• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Applied Biological Chemistry
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• v.47 no.4
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• pp.403-408
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• 2004

We studied active substances like crude cell wall components, crude protein, composing amino acids and free amino acids including orinithine cycle-related amino acids such as asparagine, ornithine and citrullin in fully ripe fruits of Korean native squash, Cucurbita moschata Poir. Crude protein content of 'Jeju 2' was the highest with $2,830\;{\mu}g/g$, while 'Sangju' was the lowest with $1,319\;{\mu}g/g$. Regarding the contents of crude cell wall components, fruit 'Kanghaw' was the highest with 2,961 mg% while 'Namhea' was the lowest with 1,582 mg%. Pectin contents of crude cell wall components were the highest in 'Kanghaw' (2,198 mg%) followed by 'Jeju 2' (2,178 mg%) and 'Jeju l' (1,461 mg%). The main contents of amino acids in squash were glutamic acid, aspartic acid, lysine, leucine and valine, which comprised to be more than 50% of total amino acid contents. Especially, in 'Jeju 2' aspartic acid and threonine were not detected. In fully ripe fruits, a total of 34 kinds of free amino acids were detected including 8 kinds of essential amino acids (histidine, isoleucine, leucine, lysine, phenylalanine, methionine, threonine and valine). More than 50% of the total free amino acids were aspartic acid and asparagine, and also all varieties were detected in ornithine, citrullin, and arginine, which are related to Ornithine cycle. There was a big difference in the contents of arginine in all varieties whereas the contents of ornithine and citrullin were very similar. 'Teaan' 29.34% was 7 times higher than 'Namhea' 4.30% in regards to arginine contents.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.619-622
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• 1993

Rumen bacterial amino acids in sheep on urea diet were monitored to assess a possible change in amino acid synthesis as a long term response to high rumen ammonia environment. A sheep was fed a semipurified diet with soybean meal, followed by a diet with urea as a main nitrogen source. Mixed rumen bacteria were harvested from ruminal fluid taken 3 h after feeding (twice in soybean meal feeding and 6 times in urea feeding) and fractionated as cell wall, proteins and protein-free cell supernatant of monitor amino acids in each fraction. Ruminal ammonia concentration at the sampling ranged from 5.7 to 39.5 mgN/dl. Cell wall and protein fractions of mixed rumen bacteria were stable in their amino acid composition regardless of nitrogen sources of diet and the feeding duration. However, protein-free cell supernatant fraction showed a higher alanine proportion with urea feeding (18.6 and 28.2 molar % of alanine for samples from sheep fed soybean meal and urea, respectively) and its duration (20.6 and 32.9 molar % for samples from sheep on urea diet for 1 and 65 days, respectively). Total free amino acid level of bacteria was depressed in the initial period of urea feeding but restored on 65th day of the feeding. These results suggest that an alanine synthesizing system may develop in rumen bacteria as urea feeding becomes longer.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.31-35
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• 1995

Using the alkalophillic Bacillus sp. SH-8 and its mutant Bacillus sp. SH-8M capable of growing at the neutral pH, the amino acid compositions of the cell wall and cell membrane were studied at varying cultivation pH's. The pattem of protein electrophoresis was also tested. It was elucidated that the amino acids consisting of the cell wall were alanine, glutamic acid, lysine, aspartic acid, and meso-diaminopimelic acid. There was not any significant difference in the amino acid compositqon betweeo`two straqns regardless of the culture pH. As the results of HPLC ssay, glutamic acid and aspartic aciu accounted for more than 50% in the amqno acid composytqon of the cell wall. By the isolatqon of the crude cell membrane and the SDS-PAGE analysis, it was found that there was a considerable difference qn the protein pattern when the straqns were cultured at the neutral pH. In addition, by the two dimensional gel electrophoresis, it was confirmed that there was a difference in the protein patterns between two strains cultivated at the neutral pH medium but no difference at the alkaline medium.

• Korean Journal of Environmental Agriculture
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• v.16 no.1
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• pp.55-60
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• 1997

Heavy metal-tolerant microorganisms, such as Pseudomonas putida, P. aeruginosa, P. chlororaphis and P. stutzeri which possessed the ability to accumulate cadmium, lead, zinc and copper, respectively, were isolated from industrial wastewaters and mine wastewaters polluted with various heavy metals. The distribution of heavy metal in the cell components, and amino acid compositions, was investigated. The distribution of heavy metal in the cell fractions of each heavy metal-tolerant microorganism grown for 20 hours in the basal medium containing 100mg/l of each heavy metal was investigated. In the case of cadmium-tolerant P. putida, lead-tolerant P. aeruginosa and copper-tolerant P. stutzeri, approximately $50{\sim}60%,\;30{\sim}40%$ and $10{\sim}17%$ of each heavy metal absorbed were distributed to cell wall, cell membrane and cytoplasm fractions, respectively. In the case of zinc-tolerant P. chlororaphis, approximately 32%, 55% and 13% of zinc were distributed to cell wall, cell membrane and cytoplasm fractions, respectively. These results indicated that the cell wall was a major adsorbing fraction of cadmium, lead and copper, and the cell membrane was that of zinc. Total amino acid content per gram of the cell grown in the culture media with heavy metal was higher than that of the cell grown in the culture media without heavy metal, and the content of acidic amino acids, such as aspartic acid(Asp.+Asn.) and glutamic acid(Glu.+Gln.) was higher than that of basic amino acids, such as histidine, lysine and arginine.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.483-487
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• 1992

Among a few number of UV-resistant isolated form various environmental sources (10), we made a comparative physio-chemoanalytical study on one of spherical bacteria isolated from air dust, presumably Deinococcus sp. (CM strain 29) with an UV resistant bacterium, Deinococcus radiophilus ATCC 27603 as the reference strain. Our isolate of UV resistant coccus, Deinococcus sp. CM 29 and D. radiophilus ATCC 27603 showed more than 75% matching coefficient in metabolic activity of various substrates. The most predominant cellular fatty acid of both strains was palmitoleic acid (C 16 :1, cis 9), but the detail fatty acid profiles were slightly dissimilar to each other. Cell-bound arange pigment seemed to be an identical chemicals on spectrophotometric analysis. L-ornithine was detected as cell-wall amino acid in both strains. Galactose was detected as cell-wall sugar in D. radiophilus ATCC 27603, whereas glucose in Deinococcus sp. CM 29. G-C molar ratio of both strains was comparable, 63-65%.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1142-1149
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• 2004

Streptococcus mutans has the capacity of inducing dental caries. Thus, to develop a novel way of preventing dental caries, a cell wall hydrolase-producing strain was isolated and its characteristics were investigated. Among 200 alkalophilic strains isolated from soil, 8 strains exhibited lytic activities against Streptococcus mutans. However, strain YU5215 with the highest cell wall hydrolase activity was selected for further study. Strain YU5215 was identified as a novel strain of Bacillus based on analyzing its 16S rDNA sequence and Bergey's Manual of Systematic Bacteriology, and thus designated as Bacillus mutanolyticus YU5215. The optimal conditions for the production of the cell wall hydrolase from Bacillus mutanolyticus YU5215 consisted of glucose ($0.8\%$), yeast extract ($1.2\%$), polypeptone ($0.5\%$), $K_{2}HPO_{4}\;(0.1\%$), $MgSO_{4}{\cdot}7H_{2}O$ ($0.02\%$), and $Na_{2}CO_{3}\;(1.0\%$) at pH 10.0. Bacillus mutanolyticus YU5215 was cultured at 30^{circ}C for 72 h to produce the cell wall hydrolase, which was then purified by acetone precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined as 22,700 Da by SDS-PAGE. When the cell wall peptidoglycan of Streptococcus mutans was digested with the lytic enzyme, no increase in the reducing sugars was observed, while the free amino acids increased, indicating that the lytic enzyme had an endopeptidase-like property. The amino terminus of the cell wall peptidoglycan digested by the lytic enzyme was determined as a glutamic acid, while the lytic site of the lytic enzyme in the Streptococcus mutans peptidoglycan was identified as the peptide linkage of L-Ala and D-Glu.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.633-637
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• 2004

The experiment of silage for preservation of fresh Italian ryegrass (Lolium multiflorum) was carried out to examine whether the fermentation quality and microbial degradation in the rumen can be altered by the treatment of amino acids fermentation byproduct (AFB). The plant was ensiled for 40 days with 4 treatments of different ratios of AFB and sugarcane molasses (SCM) mixture. The treatment 2 (T2, AFB:SCM=100:0) and treatment 3 (T3, AFB:SCM=40:60) silages showed higher (p<0.05) concentrations of lactic acids, lower (p<0.05) pH and dry matter (DM) losses than the Control (T1, none additive) and treatment (T4, AFB:SCM=0:100) silages. The treatments 2 and 3 contained higher (p<0.05) DM and crude protein contents in silages compared to treatments 1 and 4 silages. The NDF, ADF and cellulose contents were also lower (p<0.05) in T2, T3 and T4 silages than T1 silage and fresh material before ensiled. The in situ rumen DM, NDF, ADF, hemicellulose and cellulose degradability was also higher (p<0.05) in T2, T3 and T4 silages than T1 silage, while the highest improvement was achieved with addition of AFB:SCM at level of 40:60 at ensiling. The result in this study indicates that the addition of AFB and SCM additives improved the silage fermentation and cell wall degradability of Italian ryegrass silage.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.496-501
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• 1995

Experiments were carried out to find out the optimal condition of SCP produced by Cellulomonas sp. KL-6 and to evaluate nutritional value for the protein of this organism Intracellular- and extracellular proteins produced by this strain were estimated to be nearly maximum, $266\;{\mu}g/m{\ell}\;and\;37\;{\mu}g/m{\ell}$, in the medium containing 0.001% of thiamin after 5 days cultivation. When used rice straw as carbon source for the cell growth of this organism after crusing them by cutting mill, and treating them with 1.0% of NaOH and 10.0% of $NH_4OH\;at\;80^{\circ}C$ for 30 minutes and neutralizing continuatively them with 85% of $H_3PO_4$, SCP production rates were very increased to $1.63\;g/{\ell}$ (NaOH) and $1.47\;g/{\ell}$ (NH4OH), respectively than $0.5\;g/{\ell}$ produced in untreated rice straw. We compared their amino acids patterns with that of FAO provisional patterns. Amino acids content of strain KL-6 was excellents. However. when intended these cell mass to use in practical animal feeding test it would be advisable that destruction or lysis of cell wall should be done.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.225-231
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• 1997

Elastase inhibitor (EI) producing microorganisms were screened by microplate-assay of culture broth using Succinyl-$(Alanine)_3$-Nitroanilide as a chromogenic substrate. The portion of microorganisms showed EI activity above 90% was about 2% of all assayed. Isolated SMF-11 showed relatively high EI activity after filtration using membrane filter of NMWL 3,000. SMF-11 was identified as Streptomyces sp. according to results of morphological or physicochemical identification. SMF-11 has both rectiflexible and spiral spore chains with smooth spore surface and cell wall contained LL-DAP, iso- and anteiso-fatty acids. The amino acids in cell wall were alanine, glutamic acid and glycine. Fifty unit characters for major cluster were tested and the data were analyzed numerically using the TAXON program. The isolate SMF-11 was identified as a strain of Streptomyces lavendulae.