• 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.2 no.1
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• pp.56-65
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• 1992

The complete nucleotide sequence of the Bacillus circulans F-2 RSDA gene, coding for raw starch digesting a-amylase (RSDA), has been determined. The RSDA structure gene consists of an open reading frame of 2508 bp. Six bp upstream of the translational start codon of the RSDA is a typical gram-positive Shine-Dalgarno sequence and the RSDA encodes a preprotein of 836 amino acids with an Mr of 96, 727. The gene was expressed from its own regulatory region in E. coli and two putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. Confirmation of the nucleotide sequence was obtained and the signal peptide cleavage site was identified by comparing the predicted amino acid sequence with that derived by N-terminal analysis of the purified RSDA. The deduced N-terminal region of the RSDA conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete amino acid sequence was deduced and homology with other enzymes was compared. The results suggested that the Thr-Ser-rich hinge region and the non-catalytic domain are necessary for efficient adsorption onto raw substrates, and the catalytic domain (60 kDa) is necessary for the hydrolysis of substrates, as suggested in previous studies (8, 9).

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.447-452
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• 2014

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for ${\alpha}$-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel ${\alpha}$-amylase from a gastrointestinal metagenomic library.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.251.2-251
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• 1996

No Abstract, See Full Text

• Proceedings of the Zoological Society Korea Conference
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• 1999.10b
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• pp.288.2-288
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• 1999

No Abstract, See Full Text

• 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.16 no.11
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1052-1056
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• 2004

A secretion signal sequence-selection vector (pGS40) was constructed based on an $\alpha$-amylase gene lacking a secretion signal and employed for selecting secretion signals from Lactococcus lactis ssp. cremoris LM0230 chromosomal DNA. Six fragments were identified based on their ability to restore $\alpha$-amylase secretion in E. coli, and among these, a fragment, S405, conferred the highest secretion activity (84%) in E. coli. Meanwhile, S407, which conferred poor secretion activity in E. coli, was quite active in L. lactis. The results suggested that the efficiency of a secretion signal depended on the host. All six fragments had an open reading frame (ORF) fused to the reporter gene, and the potential Shine-Dalgamo (SD) sequence and putative promoter sequences were located upstream of the ORF. Deduced amino acid sequences from the six fragments did not show any homology with known secretion signals. However, they contained three distinguished structural features and cleavage sites, commonly found among typical secretion signals. The characterized secretion signals could be useful for the construction of food-grade secretion vectors and gene expression in LAB.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.2000-2003
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• 2006

A gene (GenBank AF096282) coding for a $\alpha$-glucosidase (TcaAG, EC 3.2.1.20) from Thermus caldophilus GK24 was expressed in Saccharomyces cerevisiae, a generally recognized as safe (GRAS) host. The thermostable $\alpha$-glucosidase was produced inside of the GRAS host at 0.04 unit/mg-dry cell by the constitutively expressing ADH1 promoter and at 1.2 unit/mg-dry cell by the inductively expressing GALl0 promoter, respectively. No $\alpha$-glucosidase activities were found in the medium when the MF-alpha signal sequence from S. cerevisiae or $\alpha$-amylase signal sequence from Aspergillus oryzae were fused before the $\alpha$-glucosidase gene for the secretion.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1539-1545
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• 2010

In beer, glutathione works as the main antioxidant compound, which also correlates with the stability of the beer flavor. In addition, high residual sugars in beer contribute to major nonvolatile components, which are reflected in a high caloric content. Therefore, in this study, the Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and the Saccharomycopsis fibuligera ALP1 gene encoding ${\alpha}$-amylase were coexpressed in industrial brewing yeast strain Y31 targeting the ${\alpha}$-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), resulting in the new recombinant strain TY3. The glutathione content in the fermentation broth of TY3 increased to 43.83 mg/l as compared with 33.34 mg/l in the fermentation broth of Y31. The recombinant strain showed a high ${\alpha}$-amylase activity and utilized more than 46% of the starch as the sole carbon source after 5 days. European Brewery Convention tube fermentation tests comparing the fermentation broths of TY3 and Y31 showed that the flavor stability index for TY3 was 1.3-fold higher, whereas its residual sugar concentration was 76.8% lower. Owing to the interruption of the ILV2 gene and ADH2 gene, the contents of diacetyl and acetaldehyde as off-flavor compounds were reduced by 56.93% and 31.25%, respectively, when compared with the contents in the Y31 fermentation broth. In addition, since no drug-resistant genes were introduced to the new recombinant strain, it should be more suitable for use in the beer industry, owing to its better flavor stability and other beneficial characteristics.