• Journal of Microbiology and Biotechnology
• /
• v.15 no.2
• /
• pp.239-246
• /
• 2005

Some characteristics of two forms of glucoamylase (glucan 1 A-$\alpha$-glucosidase, EC 3. 2. I. 3) purified from Aspergillus coreanus NR 15-1 were investigated. The enzymes were produced on a solid, uncooked wheat bran medium of A. coreanus NR 15-1 isolated from traditional Korean Nuruk. Two forms of glucoamylase, GA-I and GA-II, were purified to homogenity after 5.8-fold and 9.6-fold purification, respectively, judged by disc- and SDS-polyacrylamide gel electrophoresis. The molecular mass of GA-I and GA-II were estimated to be 62 kDa and 90 kDa by Sephadex G-1OO gel filtration, and 64 kDa and 91 kDa by SDS-polyacrylarnide gel electrophoresis, respectively. The optimum temperatures of GA-I and GA-II were 60$^circ$C and 65$^circ$C, respectively, and the optimum pH was 4.0. The activation energy (Ea value) of GA-I and GA-II was 11.66 kcal/mol and 12.09 kcal/mol, respectively, and the apparent Michaelis constants (K_{m}) of GA-I and GA-II for soluble starch were found to be 3.57 mg/ml and 6.25 mg/ml, respectively. Both enzymes were activated by 1 mM Mn^{2+} and Cu^{2+}, but were completely inhibited by 1 mM N­bromosuccinimide. The GA-II was weakly inhibited by 1 mM p-CMB, dithiothreitol, EDTA, and pyridoxal 5-phosphate, but GA-I was not inhibited by those compounds. Both enzymes had significant ability to digest raw wheat starch and raw rice starch, and hydrolysis rates of raw wheat starch by GA-I and GA-II were 7.8- and 7.3-fold higher than with soluble starch, respectively.

• Korean Journal of Food Science and Technology
• /
• v.5 no.2
• /
• pp.78-83
• /
• 1973

The naringin hydrolyzing enzyme has been purified from the culture filtrate of the mold Aspergillus S-1 which selected to remove the bitter test of the orange or citrus fruits industrily. In a view of purity naringinase was more effectively purified in order of molecular sieving on Sephadeex G-200, starach gel electrophoresis, chromatography or a DEAE-Cellulose column and fractional precipitation by ammonium sulfate. The purified enzyme is homogeneous in paper electrophoresis from a culture filtrate by treatment fractional precipitation with ammonium sulfate, DEAE-Cellulose treatment and Sephadex-200 column chromatography and it hydrolyse only naringin to purunin.

• The Korean Journal of Malacology
• /
• v.17 no.2
• /
• pp.117-123
• /
• 2001

A genetic analysis using starch gel electrophoresis was peformed to clarify the degrees of genetic differentiation among two species of the genus Koreanomelania and Koreoleptoxis (Gastropoda, Pleuroceridae). The average genetic similarity coefficients (S) among the populations of K. nodifila was S=0.848 (0.805-0.905) and K. globus ovalis was S : 0.755 (0.666-0.860). The genetic similarity coefficients between Semisulcospira gottschei and K. nodifila, S. gottschei and K. globus ovalis, and K. nodifila and K. giobus ovalis were 0.194, 0.301, and 0.301, respectively. So between K. nodifila and K. globus ovalis seems to be genetically two distinct taxa.

• Microbiology and Biotechnology Letters
• /
• v.27 no.2
• /
• pp.129-135
• /
• 1999

EH-1 was highest at 9 days of incubation. This regrowth and enzymatic activity of Haloarcular sp. EH-1 was highest at 9 days of incubation. This amylase was purified by acetone fractionation, DEAG-Cellulose column chromatography, 1st Sephadex G-75 gel filtration, CM-Cellulose column chromatography and 2nd Sephadex G-75 gel filtration. The amylase was purified about 98.64 fold with a yield of 11.75%. The molecular weight of amylase was estimated to be about 43,000and 40,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that the enzyme was a monomer. Amylase had an optimal temperature of 4$0^{\circ}C$, and an optimum pH of 7.0, and the thermal stability was observed the above 50% at 10$0^{\circ}C$ after 1 hour, and the stable range of pH was 6.0 to 8.0. The enzymatic activity was increased in the presence of 10 mM 2-mercaptoethanol, slightly by 10 mM SnCl2.2H2O.FeCl2.4H2O.CuCl2.2H2O.HgCl2.6H2O and SDS. End products from soluble starch were glucose, maltose and maltotriose, and Km value for soluble starch was 2.5mg/ml.

• Korean Journal of Food Science and Technology
• /
• v.16 no.3
• /
• pp.322-328
• /
• 1984

These experiments were conducted to purify the glucoamylase produced by Rhizopus oryzae. Two forms of glucoamylase (GI and GII) from Phizopus oryzae were purified by $(NH_2)_2SO_4$ fractionation, acetone fractionation and successive column chromatography on DEAE-cellulose and CM-cellulose. The specific activities of GI and GII toward soluble starch were 157.6 U/㎎. protein (37.5 fold of crude extract), and 164.7 U/㎎. protein (39.2 fold of curde extract), respectively, and the yields of them were 4.3% and 3.8%, respectively. The two purified enzymes have shown a single band by polyacrylamide disc gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The protein bands of their electrophoresis gel were revealed to have glucoamylase activity by iodine staining and were proved to be glycoprotein by periodic acid Schiff's staining.

• Korean Journal of Pharmacognosy
• /
• v.17 no.2
• /
• pp.134-138
• /
• 1986

Based on a tracer study, starch phosphorylase was implicated as an agent in the starch synthesis in sweet potato roots. The enzyme was purified from the tissue as a cluster of isozymes with an average mw of 205K (fresh roots) or 159K (roots stored for 3 mon.). On SDS polyacrylamide gel electrophoresis, one large subunit of 98K mw and several small ones of 47${\sim}57K mw were observed. From the mw data and the results of peptide mapping and immunoelectrophoretic blotting using mono- and polyclonal antibodies, it was deduced that a large part of the large subunit was cleaved at the middle part of the peptide chain to give rise to the small subunits, and on storage, the enzyme molecules were further modified by proteolysis. During the course of phosphorylase purification, a proteinaceous inhibitor of the enzyme was isolated. It had a mw of 250K and was composed of 5 identical subunits of 51K mw. In the direction of starch synthesis, the inhibitor showed a noncompetitive kinetics with a Ki of $1.3{\times}10^{-6}\;M$. By immunohistochemical methods, both the enzyme and the inhibitor were located on the cell wall and amyloplast. Crossreacting materials of the inhibitor were present in spinach leaf, potato tuber and rice grain. These findings indicate the wide occurrence of the inhibitor and also imply its possible participation in regulating starch phosphorylase activity in vivo.

• Microbiology and Biotechnology Letters
• /
• v.18 no.3
• /
• pp.251-259
• /
• 1990

Asp. usumii IAM 2185 was selected as a strain producing the powerful raw starch digesting glucoamylase. The optimum initial pH, the optimum temperature and the optimum cultural time for the enzyme production on wheat bran medium were pH 6-8,25-$30^{\circ}C$ and 72 hrs, respectively. The addition of ammonium nitrate and albumin on wheat bran medium, respectively, increase slightly the enzyme production. The enzyme was purified by ammonium sulfate fractionation, CM-cellulose and DEAE-cellulose column chromatography. The specific activity of the purified enzyme was 34.3 U/mg protein and the yield of enzyme activity was 10.3%. The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 67,000 by SDS polyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pR 3.7. The optimum temperature and optimum pH were $60^{\circ}C$and pH 3.0 and the purified enzyme was stable in the pH range of 1.0-11.0. The purified enzyme was stable below $50^{\circ}C$ and its thermostability was greatly increased by the addition of $Ca^{2+}$. The purified enzyme showed a high hydrolysis rate on various raw starches such as corn, rice, yam, arrow root, sweet potato and glutinous rice.

• Microbiology and Biotechnology Letters
• /
• v.23 no.6
• /
• pp.695-701
• /
• 1995

An alkaline lipase producing bacteria was isolated from soil and identified as Serratia liquefaciens AL-11. from the results of analysis of its morphological, biochemical and physiological properties. This strain showed the highest productivity of alkaline lipase when grown at pH 9.0 and 30$\circ$C for 42 hours in the medium of 1% peptone, 0.5% tryptone, 0.9% yeast extract, 1% starch, 1% tween 80, 0.05% CaCl$_{2}$ and 0.05% NaCl. The enzyme was purified by ammonium sulfate treatment, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 column chromatography. The specific activity of the purified enzyme was 27 unit/mg protein and the yield of enzyme activity was 61.3%. The homogeneity of the purified enzyme was verified by polyacrylamide gel disc electrophoresis. Molecular weight of the purified enzyme was estimated about 53,000 by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. This enzyme is composed of 17 amino acids of which glycine, proline and glutamic acid were three miajor acids.

• Applied Biological Chemistry
• /
• v.26 no.1
• /
• pp.65-72
• /
• 1983

The composition of four rice protein groups is greatly affected by the extraction conditions. The extraction amounts of albumins and glubulines primarily depended on the temperature rather than the method of extraction. The total amount of glutelins, the major components of rice storage proteins, could be extracted by a successive extraction processes, extraction with 0.5% SDS-0.1M borate buffer(pH 8.3) followed by extraction with 0.5% SDS-0.6% ${\beta}-mercaptoethanol-0.1M$ borate buffer(pH 8.3). The extracted amounts of glutelin with these solvents were 54.1 and 45% respectively. The further purification of SDS soluble glutelins was achieved by Sephadex G-150 gel column chromatography. The molecular weight of the components in four protein groups has been estimated by SDS-polyacrylamide gel electrophoresis with or without ${\beta}-mercaptoethanol.$ The comparison of albumins and globulins by starch gel electrophoresis at pH 3.1 permitted us to identify seven rice varieties. However, at pH 8.95, the specific bands for Japonica type rice varieties were observed.

• Journal of Microbiology
• /
• v.37 no.2
• /
• pp.80-85
• /
• 1999

The purification system of glucoamylase (glucan 1,4-${\alpha}$-glucosidase, EC 3. 2. 1. 3), some characteristics of the purified enzyme and hydrolysis rate of various raw starch were investigated through several experiments. The enzyme was produced on a solid, uncooked wheat bran medium of Aspergillus oryzae NR 3-6 isolated from traditional Korean Nuruk. The enzyme was homogeneously purified 6.8-fold with an overall yield of 28.3% by the criteria of disc- and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 48 kDa by SDS-PAGE. The optimum temperature and pH were 55$^{\circ}C$ and 4.0, respectively. The enzyme was stable at a pH range of 3.0∼10.0 and below 45$^{\circ}C$. Enzyme activity was inhibited about 27% by 1mM Hg2+. The hydrolysis rate of raw wheat starch was shown to be 17.5-fold faster than the hydrolysis rate of soluble starch. The purified enzyme was identified as glucoamylase because the product of soluble starch by the purified enzyme was mainly glucose by thin layer chromatography.