• 생명과학회지
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• 제12권4호
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• pp.496-503
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• 2002

곤충에서 유래한 항균 펩티드, defensin을 항균활성이 있는 활성적인 형태로 분비하는 Pichia 균주를 개발하기 위한 일환으로서 MF$\alpha$1 prerpo sequence와 defensin 합성유전자를 pichia 발현 벡터에 재조합하여 형질전환하고 아미노산 histidine을 첨가하지 않은 최소 배지에서 형질전환체를 일차적으로 선별하였다. 선별한 형질전환체를 대상으로 항생제 G-418에 대한 내성과 M. luteus를 시험균으로 사용하여 생육환 저해를 통한 defensin의 세포 외 분비를 조사하여 4 균주를 선택하고 분석하였다. Southern hybridizaion을 통해 숙주의 염색체 DNA에 삽입한 defensin 유전자가 유지됨을 확인하였으며 전체 RNA를 분리하고 RT-PCR을 수행하여 defensin mRNA를 증폭하고 Southern hybridization을 실시한 결과 증폭된 밴드는 probe로 사용한 defensin 유전자에 의해 양성 신호가 나타났다. 배양시간에 따른 4 균주의 세포성장과 항균활성을 비교하기 위해 BMMY 배지에서 96시간 배양하였다. 세포성장은 모두 유사한 양상을 보였다. 세포성장은 48시간 동안 급격하게 증가한 후 그 이후로는 정지기에 도달되었다. 항균활성도 48시간까지 급격히 증가하였으며 항균활성이 가장 높은 형질전환체 균주 3는 72시간 배양 시 550 AU/$m\ell$을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1493-1501
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• 2015

A Bacillus species, EMD4, with strong antibacterial activity was isolated from ganjang (soy sauce) and identified as B. subtilis. B. subtilis EMD4 strongly inhibited the growth of B. cereus ATCC14579 and B. thuringiensis ATCC33679. The antibacterial activity was stable at pH 3-9 but inactive at pH 10 and above. The activity was fully retained after 15 min at 80℃ but reduced by 50% after 15 min at 90℃. The activity was completely destroyed by proteinase K and protease treatment, indicating its proteinaceous nature. The bacteriocin (BacEMD4) was partially purified from culture supernatant by ammonium sulfate precipitation, and Q-Sepharose and Sephadex G-50 column chromatographies. The specific activity was increased from 769.2 AU/mg protein to 8,347.8 AU/mg protein and the final yield was 12.6%. The size of BacEMD4 was determined to be 3.5 kDa by Tricine SDS-PAGE. The N-terminal amino acid sequence was similar with that of Subtilosin A. Nucleotide sequencing of the cloned gene confirmed that BacEMD4 was Subtilosin A. BacEMD4 showed bactericidal activity against B. cereus ATCC14579.

• KSBB Journal
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• 제31권2호
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• pp.113-119
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• 2016

A novel agar-degrading marine bacterium, SH-1 strain, was isolated from seashore of Namhae at Gyeongnam province, Korea. The SH-1 strain exhibited 98% similarity with Alteromonas species based on 16S rDNA sequencing and named as Alteromonas sp. SH-1. Alteromonas sp. SH-1 showed agarase activity of 348.3 U/L (1.67 U/mg protein). The molecular masses of the enzymes were predicted as about 85 kDa and 110 kDa by SDS-PAGE and zymogram. The enzymatic activity was optimal at $30^{\circ}C$ and the relative agarase activity was decreased as temperature increase from $30^{\circ}C$ and thus about 90% and 70% activities were shown at $40^{\circ}C$ and $50^{\circ}C$, respectively. The optimum pH was 6.0 for agarase activity in 20 mM Tris-HCl buffer and activities were less than 70% and 85% activity at pH 5.0 and pH 7.0, respectively, compared with that at pH 6. Agarase activity has remained over 90% at $20^{\circ}C$ after 1.5 hour exposure at this temperature. However, its activity was less than 60% at $30^{\circ}C$ after 0.5 h exposure at this temperature. The enzymes produced agarooligosaccharides such as agaropentaose and agarotriose from agarose, indicating that the agarases are ${\alpha}$-agarases. Thus, Alteromonas sp. SH-1 and its agarases would be useful for the industrial production of agarooligosaccharides which are known as having anticancer and antioxidation activities.

• 한국식품영양과학회지
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• 제38권4호
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• pp.509-516
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• 2009

메주로부터 곰팡이 및 세균 등에 생육 저해활성을 나타내는 균주 B. polyfermenticus CJ6을 분리 동정하였다. 분리 균주 B. polyfermenticus CJ6는 2단 대수기를 나타내는 생육 곡선상 특이점을 나타내었으며 배양 30시간 이후부터 최대 활성을 나타내었고 사멸기 이후 활성이 다소 감소되었으나 120시간까지 활성을 유지하였다. B. polyfermenticus CJ6의 항진균 활성 물질은 $70^{\circ}C$ 이상에서 활성이 감소되었으나 $121^{\circ}C$에서 15분간 열처리 시 역가가 완전히 소실되지 않았다. pH 안정성 실험에서는 pH $3.0{\sim}9.0$ 구간에서 안정한 활성을 나타내었으며, 각종 효소에 대한 영향에서 항진균 활성물질은 proteinase K, protease, ${\alpha}$-chymotrypsin 등의 단백분해효소 처리로 역가를 상실하거나 일부 감소되어 단백질성 물질임을 추정하였다. 균주의 항진균 활성 물질을 $C_{18}$ Sep-Pak column으로 부분 정제한 후 Tricine-SDS-PAGE 및 direct detection 실험을 통하여 분자량이 약 1.4 kDa의 물질임을 확인하였다. B. polyfermenticus CJ6가 생산하는 항진균 활성 물질은 기존에 거의 보고되지 않은 B. polyfermenticus 유래의 단백질성 항진균 활성 물질로서 천연보존제 및 천연항균제재로 사용이 기대되며, 이를 위하여 항진균 활성 물질들의 정제 및 구조분석 등의 연구가 필요하다.

• Journal of Animal Science and Technology
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• 제52권2호
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• pp.141-148
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• 2010

이 연구는 김치에서 분리한 항균활성이 우수한 요구르트 제조용 유산균 스타터를 개발하기 위함이다. 분리한 103개의 산 생성 균주를 PCR로 screening하여 72개의 유산균을 분리하였다. 분리균의 배양액을 paper disk method를 사용하여 병원성 미생물(E. coli, S. enteritidis, S. aureus)에 대한 항균활성을 측정하였고, 활성이 강한 균주를 선별하여 API 50CHL과 16S rRNA sequencing 방법으로 균을 동정하였다. 균은 L. plantarum으로 확인되어 L. plantarum LHB55로 명명하였다. E. coli에 대해 높은 항균효과를 나타내는 L. plantarum LHB55를 사용하여 제조한 요구르트의 미생물학적, 이화학적 특성과 관능검사 결과 대조구와의 유의적인 차이는 거의 없었다. 그 결과 김치에서 분리한 L. plantarum LHB55의 항균활성이 우수한 요구르트 제조 스타터 균주로서 사용 가능함을 확인하였다. 항균성 등 인간에게 유리하게 작용하는 스타터 균주와 발효유제품 개발은 계속적인 연구가 필요할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제30권6호
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• pp.937-945
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• 2020

N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (k_cat/K_M) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 x 10⁶ to 1.45 x 10⁶ M^-1 s^-1, with distinctly low K_M values (0.16-0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.

• Asian-Australasian Journal of Animal Sciences
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• 제23권8호
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• pp.993-999
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• 2010

Serum lysozyme gene is one of the important genes influencing the immune system as its product can cause lysis of bacterial cell wall by cleaving the peptidoglycan layer. The present investigation on the serum lysozyme gene of Indian riverine buffalo was undertaken with the objectives to identify and characterize single nucleotide polymorphic patterns by PCR-SSCP method as well as to study the effect of different genotypes on serum lysozyme activity and somatic cell count. A total of 280 animals comprising four different famous bubaline breeds (Murrah, Mehsana, Surti and Bhadawari), spread over six different farms across the country were used for this study. A 276 bp (partial intron 2, complete exon 3 and partial intron 3) fragment of lysozyme gene was screened for polymorphism using the SSCP technique. Four genotypes namely AA, AB, BC and AC were observed, out of which BC genotype was found to be the most frequent. Among these three alleles, C allele (0.38) was most prevalent in these populations. Various SSCP allelic variants were cloned for sequencing and sequences were submitted to NCBI Genbank. From the alignment of the nucleotide sequences of various allelic variants, it was found that there were differences in 12 positions among the alleles, out of which maximum variation (at 8 places) was found in the intronic region. The allele A was closer to allele-C than allele-B. Allele B was phylogenetically equidistant from both of the other alleles. Mean lysozyme activity determined in serum samples of different animals of Murrah buffalo was $27.35{\pm}2.42\;{\mu}g$ per ml of serum, whereas the mean somatic cell count was $1.25{\pm}0.13{\times}10^5$ cells per ml of milk. The SSCP pattern-wise effects of various genotypes on lysozyme activity and SCC were analyzed. Although the mean values were apparently different in various genotypes, these differences were statistically non-significant. It can be concluded that the riverine buffaloes are sufficiently polymorphic with respect to serum lysozyme gene. The absence of AA genotype in Bhadawari breed of buffalo can be considered as a marker for breed characterization. The difference of four nucleotides in exon-3 indicates high selection pressure on the gene.

• 동아시아식생활학회지
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• 제7권3호
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• pp.289-300
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• 1997

생후 2주일 되는 강아지의 위에서 카이모신을 추출하고 이온교환 크로마토그래피로 정제하였다. 카이모신 아미노산 서열의 반은 아미노산 서열 분석법으로, 또 프로카이모신의 전아미노산 서열은 프로카이모신 cDNA의 염기서열로부터 결정하였다. 강아지 프로카이모신의 아미노산 서열은 송아지와는 79%, 돼지 펩신노진 A와는 54%의 상동성을 보였다. 프로펩티드의 크기와 활성효소의 N-말단 아미노산 잔기의 위치는 다른 프로카이모신과 같았다. 강아지 카이모신의 pH 3.2에서 단백질 분해활성의 최대 값은 돼지 펩신의 pH 2에서 값의 3-4% 밖에 되지 않았으나, 웅유활성은 송아지보다 훨씬 높았다. 강아지의 위 추출물에 대한 pH 5.2에서의 한천 젤 전기이동으로 프로카이모신과 카이모신에는 두 가지의 현저한 유전적 변이형이 존재함을 확인하였다. 두 변이형은 아미노산 조성, N-말단 서열, 그리고 효소성질에서 차이가 없었다. 송아지와 강아지 카이모신의 기질결합에 관여하는 아미노산 잔기는 다음과 같이 서로 달랐다(돼지 펩신의 서열번호로 표시함) : Ser12 Thr (S$_4$), Leu30 Val (S$_1$/S$_3$), His 74 Gln (S'$_2$), Val111 Ile (S$_1$/S$_3$), Lys220 Met (S$_4$). 강아지 카이모신의 단백질 분해활성이 낮은 것은 송아지의 Asp 303이 강아지에서는 Val303으로 바뀐 때문이라고 생각된다.

• 한국축산식품학회지
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• 제30권2호
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• pp.328-335
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• 2010

이 연구는 김치에서 분리한 항균활성이 우수한 요구르트 제조용 유산균 스타터를 개발하기 위함이다. 분리한 103개의 산생성 균주를 PCR로 screening하여 72개의 유산균을 분리하였다. 분리균의 배양액을 paper disk method를 사용하여 병원성 미생물(E. coli, S. Enteritidis, S. aureus)에 대한 항균활성을 측정하였고, 활성이 강한 균주를 선별하여 API 50CHL과 16S rRNA sequencing 방법으로 균을 동정하였다. 균은 L. plantarum으로 확인되어 L.plantarum LHC52로 명명하였다. L. plantarum LHC52는 특히 E. coli에 대해 높은 항균성을 나타내었다. L.plantarum LHC52를 사용하여 제조한 요구르트의 미생물학적, 이화학적 특성과 관능검사 결과 대조구와의 유의적인 차이는 없었다. 그 결과 김치에서 분리한 L. plantarum LHC52의 항균활성이 우수한 요구르트 제조 스타터 균주로서 사용 가능함을 확인하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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• pp.131-138
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• 2009

Among rumen microbes, bacteria play important roles in the biological degradation of plant fiber due to their large biomass and high activity. To maximize the utilization of fiber components such as cellulose and hemicellulose by ruminant animals, the ecology and functions of rumen bacteria should be understood in detail. Recent genome sequencing analyses of representative fibrolytic bacterial species revealed that the number and variety of enzymes for plant fiber digestion clearly differ between Fibrobacter succinogenes and Ruminococcus flavefaciens. Therefore, the mechanism of plant fiber digestion is also thought to differ between these two species. Ecology of individual fibrolytic bacterial species has been investigated using pure cultures and electron microscopy. Recent advances in molecular biology techniques complement the disadvantages of conventional techniques and allow accurate evaluation of the ecology of specific bacteria in mixed culture, even in situ and in vivo. Molecular monitoring of fibrolytic bacterial species in the rumen indicated the predominance of F. succinogenes. Nutritive interactions between fibrolytic and non-fibrolytic bacteria are important in maintaining and promoting fibrolytic activity, mainly in terms of crossfeeding of metabolites. Recent 16S rDNA-based analyses suggest that presently recognized fibrolytic species such as F. succinogenes and two Ruminococcus species with fibrolytic activity may represent only a small proportion of the total fibrolytic population and that uncultured bacteria may be responsible for fiber digestion in the rumen. Therefore, characterization of these unidentified bacteria is important to fully understand the physiology and ecology of fiber digestion. To achieve this, a combination of conventional and modern techniques could be useful.