• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.7-12
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• 2000

The expression of the mouse $\alpha-amylase$ gene in the methylotrophic yeast, P pastoris was investigated. The mouse $\alpha-amylase$ gene was inserted into the multi-cloning site of a Pichi a expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested with SalI or BglII, and was introduced into P. pastoris strain GSl15 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SaiII or BglII into the HIS4locus $(38\;of\;Mut^+\;clone)$ or into the AOX1 locus $(15\;of\;Mut^s\;clone)$. Southern blot was carried out in 11 transformants, which showed that the mouse $\alpha-amylase$ gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest $\alpha-amylase$ activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse $\alpha-amylase$ gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse $\alpha-amylase$ gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.209-212
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• 1986

$\alpha$-Amylase gene of Bacillus amyloliquetaciens was cloned on plasmid YEp13, S. cerevisiae-E. coli shuttle vector. Hybrid plasmid pTG17, carrying $\alpha$-amylase gene of B. amyloliquefaciens, was transformed to E. coli and the expression of it in yeast was investigated. This plasmid was unstable in E. coli and produced two minor plasmids, pTG17-1 and PTG17-2, which resulted from the segregation of it. Transformant of S. cerevisiae MC16 with pTG17-1 plasmid was not appeared on SD medium because of the Leu2 gene defection. S. cerevisiae could be transformed by the hybrid plasmid, and $\alpha$-amylase activity of the yeast transformant was detected by somogyi-Nelson method and agar diffusion method.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.166-173
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• 1992

The expression of Bacillus subtilis $\alpha$-amylase from the phoA-amyE fusion gene in recombinant E. coli was investigated under various environmental conditions. The overexpression of cloned $\alpha$-amylase caused retardations in cell growth and synthesis of alkaline phosphatase (AP) from the chromosomal phoA gene. The change of culture temperature from $37^\circ{C}$ to $30^\circ{C}$ increased the specific activities of both $\alpha$-amylase and $\beta$-lactamase by six and two times, respectively, whereas the AP activity remained unchanged. The experiments with chlorampenicol (a translation inhibitor) suggested the enhancement of $\alpha$-amylase activity at $30^\circ{C}$, and this was partly due to the stability of $\alpha$-amylase itself. The further decrease of the temperature to $25^\circ{C}$ slowed down both the cell growth and cloned-gene expression rate. The $\alpha$-amylase activity showed a maximum at pH of 7.4 while alkaline phosphatase was most effectively produced at pH of 8.3.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• 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.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.161-168
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• 1986

A 1.95Kb Sau3Al fragment coding for $\alpha$-amylase from Bacillus amyloliquefaciens was isolated by the shotgun method using Escherichia coli as a host. The genome of Bacillus amyloliquefaciens was partially digested with the restriction endonuclease Sau3Al and joined to plasmid YRp7 cleaved with the restriction endonuclease BamHI. The $\alpha$-amylase gene present in a 1.95Kb insert was stably maintained and expressed in Escherichia coli. The amount of $\alpha$-amylase activity produced by Escherichia coli containing the hybrid plasmid pEA24 was about 65% of the activity produced by the donor Bacillus amyloliquefaciens strain. The properties of $\alpha$-amylase produced by Escherichia coli were very similar to those produced by Bacillus amyloliquefaciens as based on optimum temperature, pH, and effect of CaCl$_2$ concentration. About 70% of the $\alpha$-amylase produced by Escherichia coli was localized in the periplasmic space, whereas the remaining enzyme was localized in the inner part of the cell.

• Food Science and Biotechnology
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• v.16 no.4
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• pp.655-658
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• 2007

Bacillus polyfermenticus SCD was transformed by the recombinant shuttle vector for Bacillus and Escherichia coli containing 3 antibiotic resistant genes and an ${\alpha}$-amylase gene from Bifidobacterium adolescentis Int57. The ${\alpha}$-amylase gene fused to a secretion sequences was expressed under the control of the promoter of amylase gene from B. subtilis var. natto. The recombinant plasmid was maintained stably in the transformants producing the ${\alpha}$-amylase. The enzyme was secreted to outside of the cell and showed the similar enzyme activity as that of Bacillus subtilis BD170 under the same conditions of pH and growth temperature. Because of the relatively easy transformation and the secretion of the enzyme, the transformants of B. polyfermenticus SCD may give a new strategy in the production of foreign genes.

• Journal of Microbiology and Biotechnology
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• v.5 no.5
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• pp.257-263
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• 1995

As a first step for developing Lactobacillus strains capable of fermenting starch directly, the $\alpha$-amylase gene (amyL) from Bacillus licheniformis (Kim et al., 1988. Kor. J. Appl. Microbiol. Bioeng. 16: 369-373) was introduced into Lactobacillus casei strains and the level of $\alpha$-amylase expression in transformants was examined. 3 kb EcoRI fragments encompassing amyL were subcloned into the suitable lactococcal cloning vectors (pSA3, pMG36e, and p1L2530) and then recombinant plasmids were introduced into E. coli and L. casei strains by electroporation. Only one recombinant plasmid, $pIL2530\alpha$ was able to transform few L. casei strains tested at low efficiencies. The transformation efficiencies with the plasmid into L. casei YIT 9018 and L. casei A Tee 4646 were less than $10^2/\mu$ g pIL2530\alpha$. The level of amylase activities in L. casei was five to ten-fold lower than that in E. coli cells. $p1L2530\alpha$ was stably maintained in Lactobacillus strains in the presence of Em (5 $\mu $g/ml) but without antibiotic selection, it was unstable so more than 95$%$ of cells lost plasmids after a week of daily subculturing.

• Preventive Nutrition and Food Science
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• v.13 no.3
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• pp.190-195
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• 2008

The $\alpha$-amylase gene, amyL, from Bacillus licheniformis was expressed in Lactobacillus brevis 2.14 and Escherichia coli $DH5{\alpha}$ using two different shuttle vectors, pCW4 and pSJE. E. coli transformants (TFs) harboring either $pCW4T{\alpha}$ or $pSJET{\alpha}$ produced active $\alpha$-amylase but L. brevis TFs did not, as determined by enzyme assays and zymography. But amyL transcripts were synthesized in L. brevis TFs. In terms of plasmid stability, pSJE, a theta-type replicon, was more stable than pCW4, an RCR (rolling circle replication) plasmid, in L. brevis without antibiotic selection.