• Journal of the Korean Society of Food Science and Nutrition
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• v.19 no.6
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• pp.625-635
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• 1990

The purpose of this study was focused on investigation of biochemical properties of amylases in germinating corn(Zea mays L.) the amylase(I), (II) and (III) from germinating corn seeds were partially purified by ammonium sulfate precipitation, DEAE-Sephadex A-50 ion exchange column chromatography and Sephadex G-100 gel filtration. The last step was effective for separation of the corn amylases to a homogeneous slate. the purified amylase(I) was identified as a kind of $\alpha$-amylase from the fact that 5％ starch solution was hydrolysed into mainly maltose and maltotetrose by it, and amylase(II) and amylase(III) were enzymes producing maltotetrose as main product. The molecular weight and specific activity of the amylase(I), (II) and (III) were determined to be 54,000 and 70.47 unit/mg, 39,000 and 62.98 unit/mg, and 51,000 and 80.39 unit/mg, respectively. It showed a tendency to increase the amylases activities in presence of Ba, Ca, Co and Fe groups, but inhibits in that of Ag, Sn, Hg and Zn groups, and amylase(I), (II) and (III) remained stable at pH 5-6 and 2$0^{\circ}C$ for 40 days in containing of 1 mM CaCl$_2$. The optimum pH and optimum temperatures were pH 6, pH 5 and pH 6 and 35$^{\circ}C$, 55$^{\circ}C$ and 55$^{\circ}C$, respectively. These results suggest that the amylase(I), (II) and (III) were different amylases.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1306-1318
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• 2009

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two $\alpha$-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same $\alpha$-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during S. sclerotiorum cultivation. The structural gene of Scamy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal $\alpha$-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known $(\beta/\alpha)_8$ barrel motif in domain A, have a typical structure of $\alpha$-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal $\alpha$-amylase described until now with the smallest catalytic domain.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.4
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• pp.253-262
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• 2000

Aspergillus is considered to be an attractive host for heterologous protein production because of its safety and ability to secrete large amounts of proteins. In order to obtain high productivity, thus far promoters of amylases have been most widely used in A. oryzae. Recent progress in cloning and expression analysis, including EST sequencing, revealed that glycolytic genes represent some of those most strongly expressed in A. oryzae. Therefore, promoters of glycolytic genes could be important alternatives to promoters of amylases because lower amounts of proteases are produced in the presence of glucose. Several A. oryzae transcription factors responsible for the induction and/or maximum expression of many industrially important genes encoding amylases and proteases have been cloned and characterized. In addition to the transcriptional regulatory factors, the gene encoding the largest subunit of RNa polymerase II, constituting the basic transcription machinery, has also been cloned from A. oryzae. This recently acquired understanding of the details of transcriptional regulatory mechanisms and factors will facilitate engineering flexible controls for the expression of proteins important for the fermentation industries.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.563-568
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• 2010

The Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the $\alpha$-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant $\alpha$-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type $\alpha$-amylase, the mutant $\alpha$-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant $\alpha$-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the $\alpha$-amylase activity. Interestingly, the mutant $\alpha$-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.

• Korean Journal of Food Science and Technology
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• v.40 no.1
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• pp.70-75
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• 2008

Two extracellular amylase isozymes were purified and characterized from alkalophilic Pseudomonas sp. KFCC 10818 for the production of maltooligosaccharides. The molecular weights of the homogeneous proteins were 50 kDa and 75 kDa, respectively. The 50 and 75 kDa amylases showed optimum temperatures at 35 and $40^{\circ}C$, respectively. The optimum pH of the enzymes ranged from pH 6-8, and the enzymes were resistant to an alkaline condition of pH 12. Via the enzyme's actions, the final products from maltooligosaccharides or soluble starch were maltose and maltotriose. Calcium was a potent activator of the 50 kDa amylase. Finally, the N-terminal amino acid sequences of the 50 and 75 kDa amylases were QTVPKTTFV and DTVPGNAFQ, respectively.

• Bulletin of the Korean Chemical Society
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• v.15 no.10
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• pp.832-835
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• 1994

The ${alpha}$-amylase gene of Bacillus licheniformis has been cloned and two mutant ${alpha}$-amylase genes of which histidine 235 was changed to glutamine (H235Q) and aspartic acid 328 to glutamic acid (D328E) have been produced by site-directed mutagenesis. The kinetic parameters, optimum pH and thermostability of wild type(WT) and these two mutant amylases expressed in E. coli MC1061 have been compared after purification. The $K_m$ values of WT, H235Q and D328E ${alpha}$-amylases were 0.22%, 0.73%, and 0.80% respectively, when using starch as the substrate. The $V_max$ values of wild type ${alpha}$ -amylase and mutant ${alpha}$-amylases were 0.6-0.7%/minute, and did not show any significant differences among them. The optimum pH of D328E ${alpha}$-amylase was shifted to more acidic pH. Also, the thermostability of H235Q ${alpha}$-amylase was increased compared to the wild type ${alpha}$-amylase.

• Korean journal of food and cookery science
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• v.12 no.4
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• pp.522-534
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• 1996

The present study was conducted to investigate quality characteristics of Nochi made with malted barley flour with (Cl) and without hull (C2), comparing with Nochi that was treated with different sources of commercial amylases. There was higher level of moisture content (18.4%) in Nochi treated with fungal ${\alpha}$-amylase (FU) comparing with the other Nochi samples. However, Nochi that was treated with bacterial ${\alpha}$-amylase and ${\beta}$-amylase (BA-${\beta}$) had the lowest level of moisture content (11.2%). Nochi samples which were treated with thermostable ${\alpha}$-amylase and fungal ${\alpha}$-amylase(TE-FU) were different from traditional Nochi samples in mechanical characteristics. According to the results of sensory evaluation, Cl was similar to C2 except in cohesiveness and malt flavor. TE-FU and Bh-${\beta}$ were not different from traditional Nochi in cohesiveness, sweetness and overall desirability.

• Applied Biological Chemistry
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• v.30 no.4
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• pp.305-310
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• 1987

The enzymatic properties of ${\beta}-amylase$ from soybean and sweet potato were compared. The sweet potato enzyme consists of four identical subsunits whereas soybean enzyme has no subunit $structure^{12,\;15)}$. In the denatured state, both enzymes exhibited the same molecular weight on SDS-gel electrophoresis and on gel-filtration analysis. The spectra of circular dichroism revealed that both enzyme have almost same secondary structure but the environment of aromatic side chains are different. The chemical cleavage of soybean and sweet potato ${\beta}-amylases$ at cysteine residues and methionine residues demonstrated the homology of amino acid sequence between the enzymes. The similarity between soybean and sweet potato ${\beta}-amylase$ was also revealed by immunological method. The antibody for soybean enzyme inhibited the activity of sweet potato enzyme but it did not inhibit the activity of wheat, barley and Japanese-raddish ${\beta}-amylases$.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.127-131
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• 1991

${\alpha}-amylase\;and\;{\beta}-amylase$ were extracted from barley and wheat malt, respectively. Their kinetic parameters on gultinous and nonglutinous rice starch were examined. During the germination of barley and wheat, the increaments of ATP levels were significant after 2-day germination and the levels were reduced after 5 days. The dry weights were decreased after 3 days. The activities of amylases were the highest for 6 days in the barley and wheat malt. As for ${\alpha}-amylase$, that the substrate affinity of barley malt on nonglutionous rice starch was greater than other cases. The $V_{max}$ values of ${\alpha}-amylase$ from wheat malt on either type of rice starch showed high, and from barley malt on nonglutinous rice starch were high. The ${\beta}-amylse$ from barley malt showed high substrate affinity on the glutinous rice starch, and $V_{max}$ value of the enzyme from wheat malt on glutinous rice starch was higher than other. The substrate efficiency ($V_{max}/K_{m}$) of ${\beta}-amylase$ on the non glutinous rice strach was better than other cases.

• Journal of the Korean Society of Food Science and Nutrition
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• v.14 no.2
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• pp.188-191
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• 1985

A thermophilic and cellulolytic bacterium, Herpetosiphon geysericola CUM 317 isolated from the compost, produced ${\alpha}-amylase,\;{\beta}-amylase$, and glucoamylase. Mutual relationships on the production of the three amylases were studied by changing the cultivation conditions. ${\alpha}-Amylase$ and glucoamylase were produced highly after 40 hrs on wheat bran medium at $50^{\circ}C$ and after 30 hrs on liquid medium at $40^{\circ}C$, though ${\beta}-amylase$ was produced best at 10 hrs of initial cultivation phase. The production of the amylases was generally repressed by the addition of carbon sources in liquid medium containing polypeptone. ${\alpha}-Amylase$ production was enhanced relatively by the addition of cupric sulfate in the liquid medium, ${\beta}-amylase$ was enhanced by cadmium sulfate, and glucoamylase was enhanced by calcium chloride.