• Korean Journal of Food Science and Technology
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• v.16 no.4
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• pp.392-397
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• 1984

These experiments were conducted to investigate general enzymatic characteristics of two forms(glucoamylase I and glucoamylase II) of the purified glucoamylase produced by Rhizopus oryzae. Molecular weights of glucoamylase I and glucoamylase II estimated by Sephadex G-100gel filtration, were approximately 101,000 and 115,000, respectively, and those estimated by SDS-polyacrylamide gel electrophoresis being 120,000 and 127,000, respectively. Isoelectric points of the above enzyme were pH 7.25 and pH 7.75. The optimum temperature was $50^{\circ}C$ and the enzyme was stable below $45^{\circ}C$. Optimum pH of both glucoamylase I and glucoamylase II was about pH 5.0. The stable pH range of them were pH 3.5-8.0 and 4.5-8.0, respectively. Michaelis constants of glucoamylase I and glucoamylase II toward souluble starch were 4.545 mg/ml and 5.560 mg/ml, respectively. $Hg^{++}$, $Pb^{++}$, p-CMB and IAA were inhibitors of glucoamylase I and $Hg^{++}$, $Mn^{++}$, p-CMB and IAA were inhibitors of glucoamylase II.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.606-610
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• 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
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• v.31 no.2
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• pp.136-140
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• 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 Food Science and Technology
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• v.16 no.4
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• pp.398-402
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• 1984

These experiments were conducted to investigate the substrate specificity, the hydrolysis products on the various carbohydrates and the hydrolysis rate on the various raw starches of the two purified glucoamylase produced by Rhizopus oryzae. Both of the glucoamylases hydrolyzed amylose, amylopectin, glycogen, soluble starch, pullulan, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose, but did not act on ${\alpha}-cyclodextrin$, ${\beta}-cyclodextrin$, raffinose, sucrose and lactose. When the reaction mixture of glucoamylase and polysaccharides were incubated $37^{\circ}C$for 32 hours, glucoamylase I hydrolyzed amylopectin, soluble starch and amyloses completely, but hydrolyzing glycogen up to only about 88%. Glucoamylase II hydrolyzed the previous four polysaccharides up to about 100%. Both of the glucoamylases produced only glucose for various substrates and did not have any ${\alpha}-glucosyl$ transferase activity. Both of the glucoamylases hydrolyzed raw glutinous rice starch almost complety, wheras they acted on raw potato starch, raw green banana starch, raw arrow root starch, raw corn starch, raw yam starch and raw high amylose corn starch weakly. Glucoamylase II hydrolyzed raw starches at the higher rate than glucoamylase I.

• KSBB Journal
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• v.9 no.5
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• pp.532-540
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• 1994

Secretion efficiency is generally affected by promoter, signal sequence, characteristics of foreign protein and host. Secretion efficiencies of glucoamylase in recombinant plasmid-harboring yeast and chromosome-integrated yeast which had STA signal sequences were 74% and 65% at the 4th day of incubation, respectively. The high secretion efficiencies of the yeasts were obtained due to the fact that the expression levels were not reached at the secretory apparatus capacities of the host yeasts. In both yeasts, most of the intracellular glucoamylase were detected in cytoplasm and small portion (below 10%) of glucoamylase were located in periplasm. The characteristics of secreted heterologous glucoamylase from recombinant Saccharomyces cerevisiaes were investigated by using Western blot analysis. The secreted mature glucoamylase was heterogeneous and its molecular weight was about 200 to 300 kilodalton. The carbohydrate content of mature glucoamylase was higher than 80%, and several bands of about 55 to 65 kilodalton indicate the endoplasmic reticulum forms of intracellular glucoamylase.

• KSBB Journal
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• v.11 no.3
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• pp.311-316
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• 1996

The effects of polyethylene glycol (PEG) on glucoamylase production in recombinant Saccharomyces cerevisiae were studied. By PEG addition, the cell growth was not affected. However, the glucoamylase production was increased in all range of PEG molecular weights and concentrations. The optimal molecular weight of PEG was 6000 and the optimal concentration was 1g/L. Using these optimals, the extracellular glucoamylase activity was 23% higher in PEG-containing medium than that in medium without PEG.

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

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

• Bulletin of the Korean Chemical Society
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• v.10 no.1
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• pp.107-111
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• 1989

Glucoamylase was inactivated with 1-ethyl-2-(dimethylaminopropyl)carbodiimide (EDC) at pH 5.0. Time course of inactivation of glucoamylase was at least biphasic. From the results of the titration of SH groups with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of glucoamylase. The CD spectrum of EDC-modified glucoamylase suggested that the gross conformation of the native enzyme was retained. The inactivation of glucoamylase was reduced remarkably in the presence of maltose. The logarithm of the half-life of the inactivation of glucoamylase by EDC was a linear function of log[EDC] in each stage indicating that one carboxyl group among the modified ones was crucial for inactivation of glucoamylase. The change in the binding affinity due to modification was determined by using an affinity column. It indicates that the carboxyl group of glucoamylase seems to play a role in both, the catalysis and substrate binding in the first stage, but in the second stage the binding affinity is recovered almost up to that of native enzyme.

• The Korean Journal of Mycology
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• v.12 no.1
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• pp.21-26
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• 1984

Aspergillus awamori which produces glucoamylase was cultivated in the starch-Czapek-­Dox's medium in which sucrose was depleted. A rapid method for identification and assay of glucoamylase produced by the A. awamori in the culture was established by the use of the glucose oxidase. The levels of glucose derived from the breakdown of the starch medium were assayed by using glucose oxidase, which was proved to be effective in the screening of glucoamylase-producing fungi in terms of rapid and simple determination. After the cellulose acetate electrophoresis of the precipita ted culture broth, the glucoamylase band in the gel was contacted with 2% starch solution with glucose oxidase, and then color reaction was occurred. Also this method could be effective to identify rapidly the fungal glucoamylase.