• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.529-533
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• 2001

The ${\beta}$-Amylase cDNA fragment from the oomcete Saprolegnia parasitica was cloned by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved ${\beta}$-amylase sequences. The 5'and 3'regions of the $\beta$-amylase gene were amplified using the rapid amplification of cDNA ends (rACE) system. It consisted of an open reading frame of 1,350 bp for a protein of 450 amino acids. Comparison between the genomic and cDNA sequences revealed that the intron was not present in the coding region. The deduced amino acid sequence of the ${\beta}$-amylase gene had a 97% similarity to the ${\beta}$-amylase of Saprolegnia ferax, followed by 41% similarity to those of Arabidopsis thaliana, Hordeum vulgare, and Zea mays. The ${\beta}$-amylase gene was also expressed in Saccharomyces cerevisiae by placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.65-71
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• 2001

A gene from Paenibacillus sp. KCTC 8848P encoding ${\beta}-amylase$ was cloned and expressed in Escherichia coli. The Paenibacillus ${\beta}-amylase$ gene cosisted of a 2,409-bp open reading frame without a translational stop codon, encoding a protein of 803 amino acids. The presumed ribosime-binding site, GGAGG, was located 10 bp upstream from the TTG initiation codon. The deduced amino acid sequence of the ${\beta}-amylase$ gene had a 95% similarity to the ${\beta}-amylase$ of Bacillus firmus. The ${\beta}-amylase$ gene was introduced into wild-type strains of Saccharomyces cerevisiae using a linearized yeast integrating vector containing a geneticin resistance gene and its product was secreted into the culture medium.

• 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.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.47 no.6
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• pp.413-417
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• 2002

This study was conducted to determine $\beta$-amylase gene expression and degradation of starch granules in the endosperm near scutellar epithelium of rice cultivars under the submerged soil at hypoxia 18$^{\circ}C$, which is practically important condition for farmers in temperate regions. In case of cv. Janghyangdo, accumulation of $\beta$-amylase mRNA was detected in the aleurone layer on the ninth day after seeding. However that of cv. Suwon 287 and Norm 6 were not detected in the aleurone layer in submerged soil(hypoxia) at 18$^{\circ}C$. $\beta$-amylase of cv. Janghyangdo was synthesized de novo in aleurone cells not in the scutellar epithelium. Degradation of starch granules in the endosperm near scutellar epithelium of c.v. Janghyangdo and Ginbozu, which have a strong $\beta$-amylase activity, was greater than that of cv. Suwon 287 and Norm 6 with no $\beta$-amylase activity in submerged soil(hypoxia) at 18$^{\circ}C$. This result may indicate that $\beta$-amylase gene expression and degradation of starch granules of germinating rice seed are related to the emergence of rice under the submerged soil condition at low temperature.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.264-269
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• 2008

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.348-355
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• 1991

DNA segment encoding $\beta$-1, 4-glucanase of Bacillus subtilis was fused in frame to mouse $\alpha$-amylase signal sequence behind the alcohol dehydrogenase isoenzyme I gene (ADHI) promoter of the yeast expression vector pMS12. To enhance the expression level of the $\beta$glucanase gene in yeast, transcription terminator sequence iso-1-cytochrome c gene (CYCI) was inserted into the recombinant plasmid. The transformants harbouring such recombinant plasmids secreted $\beta$-glucanase into the culture medium. The expresstion level of the $\beta$-glucanase gene was increased about 2-fold caused by inserting the terminator. The amount of the secreted $\beta$-glucanase in culture medium was approximately 60% of the total quantity synthesized.

• 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.

• Korean Journal of Food Science and Technology
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• v.43 no.3
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• pp.369-374
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• 2011

A putative maltogenic amylase gene (DGMA) was cloned from the Deinococcus geothermalis DSM 11300 genome using the polymerase chain reaction. The gene encoded 608 amino acids with a predicted molecular mass of 68,704 Da. The recombinant DGMA was constitutively expressed using the pHCXHD plasmid. As expected, the recombinant DGMA hydrolyzed cyclodextrins and starch to maltose and pullulan to panose by cleaving the ${\alpha}$-(1,4)-glycosidic linkages, as observed for typical maltogenic amylases. Characterization of the recombinant DGMA revealed that the highest maltogenic amylase activity occurred at $40^{\circ}C$ and pH 6.0. The half-life of catalytic activity at $65^{\circ}C$ and $55^{\circ}C$ were 8.2 min and 187.4 min, respectively. DGMA mainly hydrolyzed ${\beta}$-cyclodextrin, soluble starch, and pullulan and its efficient ratio of those substrates was 9:4.5:1.

• Proceedings of the Korean Society of Crop Science Conference
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• 2000.04a
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• pp.296-297
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• 2000

• The Korean Journal of Mycology
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• v.42 no.4
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• pp.282-288
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• 2014

In this study, we attempted to isolate fungi from soybean fermented foods produced in Sunchang County and to identify Aspergillus oryzae from fungal isolates. Ten fungal isolates were identified with ${\beta}$-tubulin gene. According to the sequences of ${\beta}$-tubulin gene, ten fungal isolates were identified as A. oryzae/flavus complex. For further identification of the ten of fungal isolates, omtA gene, one gene of the aflatoxin biosynthesis gene cluster, was sequenced and the sequences were compared with those of A. oryzae and A. flavus strains from the GenBank database. In addition, identification of the ten fungal isolates was further confirmed using the PCR amplicon of norB and cypA intergenic region, in which a deletion was recognized relative to A. flavus and A. parasiticus. The amplicon size of the ten fungal isolate strains was smaller than those of A. flavus and A. parasiticus, but the same as that of the reference A. oryzae strain. These results indicated that the ten isolates should be identified as A. oryzae. The protease activity in rice koji made with 6, 13, 17, 27, 37 and 38 of strain, respectively was twice higher than that in control. The kojis made with nine of the A. oryzae isolates, respectively, did not produce aflatoxin, suggesting that the strains could possibly be used as starters for soybean products.