• Journal of Life Science
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• v.29 no.8
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• pp.916-921
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• 2019

In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase ${\delta}$ and ${\varepsilon}$ encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures ($38^{\circ}C$ and $40^{\circ}C$), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than $38^{\circ}C$. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126-Ht strains that proliferated at $40^{\circ}C$ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.256-260
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• 1991

In B. subtilis, $\alpha$-amylase synthesis is regulated by amyR located directly on the upstream of amyE. Three different amyR alleles have been reported, amyR1, amyR2 and amyR3. Strains bearing the gra-10 mutation which confers derepression for catabolite repression has GlongrightarrowA transition mutation at ＋5 of amyR1. S1 nuclease mapping demonstrated that transcription initiated at 8 bases downstream from the -10 region of putative E$\sigma^{A}$ promoter P1 in amyR1 and gra-10. In amyR2, the major transcription initiatd at the same place and the minor, 10 bases downstream from -10 of P2. The transcript from P2 contributed approximately 15-20% of total amyE mRNA. S1 nuclease protection experiment indicated that amyE mRNA levels corresponded to the rate of synthesis assumed by specific activities of $\alpha$-amylase in culture supernatants, suggesting that $\alpha$-amylase synthesis is regulated at the level of transcription.n.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.80-85
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• 2010

Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.

• Journal of Gastric Cancer
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• v.21 no.1
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• pp.30-37
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• 2021

Purpose: While the amylase concentration of the drainage fluid (dAmy) has been reported to be a predictor of postoperative pancreas-related complications (PPRC), the optimal timing for its measurement has not been fully investigated. Materials and Methods: The clinicopathological data of 387 patients who underwent elective gastrectomy for gastric cancer were reviewed. Laboratory data, including dAmy on postoperative days 1 (dAmy1) and 3 (dAmy3), and serum C-reactive protein (sCRP) concentrations on postoperative days 1 (sCRP1) and 3 (sCRP3) were compared between patients with PPRC and without PPRC. Results: Nineteen of the 387 patients (4.9%) developed PPRC. The optimal cutoff values of dAmy1, dAmy3, sCRP1, and sCRP3 were 1514 IU/L, 761 IU/L, 8.32 mg/dL, and 15.15 mg/dL, respectively. The area under the curve of dAmy1 was greater than that of dAmy3 (0.915 vs. 0.826), and that of sCRP3 was greater than that of sCRP1 (0.820 vs. 0.659). In the multivariate analysis, dAmy1 (P<0.001) and sCRP3 (P=0.004) were significant predictors of PPRC, while dAmy3 (P=0.069) and sCRP1 (P=0.831) were not. Thirteen (41.9%) of 31 patients with both dAmy1 ≥1,545 IU/L and sCRP3 ≥15.15 mg/dL had PPRC ≥Clavien-Dindo II. In contrast, among 260 patients with both dAmy1 <1,545 IU/L and sCRP3 <15.15 mg/dL, none developed PPRC. Conclusions: dAmy1 was more useful than dAmy3 in predicting PPRC. The combination of dAmy1 and sCRP3 may be a useful criterion for the removal of drains on postoperative day 3.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.151-157
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• 2010

Barley malt produces two different $\alpha$-amylase isozymes (AMY1 and AMY2), which share up to 80% of amino acid sequence identity with each other. However, their enzymatic properties differ remarkably. In this study, five chimeric enzymes between AMY1 and 2 were constructed by staggered extension process (StEP) technique, and their enzymatic properties were characterized. According to the results, chimeric AMY-D2, D8, and E12 showed the mixed or intermediate types of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY-E10 chimera could be significantly inhibited by barley $\alpha$-amylase/subtilisin inhibitor (BASI) protein. Chimera AMY-C6 showed the same calcium-dependency as AMY1, while AMY-E10 was closely similar to AMY2. As a result, it can be proposed that some amino acid residues in the region II, III, and IV of barley $\alpha$-amylases can play very important roles in the interaction with BASI, and those in III, V, VI, and VII may partly affect on the calcium-dependent activity.

• 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.

• Korean Journal of Food Science and Technology
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• v.42 no.2
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• pp.198-203
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• 2010

Although barley $\alpha$-amylase isozyme 1 (AMY1) and 2 (AMY2) share up to 80% of amino acid sequence identity, their enzymatic properties differ remarkably. In this study, the 42nd alanine residue of AMY2 was replaced with another random amino acid via saturation mutagenesis. Eight out of 370 recombinant E. coli cells showing enhanced starch-hydrolyzing activity were characterized as possessing the same proline residue instead of alanine. Even though the specific activity of AMY2-A42P is reduced to 81% of wild-type, its expression level and purification yield were enhanced by approximately 2 and 4 times that of AMY2, respectively. Characterization of its enzymatic properties confirmed that AMY2-A42P is similar to that of wild-type. However, its specificity to starch substrates is likely to be intermediate between AMY1 and AMY2.

• 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.

• 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.

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.36-41
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• 2003

In order to increase the production and secretion rate of mouse salivary $\alpha$-amylase from Saccharomyces cerevisiae, various experiments were attempted. A plasmid pCNNinv (AMY) was constructed by the substitution of ADCl promoter and native signal sequence of mouse salivary $\alpha$-amylase cDNA gene with PRBI promoter and yeast invertase leader sequence, which resulted in 25% increase in the production of $\alpha$-amylase in the culture medium. The respiratory deficient transformant carrying pCNNinv (AMY) were obtained by treating yeast cells with ethidium bromide, and the $\alpha$-amylase activities in the culture brothes of the respiratory-deficient transformants were 5-8 times higher than that of parental wild type strain. $\alpha$-Amylase activity was also increased 3 times when the 0.015% (w/v) of 2-mercaptoethanol was added to the culture medium.