• Microbiology and Biotechnology Letters
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• v.25 no.3
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• pp.271-276
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• 1997

An ascorbic acid phosphorylating enzyme, which catalyzes the formation of ascorbic acid-2-phosphate from ascorbic acid and pyrophosphate, was purified 32.7-folds to homogeneity from a cell-free extract of Cellulomonas sp. AP-7. The combination of DEAE- Sephacel ion exchange chromatography and Sephacryl S-200 get filtration was used for their purification. The molecular weight of the native protein was estimated to be 96.lkDa on high performance gel filtration chromatography. The SDS-PAGE analysis indicated that the protein consisted of four identical subunits of 24.6 kDa. The purified enzyme showed the optimal tempeature of 40$\circ$C and optimal pH of 4.5. The Km for ascorbic acid and pyrophosphate were 119 mM and 11.9 mM, respectively. The addition of 5,5'-dithiobis-(2-nitrobenzoic acid) into the reaction mixture resulted in the reduction of the enzyme activity at 51%. The enzyme also had a phosphatase activity at weakly acidic pH and the Km for ascorbic acid-2-phosphate in phosphatase activity was 7.9 mM.

• YAKHAK HOEJI
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• v.36 no.4
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• pp.345-349
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• 1992

Thymidylate synthase activity from extracts of various methotrexate-resistant strains was measured by spectrophotometric assay. Methotrexate-resistant strains of Lactobacillus, Pseudomonas sp., Micrococcus sp. HS-1, Klebsillela pneumonae, Cellulomonas fimi and Serratia marcescens elevated thymidylate synthase levels, especially, Pseudomonas sp. KL-9 resistant to $10^{-9}M$ methotrexate have a 156-fold increase in thymidylate synthase, which suggests that Pseudomonas sp. is a convenient source of thymidylate synthase. Several methotrexate strains of yeast were tested, however, their enzyme activity was generally lower than that of bacteria tested.

• Korean Journal of Agricultural Science
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• v.21 no.2
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• pp.142-147
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• 1994

Characteristics of TNP-cellulose which prepared from carboxymethyl cellulose powder, CM32, as substrate for cellulase activity assay were investigated. Enzymatic hydrolysis of TNP-cellulose occured on the cellulose moiety but not on amide bonds, following Michaelis-Menten kinetics. Three cellulase preparations from Trichoderma viride, Aspergillus niger, and Cellulomonas sp. were tested for their pH and temperature dependences and compared with the method determining the increase in reducing power. The enzyme activity was found to have the same temperature range in both methods, however the pH range was broadened in the case of using TNP-cellulose as substrate. The colorimetric method for cellulase assay using TNP-cellulose as substrate was compared with the other methods: one based on determination of the increase in reducing power; and the other based on determining the decrease in viscosity of Na-CM-cellulose solution. The activities measured by the colorimetric method showed a linear correlation with the enzyme concentration of certain range in all three enzymes tested, and the activity values were proportional to those obtained from the other methods. Depending on the enzyme, however, the activity values from this method were not always in proportion to those from the viscometric method. suggesting that this method was not specific for determination of the endo-type cellulase.

• Korean Journal of Soil Science and Fertilizer
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• v.30 no.2
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• pp.196-199
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• 1997

We studied the effect of inoculation of microorganisms known to produce plant growth promoting substances, on the growth and yield of cucumber(Cucumis sativa L.), through a field experiment. The microorganisms used were isolated from the forest soil and consisted of Micrococus sp., Baccilus sustilis, Enterobacter agglomerans, Baccilus megaterium, Pseudomonas putida, Cellulomonas sp. and Staphylococus xyposus. Fotr the multiplication, microorganisms were cultured in liquid media of Pseudomonas P and Sabouraud dextrose. Inoculation of microorganisms was done by spraying the culture media after the culture of them to soil and cucumber plants, three times during the growth of cucumber at the rate of 10l/ha. The inoculation of microorganisms tended to promote the growth of cucumber plant and increase the yield of it. No sign of significant improvement of soil chemical and physical properties were observed after the harvest of crop. The population of bacteria and actinomycetes tended to be higher in the inoculated plots than in not inoculated plots, while opposite was the case in the population of fungi.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.1-8
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• 2001

Spiders are carnivores that prey upon insects and other small arthropods through digestion of food outside the body. Although spider poison may contain proteolytic enzymes, these are thought to play an insignificant role in actual digestion. The source of active proteolytic enzymes can be either the digestive tract cells of spider, or natural microbial flora in the digestive tract of spider. In this study, digestive tracts from the spider, Nephila clavata, were screened for bacteria that have protease or lipase activity. A total of $10^3-10^5$ CFU was recovered from a spider and more than 90% of them showed protease and lipase activity respectively. Of the microbial isolates, 63.3% showed protease or lipase activity, and 50% of these showed both protease and lipase activity. Some of the isolates were characterized using a battery of chemical, phenotypic and genotypic methods. Eleven Gram negative bacteriaa (Acinetobacter calcoaceticus, A. haemolyticus, Alcaligenes faecalis, Cedecea davisae, C. neteri, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas fluorescens, Serratia marcescens, Stenotrophomonas maltophilia, Suttonella indologenes) and 11 Gram positive bacteria (Bacillus cereus, B. coagulans, B. pasteurii, B. thuringiensis, Cellulomonas flavigena, Corynebacterium martruchotii, Enterococcus durans, E. faecalis, Micrococcus luteus, Staphylococcus hominis, S. sciuri) were identified.

• Microbiology and Biotechnology Letters
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• v.5 no.1
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• pp.18-23
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• 1977

Experiments were carried out to pursue the availability and the feasibility of utilizable cellulosic materials as substrate for the production of cellulosic single cell protein. The resuluts were obtained as fellows. 1. Effects of carbolydrates as a sole carton source on the growth of Cellulomonas flavigena KIST 321 were examined. The result showed that cellulose and xylose would be most utlizable for cell mass production. 2. Alkaline treated waste papers and clothes resulted in good growth of the organism than intact ones did. However the waste papers as substrate of cellulosic fermentation were not digestible, even if the meterial was treated with alkalies. 3. Rice straw, rape straw and panic grass appeared to be good substrates for the cell mass production. 4. Leaves were proved to be a good substrate for the cell mass production, but wood sawdust was hardly digested by merely alkaline treatment. 5. When cellulosic wastes as the substrate were examined into the concentration of alkaline solution, the result suggested that the best productivity of cell mass from cellulosic materials was obtained on treatment with 0.8∼1.0％ NaOH solution. 6. The productivity of cell mass was increased by washing out with water after alkaline treatment of newspaper, pine sawdust, lime sawdust and pine leaf.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.443-448
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• 2008

Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable ${\beta}$-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric ${\beta}$-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.

• Journal of Species Research
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• v.12 no.3
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• pp.229-239
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• 2023

We isolated and identified 25 unrecorded bacterial species belonging to the phylum Actinomycetota found in the Republic of Korea. Sequence comparison of 16S rRNA was performed using the NCBI BLAST and EzBioCloud database to identify 25 species, which had a 16S rRNA gene sequence similarity of >98.8% and were allocated as unrecorded species in the Republic of Korea. Among the 25 unrecorded bacterial strains, Streptomyces was the most common with nine species, followed by Leifsonia with two species. Isoptericola, Nocardioides, Dermacoccus, Sinomonas, Patulibacter, Marmoricola, Allobranchiibius, Aldersonia, Actinokineospora, Agromyces, Aeromicrobium, Cellulomonas, and Gordonia with one species each were also found. Twenty-five unrecorded species were excavated in various environments, such as tidal flats, ferns, soil, pine cones, moss, mud, wetlands, and plants. These isolates were characterized on the basis of their phylogenetic, biochemical properties, and morphological data, and species descriptions were provided.

• Microbiology and Biotechnology Letters
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• v.4 no.3
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• pp.99-104
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• 1976

Experiments were carried out to establish the effects of acids in neutralization after alkaline treatment of rice straw, with which cellulosic single cell protein can be produced by cellulose utilizing bacteria, Cellulomonas flavigena KIST 321, previously isolated by authors. Following results were obtained. 1. Rice straw as carbon source was pretreated with 10 volumes of 1 normality of NH$_4$OH or NaOH(NaOH/substrate：40％, and then washed with water or neutralized with H$_3$PO$_4$, H$_2$SO$_4$, HCl and CH$_3$COOH. Among the above mentioned methods, neutralization with H$_3$PO$_4$after alkaline treatment was proved to be the most effective on its digestibility and SCP production. Dry cell 12.28g/$\ell$ and 78％ digestibility were obtained. 2. When rice straw was treated with NaOH solution, the result suggested that the productibity of cell-mass was attained on treatment of rice straw with 6％ of NaOH (NaOH/substrate ratio) for 15～24hrs at room temperature. 3. When rice straw was treated with NaOH, a volume of water to substrate is adequate by two or three fold and the amount of NaOH can be economized up to 5％ for the weight of rice straw. 4. The steaming of rice straw at 121$^{\circ}C$ for 30min. in alkaline treatment of rice straw gave the similiar effectiveness to that at room temperature for 15～24hrs. and accelerated the sterilization of the substrate. 5. Finally, the level of inorganic phosphate in a medium was investigated. 11.2g of dry cell was produced at the concentration of 0.2％, phosphate (phosphorous level 0.04％) in medium even though treated rice straw was neutralized with HCI instead of H$_3$PO$_4$, and 12.2g/$\ell$ at the concentration of 0.3％ phosphate (phosphorous 0.04％) on neutralization with H$_2$SO$_4$.

• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.637-645
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• 2011

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.