• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.13 no.2
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• pp.65-80
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• 1980

Processing conditions of the krill products such as paste food, krill protein concentrate, seasoned dried krill, powdered seasoning, meat ball, and snack have been examined and the quality was evaluated chemically and organoleptically. In the processing of paste food, krill juice was yielded $71\%$ and krill scrap $29\%$. The yields of paste and broth from the krill juice showed $53\%$ and $43\%$, respectively. In amino acid composition of the krill paste, proline, glutamic acid, aspartic acid, lysine, and leucine were abundant, while histidine, methionine, tyrosine, serine and threonine were poor. The optimum condition for solvent extraction in the processing of krill protein concentrate was the 5 times repetitive extraction using isopropyl alcohol at $80^{\circ}C$ for 5 mins. The yield of krill protein concentrate when used fresh frozen materials was $10.2\%$ in isopropyl alcohol solvent and $8.8\% in ethyl alcohol, and when used preboiled frozen materials, the yield was $13.0\%$ in isopropyl alcohol and $11.8\%$ in ethyl alcohol. Amino acid composition of krill protein concentrate showed a resemblance to that of fresh frozen krill meat. In quality comparison of the seasoned dried krill, hot air dried krill was excellent as raw materials and sun dried krill was slightly inferior to hot air dried krill, but preboiled frozen krill showed the poorest quality. The result of quality evaluation for seasoning made by combination of dried powdered krill, parched powdered sesame, salt, powdered beef extract, monosodium glutamate, powdered red pepper and ground pepper showed that the hot air dried krill was good in color and sundried krill was favorable in flavor. When krill meat ball was prepared using wheat flour, monosodium glutamate and salt as side materials, the quality of the products added up to $52\%$ of krill meat was good and the difference in quality upon the results of the organoleptic test for raw materials was not recognizable between fresh frozen and preboiled frozen krill. In the experiment for determining the proper amount of materials such as dried Powdered krill, $\alpha-starch$, sweet potato starch, sugar, salt, monosodium glutamate, glycine, potassium tartarate, ammonium bicarbonate, and sodium bicarbonate in processing krill snack, sample B(containing $7.7\%$ of dried powdered krill) and sampleC (containing $10.8\%$ of dried powdered krill) showed the most palatable taste from the view point of organoleptic test. Sweet potato starch in testing side materials was good in the comparison of suitability for processing krill snack. Corn starch and kudzu starch were slightly inferior to sweet potato starch, while wheat flour was not proper for processing the snack. In the experiment on frying method, oil frying showed better effect than salt frying and the suitable range of frying temperature was $210-215^{\circ}C$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.2
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• pp.351-357
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• 1997

The cyclodextrin glycosyltransferase(EC 3.2.1.19) from Bacillus firmus was purified by precipitating with ammonium sulfate followed by, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 column chromatography. In this way, we were able to obtain the single band protein on SDS-PAGE with a yield of 12%, whose purity was 49 fold. The purified CGTase was identified as a protein having molecular weight of approximately 80,000 dalton and isoelectric point of 9.6. The optimum pH and temperature for the enzyme activity were 8.0 and $65^{\circ}C$, respectively. The enzyme was stable at between pH 5.5 and 9.0 and up to $50^{\circ}C$. After 24hr of enzyme reaction using soluble starch as substrate, the ratio of ${\alpha}-$, ${\beta}-$ and ${\gamma}-cyclodextrin$ production was 0.01 : 2.90 : 1.00, respectively. And this CGTase pro-duced mainly ${\beta}-$ and ${\gamma}-cyclodextrin$.

• Applied Biological Chemistry
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• v.27 no.3
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• pp.169-173
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• 1984

In order to determine the optimum harvesting time based on grain filling and physico-chemical qualities for malting, three cultivars were harvested at every five days from 30th day after heading date at two experimental sites in the southern part of Korea. Starch accumulation and kernel weight increment were remarkable until 45th day after heading, but negligible after that. Content of ash, crude protein and polyphenolics and ${\alpha}-amylase$ activity decreased with maturation of grains and reached to the lowest value at 45th day after heading date. Germinative energy and capacity were good enough for malting from 40th day after heading date. The optimum harvesting time were estimated at 45th day after heading date in the increment of starch accumulation and kernel weight. At this time matured and immatured kernels were in the ratio 93 : 7. However, it is estimated that early harvesting time was at 40th day after heading date.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.3
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• pp.381-387
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• 2003

This study was performed to establish the fermentation method of non-steamed brown rice vinegar using starch saccharifing enzyme. During vinegar fermentation, initial pH had increased in the higher concentration of alcohol and acetic acid. Final pH was gradually changed to pH 2.90~3.44 from 3.44~4.06. The higher total acidity of brown rice vinegar resulted from the higher alcohol concentration. The total acidity was slightly dropped after gradually increasing from the starting of fermentation. Initial pH was decreased from 3.67 to 3.16. The total acidity was gradually increased from the first day of fermentation with 1.02, it was 1.54 on the second day after fermentation and there was a tendency to decreased after the highest values with 6.53 fermentation for 12 days. In organic acid composition, oxalic, malic, acetic, citric, and succinic acid were detected. The total free amino acid was decreased to 1,121 mg%. The major amino acids were ${\gamma}$ -aminobutyric acid, $\alpha$-aminoadipic acid and alanine, and ${\gamma}$-aminobutyric acid was the highest (539 mg%). The mineral contents such as P and K was high in sample and followed by Mg, Na, Ca.

• Korean Journal of Food Science and Technology
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• v.28 no.3
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• pp.521-527
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• 1996

Some structural characteristics of kidney bean starches (3 varieties : Pink kidney bean, Red kidney bean and White kidney bean) were investigated. The amylose content and the ${\beta}$-amylolysis limit of kidney bean starches were $32.6{\sim}34.5%$ and $69.9{\sim}71.0%$, respectively. The kidney bean amylopectin was composed of super long chain of ${\overline{DP}}$ above 60 ($5.28{\sim}12.62%$), B chain of ${\overline{DP}}$ $45{\sim}60\;(29.85{\sim}33.65%)$ and A chain of ${\overline{DP}}\;10{\sim}20(22.94{\sim}29.85%).$ The chain distribution of kidney bean starches were different from variety to variety. The acid (2.2 NHCI) hydrolysis of kidney bean starches showed, as hydrolysis time increased, the patterns of three stages. The acid hydrolysis rate and iodine reaction of acid treated starches were different from variety to variety As acid hydrolysis time increased, the amylose and the ${\alpha}$-1.6-glucosidic linkage of amylopectin of amorphous state were gradually hydrolyzed. Finally, the chain of ${\overline{DP}}$ 20 of crystalline state was left in the acid treated starches.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.31-36
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• 2001

Paenibacillus sp. JB-13 producing the cyclodextrin glucan-otransferase(CGTase) [EC 2.4.1.19] that glucosylated ascorbic acid(AA) at the C-2 position was isolated form soil and the optimal conditions for the production of 2-O-$\alpha$-D- Glucopyranosl L-Ascorbic acid(AA-2G) with CGTase were investigated. CGTase produced AA-2G efficiently using dextrin as a substrate and AA as an aceptor. Several AA-2-oilgosaccharides(AA-2Gs) were also produced in this reaction mixture, and these were efficiently hydro-lyzed to AA-2G and glucose by the treatment with glucoamylase. The optimal temperature for AA-2G production was $37^{\circ}C$ and the optimal pH was around 6.5. CGTase also utilized $\alpha$-,$\beta$-,${\gamma}$-CDs, soluble starch, com statch, dia-static solution from rice and diastatic solution from malt as substrate, but not glucose. The reaction mixture for the maximal production of AA-2G was following; 15% total substrate concentration, 2,500 units/ml of CGTase and a mixing ration of 3:2(g of AA: g of dextrin). Under this condition, 56 mM of AA-2G ,which corresponded to 12.4% yield based on AA. was produced after incubation for 44 hrs at $37^{\circ}C$ and pH 6.5.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.6
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• pp.791-795
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• 2011

This study examined the inhibitory activity of Ecklonia cava (EC) against ${\alpha}$-amylase to evaluate the availability of EC extract as a functional food agent. To verify the inhibitory activity of EC against porcine pancreatic ${\alpha}$-amylase, potato starch was used as a substrate. This analysis revealed that EC ethanol extract exhibited high ${\alpha}$-amylase inhibitory activity. For potential application within the food industry, the stability of the activity of EC ethanol extract under various heat and pH conditions was examined. The ${\alpha}$-amylase inhibitory activity of EC ethanol extract was not affected by the heat and pH treatment conditions used in this study. These results suggest that EC has the potential for development as a functional food agent.

• Journal of Conservation Science
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• v.31 no.3
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• pp.227-241
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• 2015

Fungi were isolated from mural painting in tomb no.6 at songsan-ri, Gong-ju. Antifungal susceptibility of essential oils extracted from natural medicine was tested and it confirmed applicability for mural painting in tombs. 26 species of fungi collected from air-borne and wall surfaces were identified to 15 species of Ascomycetes, 2 species of Zygomycetes, 1 of Basidiomycetes. Wheat starch and gelatin degradability were evaluated as isolated fungi. SY-18, SY-23, SY-25 showed high degradability of wheat starch. SY-18, SY-21, SY-23 were decomposed into gelatin. Biochemical characteristics of decomposing fungi to wheat starch glue and cowhide glue were analyzed by using ${\alpha}-amylase$ and gelatinase activity. An Antifungal test was conducted in Anethole and Eugenol. Anethole and Eugenol mixture(1:2) showed high antifungal susceptibility. Natural adhesives help microbial growth and can cause structural damage in mural painting. The expectation of this study is the possibility to control microbial growth in wall painting using natural essential oils. It can be used as a data for conservation method to control microbial damages.