• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.123-129
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• 2000

Expression of the crylAcl gene of an acrystalliferous Bacillus thuringiensis strain under the control of the native or $\alpha$-amylase gene promoter was investigated. The crylAcl gene was cloned in a B. thuringiensis - E. coli shutle vector, pHT3101, undder the control of either the native promoter (pProAc) or the $\alpha$-amylase promoter from Bacillus subtilis (pAmyAc). These two recombinant plasmids were successfully expressed in B. thuringiensis subsp. kurstaki Cry B. The first transformant (ProAc/CB), harboring pProAc, expressed an about 130 kDa protein begining 24 hr after inoculations just as in the case of the wild type of B. thuringiensis subsp. kurstaki HD-73. The second pAmyAc-transformant (AmyAc/CB) began to express the gene just 6 hr after inoculation, but Western analysis showed that the activity of the $\alpha$-amylase promoter was relatively weaker than that of the native promoter. As expected, their toxicity against Plutella xylostella larvae was dependent on the amount of Cry1Acl protein expressed.

• Applied Biological Chemistry
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• v.41 no.1
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• pp.18-22
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• 1998

A mutant strain which hyperproduced thermostable ${\alpha}-amylase$ was obtained by chemical mutagenesis of Bacillus licheniformis. The mutant strain, SK-5, produced the enzyme about 50 times higher than the original strain. The mutant was longer and slimmer in shape, slower in growth compared to the original strain. Nucleotide sequence analysis of the SK-5 ${\alpha}-amylase$ gene revealed no changes in the the structural gene. The changes found in the promoter region might be responsible for the hyperproduction of the enzyme by the mutant. No structural changes in the enzyme structure could be observed when the secreted enzymes at various culture times were analyzed by Western blot.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.115-121
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• 1992

In order to broaden the spectrum of substrate utilization of a Gram negative bacterium Zymomonas mobilis which has a great potential as an industrial ethanol producing microorganism, cloning of $\alpha$-amylase gene into Z. mobilis ZM4 was tried. The $\alpha$-amylase gene was isolated from Bacillus stearothermophilus. By Southern blot analysis, it was proven that the $\alpha$-amylase gene fragment was originated from a naturally occuring plasmid of B. stearothermophilus ATCC 31195. To place $\alpha$-amylase gene under the control of Z. mobilis promoter, two different Z. mobilis expression vectors, pZA26 and pLOI204, were used. The truncated $\alpha$-amylase gene was then introduced into these vectors. Both qualitative and quantitative activities of $\alpha$-amylase were observed in Z. mobilis cells harboring these plasmids with the $\alpha$-amylase gene inserted. Gas chromatographic analysis of ethanol showed that one of the Z. mobilis transconjugants was capable of producing 67 mM ethanol from rich medium(RM) containing 5% soluble starch as a sole carbon source.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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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.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.68-75
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• 1998

The overproduction and high level secretion of Glucose Oxidase (GOD) from A. niger in S. cerevisiae was carried out by cloning GOD gene. For this purpose, using two different strong promoters (ADH1 promoter, GAL10 promoter) and signal sequences (${alpha}$-MF signal sequence of S. cerevisiae and ${alpha}$-amylase signal sequence of A. oryzae) and GAL7- and GOD terminator, four expression vectors were constructed. All the expression vectors were transformed in S. cerevisiae 2805 using auxotroph method. By the flask culture, transformants of pGAL expression vector series containing GAL 10 promotor showed much higher GOD productivity than transformants of pADH expression vector series containing ADH1 promoter Transformants of pGALGO2 containing GAL10 promotor and ${alpha}$-amylase signal sequence has shown the best productivity of GOD ($GOD_{total}$: 10.3 unit/mL, $GOD_{ex}$: 8.7 unit/mL) at 115 hr. This value was three fold higher than that of pGALGO1 containing GAL 10 promotor and ${alpha}$-MF signal sequence, even if the same promotor was involved. Through the ${alpha}$-amylase signal sequence of A. oryzae, GOD was secreted much more than the case of ${alpha}$-MF signal sequence from S. cerevisiae. These results suggest that signal sequence may play a important roles in not only the secretion but also the overproduction of foreign protein. Secretion rate of GOD in pGALGO1 and pGALGO2 was 89% and 84%, respectively, Because of the overglycosylation in S. cerevisiae the molecular weight of recombinant GOD in S. cerevisiae was much larger (250 kDa) than that of nature GOD in A. niger (170 kDa).

• 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.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.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.625-630
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• 1998

Using a modified yeast secretory expression vector, $\alpha$-amylase of Schwanniomyces occidentalis was produced from Saccharomyces cerevisiae. The expression vector contains the a-amylase gene (AMY) harboring its own promoter without the regulatory region and the adenine base at the -3 position from the ATG start codon, its own signal sequence, CYC1 transcription terminator, and SV40 enhancer. The expressed $\alpha$-amylase activity from cells carrying the plasmid was approximately 26 times higher than that from the cells harboring an unmodified plasmid. When Saccharomyces diastaticus was transformed with this modified vector, a 2.5 times higher level of amylolytic activity than that from Sch. occidentalis was observed.

• Journal of Microbiology
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• v.34 no.1
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• pp.74-81
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• 1996

Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the .alpha.-amylase gene from an .alpha.-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the .alpha.-amylase gene for easy replacement of various foregn structural genes. To evaluate this secretion vectors, the .betha.-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active .betha.-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each .betha.-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed .betha.- lactamases were located idn the culture medium. The amount of the secreted .betha.-lactamase was about 80% of the total secreted proteins in the culture medium.