한천분해효소의 재조합발현 : 기원, 활성조건, 분비신호와 게놈분석 등

이동근(Dong-Geun Lee),이상현(Sang-Hyeon Lee) 한국생명과학회 2020 생명과학회지 Vol.30 No.3

한천분해효소(agarase)는 기초과학영역, 한천유래 고기능성 올리고당의 생산, 해조류를 이용한 바이오에너지 생산 등에 사용될 수 있다. 본 연구진은 2012년에 한천의 분류, 기원, 생산 및 응용에 관하여 총설하였다. 이에 본고에서는 2012년부터의 agarase 재조합 발현에 대해 총설하고자 한다. Agarase의 재조합 발현에 사용된 유전자는 Agarivorans 속(genus) 세균, Flameovirga 속 세균, Pseudoalteromonas 속 세균, Gayadomonas 속 세균, Catenovulum 속 세균, Microbulbifer 속 세균, Cellulophaga속 세균, Saccharophagus 속 세균, Simiduia 속, Vibrio 속 세균 등의 19종의 세균들에서 유래하였다. 47개의 재조합 발현된 agarase 중에서 α-agarase는 2개였고 나머지는 모두 β-agarase였다. α-Agarase는 모두 agarotetraose (A4)를 생산하였고 β-agarase는 NA2부터 NA12까지 다양한 산물을 생산하였다. 최적온도는 25~60℃, 최적 pH는 3.0~8.5의 범위였다. 50℃ 이상의 최적 온도를 갖는 agarase는 14개로 이들은 한천을 가열한 후에 졸상태가 유지되는 온도에서도 활발한 활성을 보일 것이다. CBM (carbohydrate-binding module)의 조작 등의 인위적 돌연변이로 agarase의 열안정성 증가, 최적온도와 활성의 동시 증가에 관한 연구사례도 있었다. 재조합발현의 숙주로 E. coli, B. subtilis, S. lividans, S. cerevisiae 등이 활용되었으며, agarase 유전자의 분비신호, 다른 생물의 분비신호 및 riboswitch가 agarase의 재조합 발현에 사용되었다. Agarase를 정제한 후에 아미노산 서열에 기반한 유전자 재조합 이외에도 게놈서열 파악과 유사성 비교를 통해 putative agarase와 메타게놈에서 유래한 agarase의 재조합 발현에 관한 연구도 있다. 이러한 연구들은 향후 agarase 및 agarase를 이용한 한천분해산물의 응용 분야 등에 활발하게 이용될 것으로 기대된다. Agarase can be used in the field of basic science, as well as for production of agar-derived high-functional oligosaccharides and bioenergy production using algae. In 2012, we summarized the classification, origin, production, and applications of agar. In this paper, we briefly review the literature on the recombinant expression of agarases from 2012 to the present. Agarase genes originated from 19 genera, including Agarivorans, Flammeovirga, Pseudoalteromonas, Gayadomonas, Catenovulum, Microbulbifer, Cellulophaga, Saccharophagus, Simiduia, and Vibrio. Of the 47 recombinant agarases, there were only two α-agarases, while the rest were β-agarases. All α-agarases produced agarotetraose, while β-agarases yielded many neoagarooligosaccharides ranging from neoagarobiose to neoagarododecaose. The optimum temperature ranged between 25 and 60℃, and the optimum pH ranged from 3.0 to 8.5. There were 14 agarases with an optimum temperature of 50℃ or higher, where agar is in sol state after melting. Artificial mutations, including manipulation of carbohydrate-binding modules (CBM), increased thermostability and simultaneously raised the optimum temperature and activity. Many hosts and secretion signals or riboswitches have been used for recombinant expression. In addition to gene recombination based on the amino acid sequence after agarase purification, recombinant expression of the putative agarase genes after genome sequencing and metagenome-derived agarases have been studied. This study is expected to be actively used in the application fields of agarase and agarase itself.

Agarase를 생산하는 Persicobacter sp. DH-3의 분리 및 β-agarase의 특성

허다혜(Da-Hye Heo),이동근(Dong-Geun Lee),이상현(Sang-Hyeon Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.2

이 연구의 목적은 agarase를 생산하는 새로운 해양 박테리아를 분리하는 것이다. Agarase는 agarose를 가수분해하여 다양한 생리활성을 가지는 agarooligosaccharides 또는 neoagarooligosaccharides를 생성할 수 있다. 전라남도의 여수연안에서 채취한 해수로부터 DH-3 균주를 분리하였다. DH-3 균주는 16S rDNA 서열분석을 통하여 Persicobacter sp. DH-3로 명명하였다. 세포 외로 분비된 효소는 Persicobacter sp. DH-3의 배양액으로부터 얻었으며, 특성연구에 사용하였다. 20, 30, 40, 50, 60 및 70℃에서의 온도별 상대활성은 각각 50, 55, 70, 100, 90 및 50%였다. pH 5, 6, 7 및 8에서의 pH별 상대활성은 각각 75, 100, 90, 75%였다. 본 효소는 20 mM Tris-HCl 완충액에서 pH 6.0 및 50℃에서 최대활성을 나타내었다. 본 효소는 한천이 액체상태로 존재하는 50℃에서 최대활성을 나타내었으므로 산업적으로 유용한 효소로 판단된다. 본 효소는 20, 30 및 40℃에서 2시간 동안 열처리하여도 80% 이상의 잔존활성을 보였다. TLC 분석결과, Persicobacter sp. DH-3 균주의 한천분해효소는 네오한천올리고당인 neoagarohexaose와 neoagarotetraose를 생성하였으므로 β-agarase로 확인되었다. Zymogram 분석결과, Persicobacter sp. DH-3는 적어도 45, 70 및 140 kDa 크기의 3종류 이상의 한천분해효소를 생산하는 것을 확인할 수 있었다. 그러므로, Persicobacter sp. DH-3 유래 agarase는 기능성을 가진 neoagarooligosaccharides의 생산에 활용될 수 있을 것으로 기대된다. The purpose of this study was to isolate a new marine agarase-producing bacterium. Agarase can hydrolyze agar and agarose to produce agarooligosaccharides or neoagarooligosaccharides, which possess many physiological functions. Strain DH-3 was isolated from seawater collected from the coast of Yeosu at Jeollanam province, Korea. A 16S rDNA sequence analysis showed this strain to be Persicobacter sp. DH-3. Extracellular agarase was prepared from culture media of Persicobacter sp. DH-3 and used for characterization. Relative activities at 20, 30, 40, 50, 60, and 70℃ were 50, 55, 70, 100, 90, and 50%, respectively. Relative activities at pH 5, 6, 7, and 8 were 75, 100, 90, and 75%, respectively. The enzyme showed maximum activity at 50℃ in a 20 mM Tris-HCl buffer at pH 6. This enzyme could be useful, as agar is in liquid state at 50℃. Agarase activities were maintained at 80% or more for 2 hr at 20, 30, and 40℃. Thin layer chromatography analysis suggested that Persicobacter sp. DH-3 produced extracellular β-agarases as it hydrolyzed agarose to produce neoagarohexaose and neoagarotetraose. In addition, zymogram analysis confirmed that Persicobacter sp. DH-3 produces at least three agar-degrading enzymes with molecular weights of 45, 70, and 140 kDa. Therefore, it is expected that agarases from Persicobacter sp. DH-3 could be used to produce functional neoagarooligosaccharides.

해양의 Pseudomonas sp. 로부터 분리한 alginate lyase 유전자의 promoter에 의한 대장균 내에서의 β-agarase 유전자의 발현과 catabolite repression의 변화

진철호,박제현,한정현,최윤혁,이종희,이정기,공인수 한국산업미생물학회 2001 한국미생물·생명공학회지 Vol.29 No.2

Strong promoter로 밝혀진 alginate lyase유전자의 promoter부위에 대한 특성을 검토하기 위해 alginate lyase유전자의 46개 N-terminal amino acid가 포함된 promoter부분과, 같은 균으로부터 분리한 β-agarase의 유전자를 연결시켜 agarase의 activity를 평판배지상에서 보다 쉽게 확인하는 방법으로 promoter의 활성을 측정한 결과 alginate lyase유전자 promoter에 의해서 β-agarase유전자의 대량발현이 유도되고 있었으며 glucose의 존재하에서 β-agarase유전자 발현이 일어나지 않는 catabolite repression양상을 나타내고 있다. PCR로써 alginate lyase 유전자의 promoter하류에 존재하는 alginate lyase의 46개 N-terminal amino acid 부분이 순차적으로 제거된 plasmid를 제조하여 대량발현을 조사한 결과 46개의 아미노산이 제거된 후에도 β-agarase의 활성에는 변화가 없어 46개의 N-말단이 정상적인 상태에서 발현에는 영향을 미치고 있지 않음을 확인할 수 있었다. 또한 alginate lyase유전자의 promoter region에 존재하는 가능한 2개의 promoter consensus sequence PⅠ, PⅡ를 subcloning한 결과 promoter PⅡ만이 존재할 때도 대량발현이 유도되고 있음을 확인할 수 있었으며 동시에 glucose가 존재할때 catabolite repression이 역시 나타나고 있어 이 부분이 발현 및 glucose에 의한 regulation에 매우 중요하게 작용하는 부분이라는 것을 확인할 수 있었다. Promoter is a key factor for expression of the recombinant protein. There many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter and association between the promoter mutants, β-agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids of the alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using PCR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of β-agarase productivity.

한천분해세균 Agarivorans sp. KC-1의 분리 및 내열성 β-아가라제의 특성 규명

민경철(Kyung-Cheol Min),이창은(Chang-Eun Lee),이동근(Dong-Geun Lee),이상현(Sang-Hyeon Lee) 한국생명과학회 2018 생명과학회지 Vol.28 No.9

본 연구에서는 해양 한천분해세균 Agarivorans sp. KC-1과 해당균주의 agarase 특성을 조사하였다. 한천분해균인 KC-1은 경상남도 사천시 남일대 해수욕장에서 채취한 바닷물을 이용하여 Marine broth 2216 한천 배지에서 분리하였다. 분리된 균은 16S rRNA 유전자 염기서열분석을 통하여 Agarivorans 속 세균과 99% 일치하여 Agarivorans sp. KC-1으로 명명하였다. 세포외로 분비되는 agarase는 Agarivorans sp. KC-1 균주 배양액에서 획득하였으며, 이를 이용하여 특성 조사를 하였다. Agarivorans sp. KC-1 균주의 한천분해효소는 20, 30, 40, 50, 60 및 70℃에서 각각 65, 91, 96, 100, 77, 35%의 상대활성을 나타냈으며, pH 5, 6, 7, 8에서는 93, 100, 87, 82%의 활성을 나타내었다. 세포외 agarase는 50℃에서 pH 6.0인 20 mM Tris-HCl 완충용액을 사용하였을 때 최대(254 U/l)의 활성을 보였다. 이 agarase는 20, 30, 40℃에서 2시간 동안 열처리 하여도 90% 이상의 잔존활성을 보였다. 또한, 50℃에 2시간 노출된 후에도 67%의 잔존활성을 보였다. Zymogram 분석을 통하여 Agarivorans sp. KC-1 균주가 생산하는 한천분해효소의 크기는 약 130 kDa, 80 kDa, 69 kDa의 3개로 확인할 수 있었다. TLC 분석 결과, Agarivorans sp. KC-1 균주의 한천분해효소는 네오한천올리고당인 neoagarohexaose (21.6%), neoagarotetraose(32.2%) 및 neoagarobiose (46.2%)를 생성하는 것으로 보아 β-agarase로 확인되었다. 따라서 Agarivorans sp. KC-1가 생산하는 β-agarase는 전분노화 방지, 미백효과, 보습효과 및 세균생장 억제 등의 기능을 가지는 한천올리고당의 생산에 유용할 것으로 판단된다. This article reports an agar-degrading marine bacterium and characterizes its agarase. The agar-degrading marine bacterium, KC-1, was isolated from seawater on the shores of Sacheon, in Gyeongnam province, Korea, using Marine Broth 2216 agar medium. To identify the agar-degrading bacterium as Agarivorans sp. KC-1, phylogenetic analysis based on the 16S rRNA gene sequence was used. An extracellular agarase was prepared from a culture medium of Agarivorans sp. KC-1, and used for the characterization of enzyme. The relative activities at 20, 30, 40, 50, 60, and 70℃ were 65, 91, 96, 100, 77, and 35%, respectively. The relative activities at pH 5, 6, 7, and 8 were 93, 100, 87, and 82%, respectively. The extracellular agarase showed maximum activity (254 units/l) at pH 6.0 and 50℃ in 20 mM of Tris- HCl buffer. The agarase activity was maintained at 90% or more until 2 hr exposure at 20℃, 30℃and 40℃, but it was found that the activity decreased sharply from 60℃. A zymogram analysis showed that Agarivorans sp. KC-1 produced 3 agar-degrading enzymes that had molecular weights of 130, 80, and 69 kDa. A thin layer chromatography analysis suggested that Agarivorans sp. KC-1 produced extracellular β-agarases as it hydrolyzed agarose to produce neoagarooligosaccharides, including neoagarohexaose (21.6%), neoagarotetraose (32.2%), and neoagarobiose (46.2%). These results suggest that Agarivorans sp. KC-1 and its thermotolerant β-agarase would be useful for the production of neoagarooligosaccharides that inhibit bacterial growth and delay starch degradation.

신규 한천분해세균 Maribacter sp. SH-1의 분리 및 효소 특성조사

이창은 ( Chang Eun Lee ),이솔지 ( Sol Ji Lee ),이동근 ( Dong Geun Lee ),이상현 ( Sang Hyeon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.2

본 연구에서는 신규 해양성 한천분해균을 분리하고, 이 균주가 생성하는 한천분해효소의 특성을 조사하였다. 경상남도 남해군 미조면 연안에서 채취한 해수를 Marine agar 2216 배지에 도말하여 한천분해세균을 선별하였다. 선택된 한천분해균주는 16S rDNA 염기서열분석을 통해 Maribacter속 세균과 99% 유사하여 Maribacter sp. SH-1으로 명명하였다. 세포외로 분비되는 agarase는 Maribacter sp. SH-1 균주 배양액에서 획득하였으며, 이를 이용하여 특성을 조사 하였다. Maribacter sp. SH-1 균주의 한천분해효소는 20, 30, 40, 50 및 60℃에서 각각 56, 62, 94, 100, 8%의 상대활성을 나타냈으며, pH 5, 6, 7 및 8에서 각각 15, 100, 60, 21%의 상대활성을 나타냈다. 세포외 agarase는 50℃, pH 6인 20 mM Tris-HCl buffer를 사용하는 조건에서 최대활성(231 units/l)을 보였다. 한천이 sol 상태로 존재하는 50℃에서 최적활성을 보여 이 효소는 응용 가능성이 높다고 할 것이다. 효소 활성은 20, 30 및 40℃에서 30분 동안 열처리하였을 때 약 90% 이상의 상대활성을 보였다. TLC 분석 결과, Maribacter sp. SH-1 균주의 한천분해효소는 한천올리고당인 neoagarohexaose (34.8%), neoagarotetraose (52.2%) 및 neoagarobiose (13.0%)를 생성하는 것으로 보아 β-agarase로 확인되었다. 따라서 Maribacter sp. SH-1 균주와 이 균주가 생산하는 β-agarase는 보습효과, 미백효과, 세균성장 억제 혹은 전분노화 방지 등의 기능을 가지는 한천올리고당의 생산에 유용할 것으로 기대된다. In this study, we isolated a new agar-degrading marine bacterium and characterized its agarase. An agardegrading marine bacterium SH-1 was isolated from seawater, collected from the seashore of Namhae in Gyeongnam province, Korea, and cultured in marine agar 2216 media. It was identified as Maribacter. sp. SH-1 by phylogenetic analyses, based on 16S rRNA gene sequence. The extracellular agarase was extracted from culture media of Maribacter sp. SH-1 and characterized. Its relative activities were 56, 62, 94, 100, and 8% at 20, 30, 40, 50, and 60°C, respectively, whereas 15, 100, 60, and 21% relative activities were observed at pH 5, 6, 7, and 8, respectively. Its extracellular agarase exhibited maximum activity (231 units/l) at pH 6.0 and 50°C, in 20 mM Tris-HCl buffer. Therefore, this agarase would be applicable as it showed the maximum activity at the temperature at which the agar is in a sol state. Furthermore, the agarase activities remained over 90% at 20, 30, and 40°C after 0.5 h exposure at these temperatures. Thin layer chromatography analysis suggested that Maribacter sp. SH-1 produces extracellular β-agarase, as it hydrolyzes agarose to produce neoagarooligosaccharides, such as neoagarohexaose (34.8%), neoagarotetraose (52.2%), and neoagarobiose (13.0%). Maribacter sp. SH-1 and its β-agarase would be useful for the production of neoagarooligosaccharides, which shows functional properties, like skin moisturizing, skin whitening, inhibition of bacterial growth, and delay in starch degradation.

해양세균 Agarivorans sp. BK-1의 분리 및 β-아가라제의 특성 규명

안병기(Byeong-Ki Ahn),민경철(Kyung-Cheol Min),이동근(Dong-Geun Lee),김안드레(Andre Kim),이상현(Sang-Hyeon Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.11

본 연구에서는 agarose를 분해하는 세균을 분리하고 한천분해효소의 특성을 조사하였다. 한천분해세균인 BK-1은 부산광역시 수영구 광안리해수욕장에서 채취한 바닷물 시료에서 Marine agar 2216 평판배지를 이용하여 분리하였다. 분리된 균은 16S rRNA 유전자 염기서열분석을 통해 Agarivorans sp. BK-1으로 명명하였다. 세포 외 agarase는 Agarivorans sp. BK-1 균주 배양액에서 획득하였으며, 이를 이용하여 효소의 특성을 조사하였다. Agarivorans sp. BK-1 균주의 한천분해효소는 20, 30, 40, 50, 60 및 70℃에서 각각 67, 93, 97, 100, 58, 52%의 상대활성을 나타냈으며, pH 5, 6, 7, 8에서는 59, 100, 95, 91%의 활성을 나타내었다. 세포 외 agarase는 50℃에서 pH 6.0인 20 mM Tris-HCl 완충용액을 사용하였을 때 최대의 활성을 보였다. 이 agarase는 20, 30, 40℃에서 2시간 동안 열처리하여도 90% 이상의 잔존활성을 보였다. 또한, 50℃에서 2시간 동안 노출된 후에도 56%의 잔존활성을 보였다. 60과 70℃에서 30분 동안 노출된 후에는 효소활성 대부분이 사라졌다. Zymogram 분석을 통하여 Agarivorans sp. BK-1 균주가 약 110, 90, 55 kDa 크기의 한천분해효소들을 생산하는 것을 확인할 수 있었다. TLC 분석 결과, Agarivorans sp. BK-1 균주의 한천분해효소는 네오한천올리고당인 neoagarobiose (46.8%), neoagarotetraose (39.7%) 및 neoagarohexaose (13.5%)를 생성하는 것으로 보아 β-agarase로 확인되었다. 따라서 Agarivorans sp. BK-1가 생산하는 β-agarase는 보습효과, 세균성장 억제, 미백효과, 식품, 화장품 및 전분노화 방지 등의 기능을 가지는 네오한천올리고당의 생산에 유용할 것으로 판단된다. The purpose of this study was to isolate an agar-degrading marine bacterium and characterize its agarase. Bacterium BK-1, from Gwanganri Beach at Busan, Korea, was isolated on Marine 2216 agar medium and identified as Agarivorans sp. BK-1 by 16S rRNA gene sequencing. The extracellular agarase, characterized after dialysis of culture broth, showed maximum activity at pH 6.0 and 50℃ in 20 mM Tris-HCl buffer. Relative activities at 20, 30, 40, 50, 60, and 70℃ were 67, 93, 97, 100, 58, and 52%, respectively. Relative activities at pH 5, 6, 7, and 8 were 59, 100, 95, and 91%, respectively. More than 90% of the activity remained after a 2 hr exposure to 20, 30, or 40℃; about 60% of the activity remained after a 2 hr exposure to 50℃. Almost all activity was lost after exposure to 60 or 70℃ for 30 min. Zymography revealed three agarases with molecular weights of 110, 90, and 55 kDa. Agarose was degraded to neoagarobiose (46.8%), neoagarotetraose (39.7%), and neoagarohexaose (13.5%), confirming the agarase of Agarivorans sp. BK-1 as a β-agarase. The neoagarooligosaccharides generated by this agarase could be used for moisturizing, bacterial growth inhibition, skin whitening, food treatments, cosmetics, and delaying starch degradation.

Isolation of a Novel Tenacibaculum sp. JS-1 and Characterization of Its β-Agarase

Kim Jin Sun,Woo Young Min,Lee Dong-Geun,Kim Andre,Lee Sang-Hyeon 한국미생물·생명공학회 2024 한국미생물·생명공학회지 Vol.52 No.2

This study reports the isolation of a bacterium capable of degrading agar and the characterization of its agarase. An agar-degrading marine bacterium JS-1 was isolated using Marine agar 2216 media from seawater collected from the seashore of Angolpo, Changwon, Gyeongnam Province, Republic of Korea. An agardegrading bacterium was named as Tenacibaculum sp. JS-1 by phylogenetic analysis based on 16S rRNA gene sequence. The extracellular crude agarase was prepared from the culture media of Tenacibaculum sp. JS-1 and used for characterization. Relative activities at 20, 30, 40, 50, and 60℃ were 39, 73, 100, 74, and 53%, respectively. Relative activities at pH 5, 6, 7, and 8 were 46%, 67%, 100%, and 49%, respectively. Its extracellular agarase showed maximum activity (164 U/l) at pH 7.0 and 40℃ in a 20 mM GTA buffer. The residual activities after heat treatment at 20, 30, and 50℃ for 30 min were 84, 73, and 26% or more, respectively. After 2 h heat treatment at 20, 30, 40, and 50℃, the residual activities were 80, 64, 52 and 21%, respectively. Thin layer chromatography analysis suggested that Tenacibaculum sp. JS-1 produces extracellular β-agarases that hydrolyze agarose to produce neoagarooligosaccharides, including neoagarohexaose (12.3%), neoagarotetraose (65.1%), and neoagarobiose (22.6%) at 6 h. Tenacibaculum sp. JS-1 and its β-agarase could be valuable for producing neoagarooligosaccharides with a variety of functional properties. These properties include inhibiting bacterial growth, slowing down starch degradation, and whitening, which are of interest for pharmaceuticals, food, cosmeceuticals, and nutraceuticals.

한천분해세균 Agarivorans sp. JS-1의 분리 및 β-아가라제의 특성 규명

김진선,이동근,여고운,박민주,이상현 한국생명과학회 2023 생명과학회지 Vol.33 No.4

This report looks at an agar-degrading marine bacterium and characterization of its agarase. Agar-degrading marine bacterium JS-1 was isolated with Marine agar 2216 media from seawater from the seashore of Sojuk-do, Changwon in Gyeongnam Province, Korea. The agar-degrading bacterium was named as Agarivorans sp. JS-1 by phylogenetic analysis based on 16S rRNA gene sequencing. The extracellular agarase was prepared from the culture media of Agarivorans sp. JS-1 and used for characterization. Relative activities at 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C were 70%, 74%, 78%, 83%, 87%, 100%, 74%, and 66%, respectively. Relative activities at pH 5, 6, 7, and 8 were 91%, 100%, 90%, and 89%, respectively. Its extracellular agarase showed maximum activity (207 units/l) at pH 6.0 and 50°C in 20 mM Tris-HCl buffer. The residual activity after heat treatment at 20°C, 30°C, and 50°C for 30 minutes was 90%, 70%, and 50% or more, respectively. After a 2-hour heat treatment at 20°C, 30°C, 35°C, 40°C, and 45°C, the residual activity was 80%, 68%, 65%, 63%, and 57%, respectively. At 50°C and above, after heat treatment for 30 minutes, the residual activity was below 60%. Thin layer chromatography analysis suggested that Agarivorans sp. JS-1 produces extracellular β-agarases as they hydrolyze agarose to produce neoagarooligosaccharides such as neoagarohexaose (20.6%), neoagarotetraose (58.5%), and neoagarobiose (20.9%). Agarivorans sp. JS-1 and its thermotolerant β-agarase would be useful in the production of neoagarooligosaccharides, showing functional activity such as inhibition of bacterial growth and delay of starch degradation.

해양성 Simiduia sp. SH-1 균주의 분리 및 한천분해효소의 특성조사

이솔지(Sol-Ji Lee),오수정(Soo-Jeong Oh),이동근(Dong-Geun Lee),이상현(Sang-Hyeon Lee) 한국생명과학회 2015 생명과학회지 Vol.25 No.11

경상남도 남해군 연안의 해수를 이용하여 신규의 한천분해 해양성세균을 분리하고 한천분해효소의 특징을 분석하였다. Marine agar 2216 배지에서 분리한 SH-1 균주는 16S rRNA 유전자 염기서열분석을 통해 Simiduia 속 세균과 99% 유사하여 Simiduia sp. SH-1으로 명명하였다. Simiduia sp. SH-1 균주가 생성하는 한천분해효소는 성장의존성 산물로 판단되었으며 정체기부터 효소활성이 감소되었다. 한천분해효소는 pH 7.0(20 mM Tris-HCl buffer), 30℃에서 최대활성(698.6 units/l)을 나타내었다. 효소의 활성은 30℃에서 최적이었고 이후 온도가 증가함에 따라 활성이 감소하였으며, 40℃와 50℃에서 각각 약 90%와 75%의 상대활성을 보였으나 내열성은 보이지 않았다. 최적 pH인 pH 7.0에 비해 pH 6.0에서는 오차범위 내에서 조금 낮은 활성을 보였으며 pH 5.0과 pH 8.0에서는 각각 80%와 75% 정도의 상대활성을 보였다. TLC 분석을 통하여 Simiduia sp. SH-1 균주가 생성하는 한천분해효소는 agarose를 분해하여 피부의 미백활성, 전분노화의 방지 및 세균성장의 억제 등의 유용한 효과를 가지는 기능성 한천올리고당인 neoagarotetraose와 neoagarobiose를 최종적으로 생성하는 β-agarase로 확인되었다. 따라서, Simiduia sp. SH-1 균주와 이 균주가 생산하는 β-agarase는 산업적 생산에 유용하게 활용될 수 있을 것으로 기대된다. Agarase from a novel agar-degrading bacterium isolated from seawater in Namhae at Gyeongsangnamdo province of Korea was characterized. The SH-1 strain was selected from thousands of colonies on Marine agar 2216 media. Almost full 16S rRNA gene sequence of the agarolytic SH-1 strain showed 99% similarity with that of bacteria of Simiduia genus and named as Simiduia sp. SH-1. Agarase production was growth related, and activity was declined from stationary phase. Secreted agarase was prepared from culture media and characterized. It showed maximum activity of 698.6 units/L at pH 7.0 and 30°C in 20 mM Tris-HCl buffer. Agarase activity decreased as the temperature increased from an optimum of 30° C, with 90% and 75% activity at 40°C and 50°C, respectively. Agarase was not heat resistant. Slightly lower agarase activity was observed at pH 6.0 than at pH 7.0, without statistical difference, and 80% and 75% activity were observed at pH 5.0 and 8.0, respectively. Neoagarotetraose and neoagarobiose were the main final products of agarose, indicating that it is β-agarase. Simiduia sp. SH-1 and its β-agarase would be useful for the industrial production of neoagarotetraose and neoagarobiose, which have a whitening effect on skin, delaying starch degradation, and inhibiting bacterial growth.

Isolation of a Novel Freshwater Agarolytic Cellvibrio sp. KY-YJ-3 and Characterization of Its Extracellular β-Agarase

( Do Youn Jun ),( Young Ho Kim ),( Won Chan Kim ),( Cho Rong Han ),( Rhee Young Joon ),( In Ku Rhee ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.10

A novel agarolytic bacterium, KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of Gramstaining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and Mono- Q column chromatographies, the extracellular agarase in the culture fluid could be purified 120.2-fold with a yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were 35oC and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose hydrolysis catalyzed by the purified agarase using thinlayer chromatography showed that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular β-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.