• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1586-1592
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• 2017

Some promoters were isolated and characterized from the genome of Leuconostoc mesenteroides SY2, an isolate from kimchi, a Korean traditional fermented vegetable. Chromosomal DNA of L. mesenteroides SY2 was digested with Sau3AI and ligated with BamHI-cut pBV5030, a promoter screening vector containing a promoterless cat-86. Among E. coli transformants (TFs) resistant against Cm (chloramphenicol), 17 were able to grow in the presence of $1,000{\mu}g/ml$ Cm and their inserts were sequenced. Transcription start sites were examined for three putative promoters (P04C, P25C, and P33C) by primer extension. Four putative promoters were inserted upstream of a promoterless ${\alpha}$-amylase reporter gene in $pJY15{\alpha}$. ${\alpha}$-Amylase activities of E. coli TFs containing $pJY15{\alpha}$ (control, no promoter), $pJY03{\alpha}$ ($pJY15{\alpha}$ with P03C), $pJY04{\alpha}$ (with P04C), $pJY25{\alpha}$ (with P25C), and $pJY33{\alpha}$ (with P33C) were 66.9, 78.7, 122.1, 70.8, and 99.3 U, respectively. Cells harboring $pJY04{\alpha}$ showed 1.8 times higher activity than the control. Some promoters characterized in this study might be useful for construction of food-grade expression vectors for Leuconostoc sp. and related lactic acid bacteria.

• BMB Reports
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• 제34권4호
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• pp.347-354
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• 2001

$\alpha$-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and $90^{\circ}C$, and was stable from pH 6.0 to 85 and up to $90^{\circ}C$. The enzyme had a half-life of 85 minutes at $90^{\circ}C$. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of $\alpha$-1,6-glucosidic linkages of isomaltosaccharides and panose, $\alpha$-1,3-glycosidic bond of nigerose and turanose, and $\alpha$-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the $\alpha$-amylase family. We suggest that this monomeric, thermostable, and broad-acting $\alpha$-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel $\alpha$-glucosidase.

• 미생물학회지
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• 제26권2호
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• pp.73-81
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• 1988

$\alpha$-Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competancy by treating with $CaCl_{2}$. As the result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine. $\alpha$-Amylases from both N.crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel foltration column. As the result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1u/mg and 85u/mg respectively. The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were $70^{\circ}C$ and optimal pH were about 6 and 10.

• Mycobiology
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• 제39권1호
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• pp.33-39
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• 2011

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 ${\alpha}$-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 ${\alpha}$-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 ${\alpha}$-amylase production, and fermented alcohol better than commercial wine yeasts.

• Asian-Australasian Journal of Animal Sciences
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• 제2권3호
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• pp.499-500
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• 1989

• 한국동물학회:학술대회논문집
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• 한국동물학회 1998년도 한국생물과학협회 학술발표대회
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• pp.323.2-323
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• 1998

No Abstract, See Full Text

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.399-400
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• 2005

• 한국동물학회:학술대회논문집
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• 한국동물학회 1997년도 한국생물과학협회 학술발표대회
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• pp.299.2-299
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• 1997

No Abstract, See Full Text

• 한국동물학회:학술대회논문집
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• 한국동물학회 1999년도 한국생물과학협회 학술발표대회
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• pp.284.2-284
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• 1999

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.149-152
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• 2006

Leuconostoc mesenteroides SY1, a strain isolated from cabbage Kimchi, was transformed with pCW4, a shuttle vector based on a cryptic plasmid from Lactobacillus paraplantarum C7. $\alpha-Amylase$ gene, amyL, from Bacillus licheniformis was cloned into pCW4, resulting in $pCW4T{\alpha},\;and\;pCW4T{\alpha}$ was introduced into SY1 by electroporation. Transformation efficiency was $10^2cells/{\mu}g$ plasmid DNA. L. mesenteroides cells harboring $pCW4T{\alpha}$ did not show amylase activity, although amyL transcript was synthesized as determined by slot blot experiment. $pCW4T{\alpha}$ was stably maintained in SY1 in the presence of erythromycin (Em, $5\;{\mu}g/ml$) but rapidly lost when Em was omitted. Less than $1\%$ of the cells maintained $pCW4T{\alpha}$ after 5 days at $30^{\circ}C$.