• KOREAN JOURNAL OF CROP SCIENCE
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• v.38 no.3
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• pp.275-282
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• 1993

The objective of this study was to clone a partial fragment of $\alpha$-amylase from Korean maize. We designed and synthesized an oligonucleotide probe and two kinds of PCR primers based on cDNA conserved region of $\alpha$-amylase sequences from other plants. Total RNA from 3-day-old maize seedling was used as template for 1st strand cDNA synthesis and RNA-DNA hybrid was used as template for polymerase chain reaction(PCR). The product of PCR was about 0.5 kb long and inserted into pUC19. We named this recombinant plasmid as pZM$\alpha$'. The cloned fragment was certified by Southern blot analysis using labeled synthetic oligonucleotide as probe.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1307-1313
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• 2010

An extracellular ${\alpha}$-amylase from Lactococcus lactis IBB500 was purified and characterized. The optimum conditions for the enzyme activity were a pH of 4.5, temperature of $35^{\circ}C$, and enzyme molecular mass of 121 kDa. The genome analysis and a plasmid curing experiment indicated that $amy^+$ genes were located in a plasmid of 30 kb. An analysis of the phylogenetic relationships strongly supported a hypothesis of horizontal gene transfer. A strong homology was found for the peptides with the sequence of ${\alpha}$-amylases from Ralstonia pikettii and Ralstonia solanacearum. The protein with ${\alpha}$-amylase activity purified in this study is the first one described for the Lactococcus lactis species, and this paper is the first report on a Lactococcus lactis strain belonging to the amylolytic lactic acid bacteria (ALAB).

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.254-263
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• 2014

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative ${\alpha}$-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in high-level expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at $40^{\circ}C$. The ${\alpha}$-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its ${\alpha}$-amylase can be useful in lowering EP molecular weight and in starch processing.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.213-218
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• 1986

Hybrid plasmid pEA24, shuttle vector YRp7 carrying amylase gene of Bacillus amyloliquefaciens, was transformed to yeast Saccharomyces cerevisiae, and the expression of B. amyloliquefaciens amylase gene in yeast was investigated. The frequency of transformation to S. cerevisiae DBY747 with YRp7 was increased by treatment of 40% polyethylene glycol (MW 4, 000), PH 7.0, at 3$0^{\circ}C$, and by regeneration used 2% top agar. The amount of cellular amylase activity produced by S. cerevisiae containing pEA24 was 2% of that secreted from B. amyloliquefaciens, but in case of S. cerevisiae transformant, the amylase secreted was not detected. A comparison of genetic stability of pEA24 and YRp7 plasmids in yeast was carried out by cultivation of transformants in tryptophan-supplement-medium. The pEA24 plasmid was more unstable than YRp7 in S. cerevisiae. The size of pEA24 extracted from S. cerevisiae transformants was found to be identical with that from E. coli transformants by agarose gel electrophoresis.

• Preventive Nutrition and Food Science
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• v.6 no.4
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• pp.206-210
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• 2001

Starch is an abundant resource in plant biomass, and it should be hydrolyzed enzymatically into fermentable sugars for ethanol fermentation. A genetic recombinant yeast, Saccharomyces cerevisiae GA-7458, was constructed by integrating the structural gene of both $\alpha$-amylase from Bacillus stearothermophilus and the gene (STA1) encoding glucoamylase from S. diastaticus into the chromosome of S. cerevisiae SH7458. The recombinant yeast showed active enzymatic activities of $\alpha$-amylase and glucoamylase. The productivity of ethanol fermentation from the pH-controlled batch culture (pH 5.5) was 2.6 times greater than that of the pH-uncontrolled batch culture. Moreover, in a fed-batch culture, more ethanol was produced (13.2 g/L), and the production yield was 0.38 with 2% of corn starch. Importantly, the integrated plasmids were fully maintained during ethanol fermentation.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1390-1401
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• 2023

In this study, a 12-week feeding experiment was conducted to characterize the effects of exogenous α-amylase on the growth, feed utilization, digestibility, plasma α-amylase activity, feed degradation rate, and fecal particle size of olive flounder (Paralichthys olivaceus). Diet was supplemented with 0 (AA₀; control), 100 (AA₁₀₀), 200 (AA₂₀₀), or 400 (AA₄₀₀) mg/kg of α-amylase, respectively. Fish (273.1 ± 2.3 g) were stocked into 12 tanks (25 fish/1,000-L tank) and 3 tanks were randomly selected for each diet group. As a result, α-amylase was found to have no significant effects (p ≥ 0.05) on the growth, feed utilization parameters, and whole-body proximate compositions. α-Amylase-treated fish exhibited only a significant increase in the apparent digestibility coefficient of carbohydrates compared to the controls. In addition, in vitro analyses revealed that α-amylase dose-dependently increased (p < 0.05) the feed degradation rate, while photographs of the intestinal content after 2, 4, and 8 h of feeding demonstrated an improved degradation rate in the α-amylase-treated groups. Plasma α-amylase content was higher in the AA₂₀₀ and AA₄₀₀ groups, whereas the control group produced significantly larger-sized fecal particles (90% size class) than these two groups. In the intestine, no changes were observed in the expression levels of the immune-related TNF-α, IL-1β, IL-2, immunoglobulin-M, HSP-70, lysozyme, and amylase alpha-2A. However, growth-related genes IGF-1, IGF-2, TGF-β3, and growth hormone genes were upregulated in muscle tissues. Collectively, exogenous α-amylase has positive roles in the modulation of the digestibility coefficient, blood α-amylase concentration, growth-related gene expression, and diet degradation for improved digestion in olive flounder.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1013-1021
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• 2005

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular $\alpha$-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5$\alpha$ and sequenced. The $\alpha$-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to $59\%$ sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and $40\%$. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5$\alpha$ was enhanced in the presence of $Mg^{2+}$ (20 mM) and $Ca^{2+}$ (30 mM) and inhibited in the presence of $Fe^{2+}$ and $Cu^{2+}$. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

• Journal of Microbiology
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• v.42 no.4
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

• Journal of Microbiology and Biotechnology
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• v.4 no.1
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• pp.7-12
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• 1994

A yeast strain secreting glucoamylase was transformed with an expression vector (pMS12) containing the promoter of yeast alcohol dehydrogenase I gene ADC1, mouse salivary $\alpha$-amylase cDNA, and a segment of yeast $21\mu m$ plasmid. The transformed strain could produce ethanol from starch (4%, w/v) through a direct one-step process with the conversion efficiency of 93.2%, during 5 days of fermentation, while the original, untransformed strain exhibited a conversion efficiency of 38.1% under the same condition. When the regulatory site of the ADC1 promoter region was removed, the production of ethanol increased to 29~37% in the presence of exogenous 3%(v/v) ethanol in the fermentation medium.

• 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.