• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.217-217
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• 2022

Pullulanase (PUL), a debranching enzyme, has been utilized in hydrolyzing the a-1,6 glucosidic linkages in starch, amylopectin, pullulan, as well as related oligosaccharides. It has also been indicated that PUL is a novel indicator of inherent RS (Resistant Starch) formation in rice. In this study, we performed haplotype analysis on 320 bred rice accessions, and additional 54 wild accessions were added to study genetic diversity along with other population-based analyses of the PUL gene. Through these investigations, we summarized a total of 10 functional (non-synonymous) SNPs from 7 different exons on chromosome 4. There were 10 haplotypes, of which only six haplotypes were functional, implicating different subpopulations. Diversity reduction was noticed in temperate japonica (0.0005) compared to the highest one (aus, 0.0154), illustrating their higher genetic differentiation by F_ST-value (0.926). The highest Tajima^ D value was observed in indica (3.6613), indicating PUL gene domestication signature under balancing selection, while the lowest Tajima's D value was found in temperate japonica (-2.2191) which might have undergone under positive selection and purified due to the excess of rare alleles. PCA, population structure, and phylogenetic analyses provide information on the genetic relatedness between and or among the cultivated subpopulations and the wild based on PUL genomic region.

• KSBB Journal
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• v.24 no.6
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• pp.579-583
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• 2009

The effects of cell lytic enzyme treatment on total phenolic content, antioxidant and antityrosinase activities of lotus leaf were investigated. The dried lotus leaves were hydroyzed by cell lytic enzymes such as Promozyme, Ceremix, Pectinex, Ultraflo, Celluclast, Pentopan, Tunicase, Viscozyme at their optimum pHs (pH 5-8) at $50^{\circ}C$ for 4 hrs. Depending on the enzymes used, total phenolic compounds content was measured as $1,079-1,476{\mu}g$/mL, and antioxidant activities and whitening activities were increased by 5~10% and 20%, respectively Among the tested hydrolytic enzymes, Promozyme (pullulanase) was selected as the most suitable enzyme for the extraction of total polyphenol from lotus leaf. The optimal dosage of Promozyme were found to be 1-2% (w/w). By Promozyme treatment, total phenolic compounds content of the lotus extract significantly increased compared to the extraction without enzyme treatment.

• 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.

• The Korean Journal of Food And Nutrition
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• v.10 no.2
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• pp.180-185
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• 1997

Sikye was produced from glutinous rice. The glutinous rice Sikhye was found to contain 7.3% of limit dextrin, 10.1% of maltose, 1.3% of maltotriose and 1.75% of rice residue. Limit dextrin in glutinous rice Sikhye was purified by ethanol fractionation followed by gel chromatography on Biogel P-2. The purified limit dextrin showed both signal of $\alpha$-1,4- and $\alpha$-1,6-glucosidic linkage with its estimation ratio of 5:1 by 1H-NMR analysis. Limit dextrin was digested with enzymes(30units/ml) of $\alpha$-amylase, $\alpha$-glucosidase and glucoamylase from Aspergillus awamori, sweet potato $\beta$-amylase and human salivary $\alpha$-amylase at 37$^{\circ}C$ for 1 hour, respectively. Hydrolysis rates of these amylases on it were similar that of rice Sikhye. $\alpha$-Glucosidase plus human salivary $\alpha$-amylase hydrolyzed it to 18%. The results suggest that glutinous rice is more effective to produce high level of branched maltooligosaccharide compared with rice as raw material for Sikye making.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.4
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• pp.354-362
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• 1991

To investigate nutritional and biochemical values of buckwheat, amino acids and minerals in buckwheat harvested from Kangweon-do were analysed. Mixed flour between buckwheat and wheat were made to be used for buckwheat noodle and were also analysed minerals and major nutrients in composite flours. When three different levels of flour mix were compared, major nutrients were about same among those mixes but minerals were higher by adding more buckwheat flour, especially by adding more imported buckwheat flour. From this mixed flour, 9 minerals and 16 amino acids including 9 essential amino acids were analysed. Contents of selenium, sodium and magnessium were very high in buckwheat flour, Therefore, nutritional value of domestic buckwheat was highly evaluated. BAP method was used to determine the change of gelatinization in the noodles prepared by extruder at 8$0^{\circ}C$, and in the noodles during freeze drying and refrigeration. 70% of gelatinization was done during noolding process and retrogradation was severe during refrigeration.

• Korean Journal of Food Science and Technology
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• v.23 no.5
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• pp.554-560
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• 1991

The physicochemical properties of Korean yam starches (D. aimadoimo, D. batatas and D. japonica) were investigated. The mean granular size of starches were 23.5 μm for D. aimadoimo, 23.9 μm for D. batatas and 18.2 μm for D. japonica. Amylose content, blue value and water binding capacity was $29{\sim}33%,\;0.42{\sim}0.51%\;and\;109.9{\sim}118.3%$, respectively. The optical transmittance of 0.3% (dry basis) yam starch suspensions were increased at $70{\sim}75^{\circ}C$ and D. japonica showed typical two-step transmittance curve. The swelling power and solubility patterns increased over $60^{\circ}C$, and D. aimadoimo was the highest values. Amylogram patterns of 5% (dry basis) yam starch suspensions, determined by Brabender amylograph, were similar to that of yam flours and the viscosity of D. aimadoimo had 630 BU, which was about 5 times higher than 130 BU for D. batatas and D. japonica. Observation under scanning electron microscope lefted marks of resistance to glucoamylase because these surfaces were similar to the natural granules. In rates of solubiliazation by dimethyl sulfoxide, D. aimadoimo showed the highest value. (3-Amylolysis limits of yam starches and their amylose were $71.8%{\sim}75.5%\;and\;90.2{\sim}92.1%$, respectively. Gel filtration patterns of debranched amylopectin by pullulanase were divided into 3 peaks. The weight ratios of peak III to peak II in yam starches were $2.15%{\sim}2.42%$.

• Korean Journal of Food Science and Technology
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• v.27 no.6
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• pp.1017-1027
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• 1995

This study was attempted to investigate physicochemical properties, molecular structural properties of native and acid-treated chestnut starch and chestnut starch gel. The amylose content was 18.9% and X-ray diffraction pattern showed Cb type. Swelling power was increased abruptly in the range of $65^{\circ}C{\sim}75^{\circ}C$ but increased slowly after that and solubility was increased abruptly until $70^{\circ}C$ but increased slowly after that. In amylograms which have different heating temperatures, cooling viscosity at $50^{\circ}C$ was reduced as heating temperature was increased. In molecular structural properties of amylose, ${\lambda}_{max}$ was 640 nm, ${\beta}-amylolysis$ limit was 84.2% and the degree of polymerization was 951 and in those of amylopectin, ${\lambda}_{max}$ was 570 nm, ${\beta}-amylolysis$ limit was 58.2%, the degree of polymerization was 1371 and average chain length was 22.6. In gel chromatography elution profiles of starch and amylose, 4.0% and 11.5% of low molecular weight-molecules($<5{\times}10^5$) were leached out. In gel chromatography elution profiles of soluble starch, the higher heating temperature was, the more high molecular weight-starches were leached out. The elution profiles after debranching amylopectin with pullulanase showed 2.2 of the ratio of peakIII(DP 10-15) to peakII(DP 35-45). Acid hydrolysis extent of 2.2 N HCI-treated starch at $35^{\circ}C$ for 10 days was 96% and hydrolysis rate showed two step pattern which had border line at 4 days. In elution profiles of acid treated chestnut starch, amylopectin peak was disappeared compeletly after 6 hrs and converted short chains of DP 10-15. Amylose content was increased until 6 hrs but decreased after that. Hardness of starch gel made at $75^{\circ}C$ of heating temperature and cohesiveness of starch gel made at $85^{\circ}C$ of heating temperature were the highest. Retrogradation rate of starch gels were relatively high, especially for the starch gel made at $75^{\circ}C$ of heating temperature.

• Journal of Life Science
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• v.10 no.3
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• pp.307-316
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• 2000

Bacillus circulans S-1 extracellular pullulan 6-glucanohydrolase (EP) (EC 3.2.1.41) has been characterized with a purified enzyme of 140 kDa. The N-terminal amino acid sequence of the purified enzyme was P-L-N-M-S-Q-P. The enzyme displayed a temperature optimum of around $60^{\circ}C$ and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at $4^{\circ}C$ for 48h. The presence of substrate allowed the protection of the enzyme from heat inactivation. The activity of the enzyme was stimulated by several metal ions such as Mn2+ and Ca2+. The enzyme had an apparent Km of 7.92 mg/ml for pullulan. The purfied enzyme completely hydrolysed pullulan to maltotriose.

• Applied Biological Chemistry
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• v.43 no.4
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• pp.241-246
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• 2000

In order to investigate structural properties of Chindo black rice(grown in Chindo, Chonnam) starch and its amylopectin, Chindo black rice was investigated in comparison to Shinsun waxy rice. The maximum absorbance wave and intrinsic viscosity of Chindo black rice starch and Shinsun waxy rice starch were 523 nm, 521 nm and 183 ml/g, 178 ml/g, respectively. ${\beta}-amylolysis$ limit(%) of Chindo black rice and Shinsun waxy rice starch were 62.8% and 60.3%, respectively. Chindo black rice was determined to be a waxy rice due to the results of iodine reaction and elution profile on Sephroce CL-2B. The chain of amylopectins in Chindo black rice distributed fraction 1$(F_1)$ of above degree of polymerization$({\overline{DP}})$ 55, fraction 2$(F_2)$ of ${\overline{DP}} $40{\sim}50$ and fraction 3$(F_3)$ of ${\overline{DP}} $15{\sim}20$, and the ratio of $F_3$ to $F_2$ for Chindo black rice was higher than that for Shinsun waxy rice. The super long chain of amylopectin in Chindo black rice was consisted much more than that of Shinsun waxy rice. ${\beta}-limit$ dextrins in Chindo black rice amylopectin distributed $F_1$ of above ${\overline{DP}} 55, $F_2$ of ${\overline{DP}} $30{\sim}45$ and $F_3$ of ${\overline{DP}} $10{\sim}20$. Little difference was shown between elution patterns of the pullulanase treated ${\beta}-limit$ dextrins of Chindo black rice amylopectin and Shinsun waxy rice amylopectin. These results suggest that Chindo black rice starch was similar to Shinsun waxy rice starch.