• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.33-43
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• 2015

GAM-1 and GAM-2, two themostable glucoamylases from Aspergillus niger B-30, possess different molecular masses, glycosylation, and thermal stability. In the present study, the effects of additives on the thermal inactivation of GAM-1 and GAM-2 were investigated. The half-lives of GAM-1 and GAM-2 at 70℃ were 45 and 216 min, respectively. Data obtained from fluorescence spectroscopy, circular dichroism spectroscopy, UV absorption spectroscopy, and dynamic light scattering demonstrated that during the thermal inactivation progress, combined with the loss of the helical structure and a majority of the tertiary structure, tryptophan residues were partially exposed and further led to glucoamylases aggregating. The thermal stability of GAM-1 and GAM-2 was largely improved in the presence of sorbitol and trehalose. Results from spectroscopy and Native-PAGE confirmed that sorbitol and trehalose maintained the native state of glucoamylases and prevented their thermal aggregation. The loss of hydrophobic bonding and helical structure was responsible for the decrease of glucoamylase activity. Additionally, sorbitol and trehalose significantly increased the substrate affinity and catalytic efficiency of the two glucoamylases. Our results display an insight into the thermal inactivation of glucoamylases and provide an important base for industrial applications of the thermally stable glucoamylases.

• 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.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.383-390
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• 2010

A newly isolated active producer of raw-starch-digesting amyloytic enzymes, Rhizopus microsporus var. chinensis CICIM-CU F0088, was screened and identified by morphological characteristics and molecular phylogenetic analyses. This fungus was isolated from the soil of Chinese glue pudding mill, and produced high levels of amylolytic activity under solid-state fermentation with supplementation of starch and wheat bran. Results of thin-layer chromatography showed there are two kinds of amyloytic enzymes formed by this strain, including one $\alpha$-amylase and two glucoamylases. It was found in the electron microscope experiments that the two glucoamylases can digest raw corn starch and have an optimal temperature of $70^{\circ}C$. These results signified that amyloytic enzymes secreted by strain Rhizopus microsporus var. chinensis CICIM-CU F0088 were types of thermostable amyloytic enzymes and able to digest raw corn starch.

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

• BMB Reports
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• v.28 no.5
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• pp.375-381
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• 1995

Two forms of glucoamylase (GI and GII) from starch-grown Lipomyces kononenkoae CBS 5608 mutant were purified to apparent homogeneity by means of ultrafiltration, Sephacryl S-200 gel filtration and DEAE Sephadex A-50 chromatography. The apparent molecular weight was calculated as ca. 150 kDa for GI and ca. 128 kDa for GII, respectively. Both enzymes were glycoproteins with isoelectric points of 5.6 (GI) and 5.4 (GII). They had a pH optimun of 4.5 and were stable from pH 5 to 8. The temperature optimum for both enzymes was $60^{\circ}C$, but they were rapidly inactivated above $70^{\circ}C$. The $K_m$ values toward starch were estimated to be 6.57 mg per ml for GI and 4.52 mg per ml for GII, and the $V_{max}$ values were 16.28 ${\mu}M$ per mg for GI and 32.25 ${\mu}M$ per mg for GII, respectively. The $K_m$ and $V_{max}$ values of GII for ${\alpha}-$ or ${\beta}-cyclodextrin$ were estimated to be 0.15 mg per ml and 2.0 mg per ml, respectively ($K_m$) and 1.02 ${\mu}M$ per mg or 1.02 ${\mu}M$ per mg, respectively ($V_{max}$). Neither enzyme exhibited pullulanase activity but they released only glucose from starch or cyclodextrin. Amino acid analysis indicated that both glucoamylases were enriched in proline and acid amino acids. Glucoamylase GII strongly cross-reacted with a monoclonal antibody raised against GI enzymes, and the two enzymes shared very similar amino acid composition. Western blot analysis indicated that L. kononenkoae CBS 5608 mutant produced two forms of glucoamylase on starch, and that synthesis of them was subject to glucose repression.

• Applied Biological Chemistry
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• v.28 no.4
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• pp.233-238
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• 1985

Glucoamylases catalyze a stepwise hydrolysis of starch with the production of glucose. In order to make an efficient conversion of starch into glucose, glucoamylases prepared from Rhizopus spp. (Sigma Co.) were attached to a porous glass and immobilized by glutaraldehyde-induced crosslinking. The porous glass used in this study was $ZrO_2$ coated, $40{\sim}80$ mesh, 550 A pore diameter. Using the forgoing glass, we could couple as much as 50mg of protein per gram of carrier. Substrate for the glucoamylase was an enzyrne-modified thin-toiling 30% cornstarch solution used where greater solubility and low viscosity are desired. Immobilized glucoamylase had an optimum pH 7.0 to the alkaline side of soluble enzyme. Km values of immobilized and soluble enzyme were 1.04 mM and 1.25mM, respectively. The thermal stability of glucoamylase was increased by immobilization and the immobilized enzyme showed an optimum temperature at $40{\sim}60^{\circ}C$. The continuous conversion of cornstarch to glucose by use of immobilized glucoamylase resulted in the production of a more than 90 DE product.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.529-532
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• 1989

A strain of Streptomyces sp. (YS-221-B) extracellularly produced an inhibitory substance for $\alpha$-D-Glucosidase. The substance was purified 96-fold from culture filtrate by dialysis, heat treatment, adsorption on active carbon, Bio-Gel P-10 and Sephadex G-75 column chromatography with yield of 9.2%. The substance was stable in pH range from 7.0 to 11.0 at 37$^{\circ}C$, and a treatment at 10$0^{\circ}C$ for 20 min diminished only 15% of the original activity. The inhibitor was not inactivated by the treatment of $\alpha$-, $\beta$-amylases, glucoamylases, trypsin and chymotrypsin but inactivated by pyoteases from Streptomyces griseus and Tritirachium album.

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

• Food Science and Biotechnology
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• v.14 no.6
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• pp.860-865
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• 2005

A gene for a putative glucoamylase, stg, of a hyperthermophilic archae on Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The recombinant glucoamylase (STGA) had an optimal temperature of $80^{\circ}C$ and was extremely thermostable with a D-value of 17 hr. The pH optimum of the enzyme was 4.5. Being different from fungal glucoamylases, STGA hydrolyzed maltotriose (G3) most efficiently. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis showed that the enzyme existed as a dimer. STGA was stable enough to hydrolyze liquefied com starch to glucose in 4 hr at $90^{\circ}C$ with a yield of95%. Comparison of the $k_{cat}$ values for the hydrolysis and the reverse reaction at $75^{\circ}C$ and $90^{\circ}C$ indicated that glucose production by STGA was more efficient at $90^{\circ}C$ than $75^{\circ}C$. Therefore, STGA showed great potential for application to the industrial glucose production process due to its high thermostability.

• Korean Journal of Microbiology
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• v.18 no.4
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• pp.161-171
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• 1980

A mutant strain having increased productivity of both enzymes, protease and amylase, was obtained from A. flavus KU 153, isolatd from South Korea for its high protease production by successive ultra-violet light irradiation, Two glucoamylases from the mutant strain selected were purified from wheat branculture by successive salting out, followed by dialysis and column chromatography, and their characteristics were compared with those of the wild strain. Glucoamylase production of the mutant selected was increased about 3.3 times compared with the wild strain, and 2.1 times compared with the parental strain, ${\alpha}-amylase$ activity of the mutant selected was about 2 times hugher than that of the wild strain or the parental strain. Protease and cellulase productivities of the muant selected were all alike compared with those of the highly proteolytic mutant, the parental strain. Therefore, it was considered that the back mutation on the protease production did not occurred in the formation process of the glucoamylase producing mutant. Total activities of glucoamylase I and II from the mutant selected were 2.86 and 3.65 times higher compared with those from the wild strain, respectively. Considering the optimal pH-thermal stability and Km-Vmax value of glucoamylase I and II from both strains, wild and mutant, it was deduced that the characteristics of glucoamylase I and II from the wild strain did not altered during the mutation process. Therefore, it was concluded that the selected mutant did not induce the formation of another glucoamylase isozyme, or the changes in the characteristics of the glucoamylase, but induce the productivity of the same glucoamylase I and II by the action of regulatory gene.