinant Escherichia coli. The enzyme showed the highest activity at pH 7.0 and 37℃. licd could hydrolyze β-cyclodextrin, starch, and maltotriose to mainly maltose, and it cleaved pullulan to panose. It could also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. In particular, it showed significantly higher activity towards β-cyclodextrin and maltotriose than towards starch and acarbose. licd also showed transglycosylation activity, producing α-(1,6)- and/or α-(1,3)-linked transfer products from the acarbose donor and α-methyl glucopyranoside acceptor.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.415-420
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• 1986

Alcohol fermentation of uncooked naked barley was carried out by the combined action of the maceration enzyme from black Aspergillus niger and the glucoamylase from Rhizopus sp. The combined enzyme preparation was found to be effective in maceration and saccharification of the raw naked barley starch. The Hydrolysis rate measured by the amount of glucose liberated reached more than 70% at pH 4.5 and 3$0^{\circ}C$ after 76 hrs. For alcohol fermentation without cooking, the naked barley mash of 18% initial total sugar was pretreated with concentrated sulfuric acid (0.15 weight % of the mash volume) at 55$^{\circ}C$ for 2hr, and used for alcohol fermentation. A simultaneous saccharification and fermentation was carried out at pH 4.8 and 3$0^{\circ}C$ for 96 hrs. Under this fermentation condition, 3.5% increase in alcohol yield together with 2.0% increase in alcohol concentration were obtained when compared with the conventional cooking fermentation.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.765-775
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• 2019

A new ${\alpha}$-amylase-encoding gene (amySL3) of glycoside hydrolase (GH) family 13 was identified in soda lake isolate Alkalibacterium sp. SL3. The deduced AmySL3 shares high identities (82-98%) with putative ${\alpha}$-amylases from the genus Alkalibacterium, but has low identities (<53%) with functionally characterized counterparts. amySL3 was successfully expressed in Escherichia coli, and the recombinant enzyme (rAmySL3) was purified to electrophoretic homogeneity. The optimal temperature and pH of the activity of the purified rAmySL3 were determined to be $45^{\circ}C$ and pH 7.5, respectively. rAmySL3 was found to be extremely halophilic, showing maximal enzyme activity at a nearly saturated concentration of NaCl. Its thermostability was greatly enhanced in the presence of 4 M NaCl, and it was highly stable in 5 M NaCl. Moreover, the enzyme did not require calcium ions for activity, and was strongly resistant to a range of surfactants and hydrophobic organic solvents. The major hydrolysis products of rAmySL3 from soluble starch were maltobiose and maltotriose. The high ratio of acidic amino acids and highly negative electrostatic potential surface might account for the halophilic nature of AmySL3. The extremely halophilic, calcium-independent, and surfactant-resistant properties make AmySL3 a promising candidate enzyme for both basic research and industrial applications.