• Journal of Microbiology and Biotechnology
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• 제11권3호
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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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• 제47권4호
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• pp.403-408
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• 2004

한국산 재래종 호박 완숙과의 총세포벽 물질, 펙틴, 총단백질, 단백질을 구성하고 있는 아미노산 및 유리아미노산의 함량을 비교 조사하였다. 과육의 단백질 함량은 ‘제주 2’가 $2,830\;{\mu}g/g$으로 가장 높았고 '상주'는 $1,319\;{\mu}g/g$으로 가장 낮았다. 세포벽물질은 ‘강화’가 2,961 mg%로 가장 높았고, '남해'는 1,582 mg%로 가장 낮았다. 또한 세포벽 물질 내 펙틴의 함량은 ‘강화'(2,198 mg%), '제주 2'(2,178 mg%) 및 ’제주 1'(1,461 mg%)의 순서로 높았다. 또한 세포벽 물질 내 펙틴은 수용성과 불용성 펙틴이 주류를 이루고 있음을 알 수 있었다. 아미노산 함량조성은 품종에 따라 다소 차이는 있지만 glutamic acid, aspartic acid, lysine, leucine 및 valine이 주를 이루어 전체 아미노산 함량의 50% 이상을 차지하였고, 특이하게도 '제주 2'는 aspartic acid와 threonine이 검출되지 않았다. 한편 완숙과 호박의 과육 내 유리 아미노산은 8종의 필수아미노산(histidine, isoleucine, leucine, lysine, phenylalanine, methionine, threonine 및 valine)을 비롯해 총 34종류의 유리아미노산이 검출되었고 aspartic acid와 asparagine의 함량이 전체 유리아미노산 중 50% 이상으로 가장 높았다. 또한 이뇨작용과 관련된 ornithine, citrulline, arginine이 모든 품종에서 검출되었으며 ornithine과 citrulline은 품종간에 큰 차이가 없었으나 arginine은 ‘남해’가 4.3 mg%이었으며, ‘태안’이 29.34 mg%로 품종간에 7배의 큰 차이를 보였다.

• Asian-Australasian Journal of Animal Sciences
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• 제6권4호
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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.

• 한국미생물·생명공학회지
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• 제23권1호
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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.

• 한국환경농학회지
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• 제16권1호
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• pp.55-60
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• 1997

광산폐수, 산업폐수등으로 부터 Cd, Pb, Zn 및 Cu 등 중금속에 강한 내성을 지니고 있을 뿐만 아니라 균체내 중금속 축적능력이 우수한 중금속 내성 미생물 균주 Pseudomonas putida, P. aeruginosa, P. chlororaphis 및 P. stutzeri를 각각 분리하여, 세포 구성성분별 중금속 분포 및 중금속 처리 유무에 따른 균체내 amino acid 조성변화등을 조사한 결과는 다음과 같다. 중금속이 100mg/l 농도로 첨가된 배지에서 20시간 배양한 중금속 내성균주들의 균체내 축적된 중금속의 세포 구성 성분별 분포도를 조사한 결과, Cd, Pb 및 Cu 내성균은 cell wall에 약 $50{\sim}60%$가 분포되어 있었고 cell membrane 및 cytoplasm에 각각 약 $30{\sim}40%$ 및 $10{\sim}17%$가 분포되어 있었다. 그러나 Zn 내성균주는 cell wall, cell membrane 및 cytoplasm에 각각 32%, 56% 및 13%가 분포되어 있었다. 중금속이 처리된 배지에서 배양한 중금속 내성균체의 g당 총 아미노산 함량은 중금속이 처리되지 않은 배지에서 배양한 균체에 비하여 높게 나타났으며, 산성 아미노산인 aspartic acid(Asp.+Asn.) 및 glutamic acid(Glu.+Gln)의 함량이 염기성 아미노산인 histidine, lysine, arginine에 비하여 많이 함유되어 있었다.

• 미생물학회지
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• 제30권6호
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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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• 제14권6호
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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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• 제17권5호
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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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• 제38권6호
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• pp.496-501
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• 1995

Cellulomonas sp. KL-6 균주의 균체내 외 단백질은 배양 5일째에 $266\;{\mu}g/m{\ell},\;37\;{\mu}g/m{\ell}$로 가장 많이 생산하였다. 본 실험에서 사용한 KL-6 균주는 볏짚을 분쇄 후 1.0% NaOH와 10% $NH_4OH$로 $80^{\circ}C$에서 30분간 처리한 후 $H_3PO_4$로 중화하여 탄소원으로 이용하였을 때 각각 $1.63\;g/{\ell}$ 및 $1.47\;g/{\ell}$의 SCP가 생산되어 처리하지 않은 볏짚의 $0.5\;g/{\ell}$ 보다 균체수율이 증가되었다. KL-6 균주가 생산한 SCP의 필수아미노산의 함량이 FAO 표준단백질에 비해 많아 영양적 가치가 매우 우수하였다. 그러나 실제 동물사육에 이 균체를 이용코저 할 때는 균체의 세포벽을 파쇄후 사용해야 되리라 사료된다.

• 미생물학회지
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• 제33권4호
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• pp.225-231
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• 1997

Elastase 저해물질(EI) 생성균주로 토양분리주 SMF-11을 선별하였다. 분리주 SMF-11는 매끈한 표면의 rectiflexible 및 spiral의 포자 사슬을 동시에 형성하고 세포벽 성분으로 LL-DAP, ios- 및 anteiso-fatty acid($C_{14}-C_{17}$), 세포벽 아미노산으로 alanine, glutamic acid, glycine을 가지고 있어 방선균속으로 분류되었다. 종 수준의 동정을 위하여 화학적 시험과 50가지의 단위형질에 대한 특성을 조사하고 TAXON program으로 수리분류한 결과, SMF-11 분리주는 방선균 주군집 61의 중심균주인 Streptomyces lavendulae로 동정하였다.