• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.5
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• pp.779-784
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• 1995

The effective production of 5'-GMP(5'-Guanylic acid) by immobilized 5'-GMP producing fusant RC102(intergeneric protoplast fusion between Brevibacterium ammoniagenes ATCC21263 and Corynebacterium glutamicum ATCC21171) was investigated. The Fusant RC102 was immobilized by entrapping in -carrageenan, agar, polyacrylamide or Ca-alginate. 3% k-carrageenan was selected as the most suitable matrix. In the production of 5'-GMP using the immobilized whole cells of fusant RC102, the optimum conditions were $32^{\circ}C$, pH 8.0, $30\mu\textrm{g}/L\;of\;Mn^{2+},\;1{\times}10^{-6}%\;of\;Zn^{2+}$. In order to use fermentation medium containing CSL(Corn Steep Liquor) plentiful in $Mn^{2+}$, the optimum conditions of penicillin G, D-cycloserine and POESA(polyoxyethylene stearylamine) for production of 5'-GMP were 0.8unit/ml, 0.8unit/ml, 0.8unit/ml and 5mg/ml, respectively. Cationic surfactant, POESA was effective and superior to the antibiotics, penicillin G or D-cyloserine in 5'-GMP productivity. The condinuous fermentation using immobilized fusant RC102 showed that 5'-GMP productivity was stable for more than 15 days.

• YAKHAK HOEJI
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• v.32 no.5
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• pp.295-303
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• 1988

For microbial production of acetaminophen, a popular analgesic-antipyretic from aniline, we screened various fungi and bacteria. And we succeeded to some extents in acetaminophen production by successful protoplast fusion between S. lividans and S. globisporus and also between S. rimosus and S. aureofaciens. However, more fertile results might be brought via performing the cloning of acetanilide p-hydroxylation genes of Streptomyces in yeast. This study was initiated to determine whether the acetanilide p-hydroxylase of Streptomyces is cytochrome P-450 species or non-heme iron protein species. The p-hydroxylationactivity on acetanilide in S. aureofaciens ATCC 10762 was found to be unstable on exposing to the air. However, 100,000xg supernatant of the cell free extracts which were prepared in $N_2$ atmosphere showed the p-hydroxylation activity. Characteristic absorption peak of cytochrome P-450 after reduction with dithionite and addition of CO was not observed in the region of 450nm. Moreover, metyrapone and 2, 6-dichloroindophenol did not affect this enzyme activity, but sodium azide, sodium cyanide, cupric sulfate, cadmium chloride, ${\alpha}$, ${\alpha}'-dipyridyl$, and o-phenanthroline reduced p-hydroxylase activity considerably. S. fradiae NRRL 2702 was shown to have strong p-hydroxylation activity in intact cells. This activity disappeared in its cell free extracts. In its 100,000xg supernatant, however, characteristic absorption peak of cytochrome P-450 after reduction with dithionite and addition of CO was observed at 446nm. Thus, the results herein presented suggest that acetanilide p-hydroxylase of Streptomyces aureofaciens is not related to cytochrome P-450 and may include non-heme iron protein for its activity. However, it is not clear whether acetanilide p-hydroxylase in S. fradiae belongs to the same category of S. aureofaciens.

• Korean Journal of Food Science and Technology
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• v.41 no.5
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• pp.541-546
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• 2009

The objectives of this study were to evaluate the physico-chemical and sensory characteristics of a Korean traditional alcoholic beverage (yakju) prepared using different nuruk (Korean-style koji) concentrations and yeasts such as the fusant FA776 and Saccharomyces cerevisiae KOY-1, respectively. The fusant FA776, which has alcohol-fermenting and starch-utilizing properties, was formed by Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC1804. The fermentation trial was conducted in a 5 L lab-scale jar at $25^{\circ}C$. The maximum alcohol production of the K-100 and F-50 reached levels of 135.0 mg/mL and 119.4 mg/mL, respectively. The pH values were in a range of 4.3-4.5. Total acidity was in a range of 0.47-0.60%. Organic acids and amino acids were analyzed in order to evaluate variations in its composition and content via HPLC analysis. Organic acids including lactic acid, citric acid, malic acid, and pyruvic acid, and 16 kinds of amino acids, including aspartic acid, were detected in all treatments. K-100 showed the highest amino acid contents, whereas F-50 exhibited the lowest amino acid contents. Volatile flavor components such as phenylethyl alcohol, isoamyl alcohol, 2-methylthiophane, isobutyl alcohol, and ethyl succinate were detected as a major component in all treatments, as determined via gas chromatography. The results of our sensory evaluation demonstrated that Yakju fermented by the FA776 fusant yielded more favorable results than S. cerevisiae KOY-1.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.143-151
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• 1997

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.

• 한국균학회소식:학술대회논문집
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• 2014.05a
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• pp.13-13
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• 2014

Two kinds of mushrooms, Gumji (金芝; Ganoderma) and Soji, were described in old book of Samguksagi (History of the three kingdoms; B.C 57~A.D 668; written by Bu Sik Kim in 1145) in Korea-dynasty. Many kinds of mushrooms were also described in more than 17 kinds of old books during Chosun-dynasty (1392~1910) in Korea. Nowadays, mushroom cultivation has been increased through out the world last decade years. Production of mushrooms has also been increased 10-20% and many varieties have been cultivated. Similar trends were also observed in Korea. Approximately two hundred commercial strains of 37 species in mushrooms were developed and distributed to cultivators. Somatic hybrid variety of oyster mushroom 'Wonhyeong-neutari' were developed by protoplast fusion, and distributed to grower in 1989. The fruiting body yield index of somatic hybrids of Pleurotus ranged between 27 and 155 compared to parental values of 100 and 138. In addition, more diverse mushroom varieties such as Phellinus baumi, Auricularia spp., Pleurotus ferulae, Hericium erinaceus, Hypsizigus marmoreus, Grifola frondosa, Agrocybe aegerita and Pleurotus cornucopiae have been attempted to cultivate in small scale cultivation. Production of mushrooms as food was 190,111 metric tons valued at 800 billion Korean Won (one trillion won if include mushroom factory products; 1dollar = 1,040 Won) in 2011. Major cultivated species are Pleurotus ostreatus, Pleurotus eryngii, Flammulina velutipes, Lentinula edodes, Agaricus bisporus, and Ganoderma lucidum, which cover 90% of total production. Since mushroom export was initiated from 1960 to 1980, the export and import of mushrooms have been increased in Korea. Technology developed for liquid spawn production and automatic cultivation systems lead to the reduction of the production cost resulting in the increasement of mushroom export. However some species were imported because of high production cost for these mushrooms requiring the effective cultivation methods. Developing of effective post-harvest system will be also directly related to mushroom export. In academic area, RDA scientists have been conducting mushroom genome projects. One of the main results is the whole genome sequencing of Flammulina velutipes for molecular breeding. An electrophoretic karyotype of of F. velutipes was obtained using CHEF with 7 chromosomes, with a total genome size of approximately 26.7 Mb. The mususcript of the genome of F. velutipes was published in PLOS ONE this year. For medicinal mushrooms, we have been conducting the genome research on Cordyceps and its related species for developing functional foods using this mushroom. In 2013, Korea Food and Drug Administraion (KFDA) approved Cordyceps mushroom for its value as an immune booster.

• Microbiology and Biotechnology Letters
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• v.39 no.2
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• pp.111-118
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• 2011

For the strain improvement of the industrial polyploid yeast strain through hybridization and protoplast fusion, a dominant selection marker other than a recessive marker such as the auxotrophic marker was required for the selection of the resulting hybrids. In the present investigation, the aureobasidin A resistant gene was tested in relation to whether it can be used as the dominant selectable marker for the isolation of hybrids of the yeast Saccharomyces. The plasmid pAUR112, carrying the gene responsible for resistance to aureobasidin A, was introduced into the haploid yeast strain K114/YIp. From the rare-mating between polyploid C6 and haploid K114/YIp carrying pAUR112, many hybrids were obtained from the agar medium containing 0.5 ${\mu}g$/ml of aureobasidin A. The hybrids exhibited characteristics derived from both of the parental strains; and the cell sizes of the hybrids were larger than those of the parental strains. These results showed that the aureobasidin A resistant gene could be successfully used as the dominant selectable marker for the isolation of yeast hybrids resulting from rare-mating.

• Molecules and Cells
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• v.28 no.5
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• pp.431-439
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• 2009

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-of-function and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

• Journal of Mushroom
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• v.14 no.4
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• pp.142-154
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• 2016

Worldwide production of mushrooms has been increasing by 10-20% every year. Recently, Pleurotus eryngii and P. nebrodensis have become popular mushroom species for cultivation. In particular, China exceeded 8.7 million tons in 2002, which accounted for 71.5% of total world output. A similar trend was also observed in Korea. Two kinds of mushrooms-Gumji (金芝; Ganoderma) and Seoji-are described in the ancient book 'Samguksagi' (History of the three kingdoms; B.C 57~A.D 668; written by Bu Sik Kim in 1145) during the Korea-dynasty. Many kinds of mushrooms are also described in more than 17 ancient books during the Chosun-dynasty (1392~1910) in Korea. Approximately 200 commercial strains of 38 species of mushrooms were developed and distributed to cultivators. The somatic hybrid variety of oyster mushroom, 'Wonhyeong-neutari,' was developed by protoplast fusion, and distributed to growers in 1989. Further, the production of mushrooms as food was 199,829 metric tons, valued at 850 billion Korean Won (one trillion won if mushroom factory products are included) in 2015. In Korea, the major cultivated species are P. ostreatus, P. eryngii, Flammulina velutipes, Lentinula edodes, Agaricus bisporus, and Ganoderma lucidum, which account for 90% of the total production. Since mushroom export was initiated in 1960, the export and import of mushrooms have increased in Korea. Technology was developed for liquid spawn production, and automatic cultivation systems led to the reduction of production cost, resulting in the increase in mushroom export. However, some species were imported owing to high production costs for effective cultivation methods. In academia, RDA scientists have conducted mushroom genome projects since 1997. One of the main outcomes is the whole genome sequencing of Flammulina velutipes for molecular breeding. With regard to medicinal mushrooms, we have been conducting genome research on Cordyceps and its related species for developing functional foods. There are various kinds of beneficial substances in mushrooms; mushroom products, including pharmaceuticals, tonics, healthy beverages, functional biotransformants, and processed foods have also became available on the market. In addition, compost and feed can likewise be made from mushroom substrates after harvest.