• Korean Journal of Microbiology
• /
• v.31 no.2
• /
• pp.136-140
• /
• 1993

The A. nidulans expression vector which contained trpC marker gene from A. nidulans was constructed to produce glucoamy]ase. The recombinant plasmid was introduced into auxotrophic mutant A. nidulans B17. Southern blot analysis of the genomic DNA from transformant showed that pKHG2 DNA had integrated into the A. nidulans chromosomes. Northern analysis of the total RNA from transform ant showed that mRNA of glucoamylase gene was synthesized in induction condition. Specific activity of glucoamylase was increased in transform ants. G]ucoamylase was shown to be active in non-denaturing acrylamide gel.

• Korean Journal of Microbiology
• /
• v.28 no.3
• /
• pp.181-187
• /
• 1990

STA gene coding glucoamylase was introduced into haploid Saccharomyces cerevisiae SHY3 and polyploid Saccharomyces cerevisiae 54. We constructed the recombinant plasmid by substituting the promoter region of alcohol dehydrogenase isoenzyme I gene for that of STA gene to increase the expression of STA gene and found that the activity of glucoamylase was increased in transformants. The plasmid stability was improved remarkably when we got the STA gene into the plasmid which had centromere. The activity of glucoamylase and transformation frequency of it, however, was decreased because of low copy number. Industrial polyploid strain was transformed with the recombinant plasmid having the $2\mu$ origin of replication and STA gene. It produced more alcohol than host when fermented in liquefied starch media. The industrial strain, however, was not transformed with the autonomously replicating plasmid containing centromere.

• Journal of Mushroom
• /
• v.9 no.2
• /
• pp.53-58
• /
• 2011

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3'- and 5'-RACE PCR and RT-PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

• Microbiology and Biotechnology Letters
• /
• v.18 no.6
• /
• pp.653-659
• /
• 1990

The gene encoding glucoamylase from Schwanniomyces cagtellii CBS 2863 was cloned and expressed in Saccharomyces cerevisiae. Southern blot analysis confirmed that this glucoamylase gene was derived from the genomic DNA of Schwanniomyces ccastellii and that no DNA fragments corresponding to 5.1 or 1.3 kb of Sch. casteltii DNA were detected in S. cereuisiae. The glucoamylase activity from S. cerevisiae transformant was approximately 2,000 times less than that of donor yeast. No expression was found in E. coti. The secreted glucoamylase from S. cerevisiae transformant was indistinguishable from that of Sch. eastellii on the basis of molecular weight and enzyme properties.

• Microbiology and Biotechnology Letters
• /
• v.17 no.6
• /
• pp.606-610
• /
• 1989

For the purpose of improving glucoamylase productivity of Saccharomyces diastaticus, useful yeast in direct ethanol fermentation of starch, the effects of growth rate on the plasmid stability and glucoamylase productivity of S. diastaticus harboring recombinant plasmid pYES 18 containing glucoamylase gene STA 1 were investigated. In a selective medium, the recombinant plasmids were maintained stably at constant level but glucoamylase productivity was very low. On the other hand, in the complex medium containing starch, growth rate of the cell was stimulated by the supplementation of glucose and plasmid stability was improved by growth stimulation. We can conclude that glucoamylase productivity of S. diastaticus harboring the recombinant plasmid was increased as the maintaining of high plasmid stability in the cell.

• Korean Journal of Microbiology
• /
• v.31 no.1
• /
• pp.48-53
• /
• 1993

For the purpose to improve the glucoamylase productivity of Saccharomyces diastaticus, we integrated STA 1 gene into chromosomal DNA of S. diastaticus using YIp vector. After construction of Ylp-STA by the subcloning of STAI (5.3 kb) into YIp5 vector, S. diastaticus GMT-II(a. ura3. STAJ) was transformed by Ylp-STA through homologous recombination at the chromosomal STAJ gene. So we obtained the tram formants that glucoamylase productivity was increased maximum six fold. These strains transformed by the multi-copy integration of Ylp-STA in chromosomal DNA were confirmed by Southern hybridization. And the integrated Ylp-STA was maintained stably during 30 mitotic divisions.

• Microbiology and Biotechnology Letters
• /
• v.16 no.6
• /
• pp.489-493
• /
• 1988

To obtain a new yeast strain that is able to efficiently produce ethanol from starch, the glucoamylase gene of Saccharomyces diastaticus was transformed into S. cerevisiae without a cloning vector. The competent cells of S. cerevisiae, induced by the treatment of Li$_2$SO$_4$, were transformed with the partial BamHI-digests of chromosomal DNA of S. diastaticus, and the transformants were selected by their abilities to utilize and ferment starch. The transformants, which appeared at a frequency of 8.5$\times$10$^{-7}$, were able to withstand up to 800 ppm of copper sulfate like the recipient and retained the phenotypic expression of the recipient with the exception of the acquisition of STA gene and MAL gene, as regards fermentation of carbohydrates. The enzymatic properties of glucoamylases produced by transformants were very similar to those produced by S. diastaticus as based on optimium pH and temperature.

• Korean Journal of Microbiology
• /
• v.41 no.2
• /
• pp.146-151
• /
• 2005

To construct an amylolytic industrial strain of Saccharomyces cerevisiae, the glucoamylase cDNA gene (GAl) from Aspergillus awamori was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) and integrated into the chromosomes of industrial S. cerevisiae. An integrative cassette lacking bacterial ampicillin resistance gene but containing the GA1 gene, $\delta$ sequences of Ty1 retrotransposon as target sites for homologous recombination and S. cerevisiae aureobasidin A resistance gene (AUR1-C) as the selection marker was constructed to obtain a strain eligible for commercial use. Industrial S. cerevisiae transformed with this 15-integrative cassette efficiently secreted glucoamylase into the medium and grew on starch as the sole carbon source. The transformants were mitotically stable for 100 generations in nonselective medium.

• Korean Journal of Microbiology
• /
• v.30 no.5
• /
• pp.403-409
• /
• 1992

For the development of a glucanolytic yeast strain. the seceretion of endo-1.4-$\beta$-D-glucanase (CMCase) of Bacillus subtilis was performed in yeast using glucoamylase gene (STA1) of Saccharomyces diastaticus. A 1.7 kb-DNA fragment of STA1 gene containing authentic promoter, signal sequence, threonine serine-rich (TS) region and N-terminal region (98 amino acids) of mature glucoamylase was ligated to YEp 24. E. coli-yeast shuttle vector. And then. CMCase structural gene of B. subtilis was fused in frame with the 1.7 kb-DNA fragment of STA1 gene, resulting in recombinant plasmid pYES('24. Yeast transformant harboring pYESC24 had no CMCase activity. So. we deleted TS region and N-terminal region of mature glucoamylase existing between signal sequence and CMCase structural gene in pYESC24. consequently constructed recombinant plasmid pYESC11. The yeast transformed with the newly constructed recombinant plasmid pYESC11 efficiently secreted CMCase to extracellular medium. After 4 days culture. total CMCase activity of this transformant was 44.7 units/ml and over 93% of total CMCase activity was detected in culture supernatant.

• Journal of Microbiology
• /
• v.37 no.1
• /
• pp.35-40
• /
• 1999

Sporulation-specific glucoamylase (SGA) gene was isolated from genomic library of Saccharomyces diastaticus 5114-9A by using glucoamylase non-producing mutant of S. diastaticus as a recipient. When the glucoamylase activities of culture supernatant, periplasmic, and intracellular fraction of cells transformed with hybrid plasmid containing SGA gene were measured, the highest activity was detected in culture supernatant. SGA produced by transformant and extracellular glucoamylase produced by S. diastaticus 5114-9A differed in enzyme characteristics such as optimum temperature, thermostability, and resistance to SDS and urea. But the characteristics of SGA produced by sporulating yeast cells and vegetatively growing transformants were identical.