• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.137-144
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• 1985

Studies were conducted on the conditions for preparation of yeast protoplasts utilizing Hansenula anomala var. anomala FRI YO-32 as well as Saccharomyces cerevisiae KFCC 32356 and a lytic enzyme from the snail Helix pomatia. The cell wails of the strain FRI YO-32 and S cerevisiae were found to be resistant to activity of the snail lytic enzyme if they were not treated with thiol compounds. Dithiothreitol was found to be more effective than 2-mercaptoethanol, but the latter was considered to be practical. As factors influencing the formation of yeast protoplast, it was considered to be concentration and incubation time of 2-mercaptoethanol or the lytic enzyme, growth stages in yeast cultivation, initial number of yeast cells, and concentration of osmotic stabilizer (KCI). Optimum conditions for the preparation of yeast protoplasts were determined.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.221-224
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• 1989

Amylolytic filamentous fungus, Aspergillus oryzae and nonamylolytic sugar fermentable yeast, Saccharomyces cerevisiae were fused by protoplast fusion in order to develope microorganisms having their intergrated function. Aminoacid auxotrophic properties were used as a genetic marker of protoplast fusion, and 35% PEG 4000 was used as a fusogenic agent. Complementation frequengy of fusion was $4.6\times 10^{-6}$ Obtained fusants showed the morphology of yeast strains, the amylase activity and the ethanol productivity. Among the properties of the fusants, morphology and prototrophic property were sustained stably but their ethanol productivity from starch was reduced. Although fusant strains had 0.5-fold ethanol productivity compared to that of S. cerevisiae in glucose medium, they produced ethanol from strach by direct fermentation.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.264-269
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• 2008

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

• Korean Journal of Food Science and Technology
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• v.40 no.5
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• pp.562-567
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• 2008

The primary objective of this study was to select a probiotic starter for cheonggukjang using 60 strains isolated from rice straw. Among isolated strains, only 8 strains including strain B-59 evidenced proteolytic, amylolytic and soybean activity. These 8 strains were all gram-positive, spore-forming rods. The B-59 strain evidenced antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Pseudomonas fluorescens, and Vibrio parahaemolyticus. The antimicrobial activity of isolated B-59 was verified by its ability to inhibit the growth of S. aureus, L. monocytogenes, P. fluorescens, and V. parahaemolyticus. The selected B-59 strain was indentified as Bacillus licheniformis, as shown by a result of 99.0% homology upon API kit analysis. The selected B-59 strain also displayed viability in pH 2.5 artificial gastric juice, artificial bile acid, NaCl (2, 4, 8, 16, 32%), and ethanol (4, 8, 16, 32%). The antioxidative activity of the strain B-59 culture was assessed via a DPPH free radical scavenging activity assay. The activity increased with an increasing in the fermentation time of strain B-59 for 20 hours.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.2
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• pp.171-180
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• 2017

Objective: This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage. Methods: The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Results: Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens (B. amyloliquefaciens), B. cereus, B. licheniformis, and B. subtilis in ATMR silage and B. flexus, B. licheniformis, and Paenibacillus xylanexedens (P. xylanexedens) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens, B. licheniformis, and B. subtilis and B. licheniformis, B. pumilus, and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. Conclusion: The microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages, whereas the microbial hemicellulase participates in the hemicellulose degradation only at the early stage of ensiling.

• 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.

• Mycobiology
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• v.39 no.2
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• pp.103-108
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• 2011

Amylases and cellulases are important enzymes that can be utilized for various biological activities. Ten different wild Nigerian mushrooms (Agaricus blazei, Agaricus sp., Corilopsis occidentalis, Coriolus versicolor, Termitomyces clypeatus, Termitomyces globulus, Pleurotus tuber-regium, Podoscypha bolleana, Pogonomyces hydnoides, and Nothopanus hygrophanus) were assayed for production of these secondary metabolites. The results revealed that most of the tested wild fungi demonstrated very good amylase and cellulase activities. With the incorporation of carboxymethyl-cellulose (a carbon source) into the culture medium, Agaricus blazei had the highest amylolytic activity of 0.60 unit/mL (at $25^{\circ}C$, pH 6.8). This was followed in order by P. tuber-regium and Agaricus sp. with 0.42 and 0.39 unit/mL, respectively ($p {\leq} 0.05$). Maltose and sucrose supplementation into the submerged liquid medium made N. hygrophanus and P. hydnoides to exhibit very low amylase activities of 0.09 and 0.11 unit/mL, respectively. Introducing peptone (an organic nitrogen source) into the basal medium enhanced the ability of C. versicolor to produce a cellulase value of 0.74 unit/mL. Other organic nitrogen sources that supported good cellulase activities were yeast extract and urea. Sodium nitrate (inorganic nitrogen source) generally inhibited cellulase production in all mushrooms. The best carbon source was carboxymethyl-cellulose, which promoted very high cellulase activity of 0.67 unit/mL in C. versicolor, which was followed in order by P. tuber-regium, T. chypeatus, and C. occidentalis ($p {\leq} 0.05$). Sucrose was the poorest carbon compound, supporting the lowest values of 0.01, 0.01, and 0.14 unit/mL in P. hydnoides, A. blazei, and Agaricus sp., respectively.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.431-440
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• 2016

In 2004, bacterial spot-causing xanthomonads (BSX) were reclassified into 4 species-Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. Bacterial spot disease on pepper plant in Korea is known to be caused by both X. axonopodis pv. vesicatoria and X. vesicatoria. Here, we reidentified the pathogen causing bacterial spots on pepper plant based on the new classification. Accordingly, 72 pathogenic isolates were obtained from the lesions on pepper plants at 42 different locations. All isolates were negative for pectolytic activity. Five isolates were positive for amylolytic activity. All of the Korean pepper isolates had a 32 kDa-protein unique to X. euvesicatoria and had the same band pattern of the rpoB gene as that of X. euvesicatoria and X. perforans as indicated by PCR-restriction fragment length polymorphism analysis. A phylogenetic tree of 16S rDNA sequences showed that all of the Korean pepper plant isolates fit into the same group as did all the reference strains of X. euvesicatoria and X. perforans. A phylogenetic tree of the nucleotide sequences of 3 housekeeping genes-gapA, gyrB, and lepA showed that all of the Korean pepper plant isolates fit into the same group as did all of the references strains of X. euvesicatoria. Based on the phenotypic and genotypic characteristics, we identified the pathogen as X. euvesicatoria. Neither X. vesicatoria, the known pathogen of pepper bacterial spot, nor X. perforans, the known pathogen of tomato plant, was isolated. Thus, we suggest that the pathogen causing bacterial spot disease of pepper plants in Korea is X. euvesicatoria.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.189-194
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• 2003

Rice seeds of 4 cultivars including Whachung-giant embryonic rice and Nampung-giant embryonic rice, as a group of the non-waxy rice cultivars, and Shinsunchal-giant embryonic rice and Whachungchal-giant embryonic rice, as that of the waxy rice cultivars, were germinated at $27^{\circ}C$ for 3 days to compare the changes in some physicochemical properties of the starch granules and the starch-hydrolysing enzyme activities during germination, respectively. ${\alpha}-Amylase$ activity of rices germinated for 3 days found to be higher than that of malt. Especially, Whachung-giant embryonic rice and Shinsunchal-giant embryonic rice were greater in activity than other rice cultivars and possessed the activities double that of malt. In contrast, ${\beta}-amylase$ of germinated rice found to be considerably less active than malt, although the giant embryonic rice group showed prevalent activity as compared o the normal rice group. With the starch granules, the amount of long glucose chains from amylose molecules were reduced in the non-waxy type giant embryonic rices, while the chain length increase was found in the waxy type giant embryonic rices. For the distribution profile of the glucose chain length from amylopectin molecules, we could observed that the chain length with DP (degree of polymerization) ranged 33 to 66 and 14 to 32 increased with the decreasing rate of that above 67 and below 13 regardless of starch waxiness. With non-waxy type of giant embryonic rices, susceptibility for glucoamylase were found to reduce along with germination, however, increase in susceptibility was observed with waxy rice types. In addition, we found the reduction in both initiation and termination temperature, and enthalpy for gelatinization.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.216-220
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• 1989

As the bsic study about protoplast fusion of amylolytic fungus Aspergillus oryze and nonamyloytic sugar fermenter, Saccaromyces cerevsisae, the intraspecific protoplast fusion of A. oryzae was carried out and the properties of the obtained fusants were investigated. For protoplast fomation from mycellia of auxotrophs, Novozyme 234 as lytic enzyme was the most effective and optimal pH was determined to be pH 5.5-6.0. When the two types of protoplasts were treated with a fusogen including 30% PEG4000, they fused effectively and most of fusants were heterokaryons. Protoplasts aggregated with 30% PEG4000 after fusion treatment were observed by the microscope. Protoplast regeneration frequency was 1.46 to 13.8% and complementation frequency of fusion was 0.12 to 0.16. Fusant strains had a 1.5-fold DNA content compared to that of parent strain. And amylase activity was intermediate between those of parent strains.