• 생명과학회지
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• 제17권1호
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• pp.70-75
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• 2007

포항 호미곶 해수에서 한천분해활성을 보이는 해양성 세균 SL-5을 분리하였으며 16S rDNA 염기서열분석으로 해양기원의 Thalassomonas 속과 가장 유사한 균주임을 확인하였다. SL-5 균주가 생성하는 한천분해효소(agarase)는 성장의존성인 것으로 확인되었고 효소활성을 위한 최적 pH는 pH 7.0(20 mM sodium phosphate 완충용액)이고 최적 온도는 $40^{\circ}C$로 나타났다. 한천분해효소는 $80^{\circ}C$까지 65%의 효소활성을 보이는 호열성 효소이지만, 내열성은 그리 높지 않았다. 한천 분해효소의 분해산물에 대한 TLC 분석결과, 본 효소가 $\beta-agarase$라는 사실을 확인할 수 있었다. 분리한 Thalassomonas sp. SL-5 균주가 생산하는 한천분해효소를 이용하여 한천으로부터 다양한 기능성 한천올리고당을 생산하는데 있어 유용하게 사용될 것으로 기대된다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.31-32
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• 2005

Neoagaro-oligosaccharides are produced only by enzymatic degradation of agarose by ${\beta}-agarase.^{1)}$ Neoagaro-oligosaccharides inhibit the growth of bacteria, slow the rate of degradation of starch, are used as low-calorie additives to improve food quality, and have macrophage-stimulating activity. Furthermore, neoagarobiose is a rare reagent that has both moisturizing effect on skin and whitening effect on melanoma $cells.^{2)}$ An agar-degrading marine bacterium was isolated from the sea water at the northeast coast in Cheju island, Korea. The strain was gram negative, aerobic, and motile rod. The 16S rRNA of the strain had the closest match of 98% homology, with that from Agarivorans albus. On the basis of several phenotypic characters and a phylogenetic analysis, this strain was designated Agarivorans sp. JA-1. In solid agar plate, Agarivorans sp. JA-1 produced a diffusible agarase that caused agar softening around the colonies. Agarivorans sp. JA-1 was cultured for 36 hr in marine broth 2216 (Difco, USA) and the supernatant that containing an extracellular ${\beta}-agarase$ was prepared by centrifugation of culture media. The enzyme exhibited relatively strong activity at $40^{\circ}C$ and was stable up to $60^{\circ}C$. Using PCR primers derived from the ${\beta}-agarase$ gene of Vibrio sp., the gene encoding ${\beta}-agarase$ from Agarivorans sp. JA-1 was cloned and sequenced. The structural gene consists of 2931 bp encoding 976 amino acids with a predicted molecular weight of 107,360 Da. The deduced amino acid sequence showed 99% and 34% homology to $agaA^{2)}$ and $agaB^{2)}$ genes for ${\beta}-agarase$ from Vibrio sp., respectively. The expression plasmid for ${\beta}-agarase$ gene of Agarivorans sp. JA-1 is being constructed and the recombinant enzyme will be biochemically characterized.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1621-1628
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• 2012

The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, $0.5-0.6{\mu}m$ wide and $2.0-2.5{\mu}m$ long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at $20^{\circ}C-37^{\circ}C$, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita $R10SW13^T$(99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, ${\alpha}$-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase, ${\beta}$-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

• 한국식품과학회지
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• 제32권2호
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• pp.284-290
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• 2000

한천분해능이 뛰어난 해양세균 Bacillus cereus ASK202를 사용하여 최적조건 하에서 배양실험을 행한 결과, 배양시간 30시간 경과시에 최대량의 한천분해효소가 생산되었으며, 그 생산량은 160.8 unit/L로 기존에 보고된 다른 균주들에 비해 한천분해효소 생산능이 뛰어남을 확인하였다. 또한, 생산된 한천분해효소를 이용하여 기질인 한천과 반응시킨 결과, 주로 중합도 2, 4, 6의 올리고당이 생성됨을 확인하였다. 그리고, 한천올리고당의 물리 화학적 특성을 기존의 제품화된 각종 올리고당과 비교 분석한 결과, 고점성, 높은 올리고당의 함유율, 뛰어난 내산 내열성, 저 감미도를 확인하였다.

• 미생물학회지
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• 제38권4호
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• pp.247-253
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• 2002

환경오염원으로서 폭약 2,4,6-trinitrotoluene (TNT)에 대한 TNT 분해세균 Stenotrophomonas sp. OK-5의 세포반응에 대하여 조사하였다. 아치사조건의 TNT농도와 노출시간에 따른 균주 OK-5의 생존율을 분석한 결과, 이 세균의 생존율은 스트레스 충격 단백질의 생성과 비례하였다. 총세포 지방산 조성분석에서 균주 OK-5는 tryp-ticase soy agar에서 자랄 때보다 TNT 배지에서 자랄 때 여러 가지 종류의 지방산이 생성되거나 사라지는 것이 밝혀졌다. 주사전자현미경하에서 TNT에 노출된 세포는 쭈글쭈글하고 불규칙적인 간상형으로 나타났다. Anti-DnaK와 anti-GroEL을 이용하여 SDS-PAGE와 Western blot을 통한 분석으로 균주 OK-5는 70 kDa DanK와 60 kDa GroEL을 포함하는 몇가지 스트레스충격단백질을 생성하는 것으로 밝혀졌다. TNT에 노출된 OK-5 배양에서 수용성 단백질 분획에 대하여 2-D PAGE를 실시하였으며, pH 3에서 pH 10의 범위에서 약 300여 개 spot들이 silver로 염색된 gel상에서 관찰되었다. 이들 가운데 TNT의 반응으로 현저하게 유도되고 발현된 10개의 spot들을 확인하였으며, 2개의 단백질, spot #1과 spot #10에 대한 내부아미노산 서열을 ESI-Q TOF로 분석한 결과, Xylella fastidiosa의 DnaK protein XF2340와 Mesorhizobium loti의 스트레스 유도단백질로 각각 밝혀졌다.

• 한국미생물·생명공학회지
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• 제31권1호
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• pp.75-82
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• 2003

환경 오염원으로서 폭약 hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX)에 대한 RDX 분해세균 Pseudomonas sp. HK-6의 세포반응과 형태변화에 대하여 조사하였다. 아치사조건의 RDX농도와 노출시간에 따른 균주 HK-6의 생존율을 분석한 결과, 이 세균의 생존율은 스트레스 충격 단백질의 생성과 비례하였다. 총세포 지방산 조성분석에서 균주 HK-6는 trypticase soy agar(TSA)에서 자랄 때 보다 RDX배지에서 자랄 때 여러 가지 종류의 지방산이 생성되거나 사라지는 것이 밝혀졌다. Anti-DnaK와 anti-GroEL을 이용하여 SDS-PAGE와 Western blot을 통한 분석으로 균주 HK-6는 70 kDa DanK와 60 kDa GroEL을 포함하는 몇가지 스트레스 충격단백질을 생성하는 것으로 밝혀졌다. RDX에 노출된 HK-6배양에서 수용성 단백질 분획에 대하여 2-D PAGE를 실시하였으며, pH 3에서 pH 10 범위에서 약 300 spots가 silver로 염색된 gel상에서 관찰되었다. 그 결과, RDX에 대한 반응으로 10여개의 spots가 현저히 유도 발현되었다. RDX(0.135mM, 12시간)에 노출된 세포는 구멍이 나타나고 표면의 불규칙적인 형태 변화가 일어나 죽게되는 것이 주사 전자현미경을 통하여 관찰되었다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.19-25
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• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.284-292
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• 2018

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

• KSBB Journal
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• 제11권1호
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• pp.37-45
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• 1996

한국의 남해안에서 한천 분해능이 뛰어난 해양 미 생물을 분리하여 통정한 결과 Pseudomonas 속으로 판명되었으며, 본 연구에서는 이 균을 Halophilic P Pseudomonas sp. W7이라 명영하였다. 이 균주는 호염성 세균으로, 한천의 존재 하에서 높은 효소활성 을 가지는 extracellular agarase를 생산해 내었다. 이 extracellular agarase를 DEAE-Cellulose ani- on exchange chromatography와 gel filtration을 통해 정제하였으며, 정제된 agarase는 SDS-PAGE 를 통해 약 89KDa의 분자량을 지니는 single protein band염을 확인하였다. 한편 agarase의 대량생 산을 위하여 host cell Eo coli JM83과 vector pUC19를 이용하여 gene cloning을 행하였다. Pseudomonas sp. W7의 chromosomal DNA가 삽 입 된 균주 중에 서 agarase activity를 나타내는 E. coli JM83/pSWl과 Eo coli JM83/pSW3를 선멸하 였으며, 전기영통 실험결과 이들은 각각 307Kb, 3.0 K Kb의 chromosomal 단편을 지니고 있음을 확인하였 다. 또한 agarase 유전자가 압입된 변이 균주(B)는 inclusion body 형태의 interacellular agarase를 세포 내에 축적하고 있음이 확인되었다.