• Journal of Applied Biological Chemistry
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• 제53권2호
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• pp.105-107
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• 2010

To overcome the limitation of E. coli expression system such as inclusion body formation and disulfide bond failure, we tried to express the heterologous protein as a secreted form. We adopted agarase signal peptide (ASP; 23 amino acid residues) from Agarivorans albus YKW-34 which is one of marine bacteia. When we used ASP to express $\beta$-agarase, about 42% activity was detected in media.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.818-821
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• 2011

An agar-degrading bacterium was isolated from the guts of spiny turban shells. It was identified as a Pseudoalteromonas species and named Pseudoalteromonas sp. JYBCL 1. The viscosity of the inoculated agar medium decreased by more than 60% after 20 h cultivation. The agarase produced by the isolate had optimal activities at $35^{\circ}C$ and pH 7. The enzyme had extremely strong resistance to ionic stress compared with other known agarases. Its molecular mass was estimated at about 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The agarase could saccharify Gelidium amansii directly, with an efficiency about half that compared with agar saccharification.

• KSBB Journal
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• 제14권5호
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• pp.572-577
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• 1999

해양으로부터 한천분해능이 뛰어난 균주를 한국의 서해(전남, 여천)으로부터 분리하여 Cytophaga sp.인 것으로 동정되었으며, 이 균주를 Cytophaga sp. AYK301이라 명명하였다. 균체성장과 효소 생산성 향상을 위한 탄소원의 영향을 단당 및 복합당류를 이용하여 살펴본 결과 본 균주는 agar기질을 사용하여 배양하였을 때 효소생산량이 가장 높은 것으로 확인되었으며, agar를 0.0~0.6%(w/v) 농도별로 첨가하여 효소생산능의 최적조건을 살펴보았다. 그 결과 0.2%(w/v) agar에서 최대효소활성을 나타내었다. 질소원의 영향은 유기질소원과 무기질소원으로 나누어 실험을 행하였는데 beef extract 0.3% (w/v), $NH_4NO_3$ 0.05%(w/v)에서 각각 최고의 효소활성을 나타내었다. 또한, 초기 pH의 영향에서 pH 7.5에서 효소활성이 가장 높게 나타났고, NaCl 농도의 영향에서는 7%(w/v)에서 가장 높은 효소활성을 나타내었으며, 각종 금속이온에 대한 효소활성은 거의 영향이 없는 것으로 확인되었다. 따라서, 본 연구에서는 최적배양조건하에서 플라스크로 배양한 결과, 231 units/L를 생한하여 기본배지를 이용하였을 때보다 약 4배 정도 효소활성이 증가하였음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• 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.

• Journal of Microbiology and Biotechnology
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• 제3권1호
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• pp.31-38
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• 1993

A bacterial strain KY-l isolated from sewage was able to produce an extracellular agarase(agarose 4-glycanohydrolase. EC 3.2.1.81). The strain KY-1 was identified as Cytophaga fermentans subsp. agarovorans based on its morphological and physiological characteristics. The agarase was purified by ammonium sulfate precipitation followed by DEAE-Sephadex A-50. Bio-Gel P-100. and CM-Cellulose column chromatography. The molecular weight of the purified enzyme was 24 kDa by SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH for the enzyme activity were 30^{circ}C and 7.5, respectively. The enzyme activity was significantly inhibited in the presence of 0.1 mM $HgCl_2$. whereas it was elevated 3 times by $MnSO_4$ at 1 mM concentration. The Km value and Vmax were 16.67 mg/ml and 3.77 unit/ml.min. The agarase gene was cloned into Escherichia coli MC1061 using the plasmid vector pBR322. A 1.4 Kb DNA fragment of PstI-digested chromosomal DNA of C. fermentans KY-l was inserted into the PstI site of pBR322. expressed in the E. coli. and up to 60% of the total enzyme was extracellularly secreted. Enzymatic properties of the extracellular agarases produced by both the transformant and the donor were very similar in terms of optimal pH and temperature.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1116-1122
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• 2011

In this study, site-directed mutagenesis was performed on the ${\beta}$-agarase AgaA gene from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutant enzymes, S63K, C253I, and S63K-C253I, were 126% (1,757.78 U/mg), 2.4% (33.47 U/mg), and 0.57% (8.01 U/mg), respectively, relative to the wild-type ${\beta}$-agarase AgaA (1,392.61 U/mg) at $40^{\circ}C$. The stability of the mutant S63K enzyme was 125% of the wild-type up to $45^{\circ}C$, where agar is in a sol state. The mutant S63K enzyme produced 166%, 257%, and 220% more neoagarohexaose, and 230%, 427%, and 350% more neoagarotetraose than the wild-type in sol, gel, and nonmelted powder agar, respectively, at $45^{\circ}C$ over 24 h. The mutant S63K enzyme produced 50% more neoagarooligosaccharides from agar than the wild-type ${\beta}$-agarase AgaA from agarose under the same conditions. Thus, mutant S63K ${\beta}$-agarase AgaA may be useful for the production of functional neoagarooligosaccharides.

• 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.

• 생명과학회지
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• 제29권11호
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• pp.1173-1178
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• 2019

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 $70^{\circ}C$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 $50^{\circ}C$에서 pH 6.0인 20 mM Tris-HCl 완충용액을 사용하였을 때 최대의 활성을 보였다. 이 agarase는 20, 30, $40^{\circ}C$에서 2시간 동안 열처리하여도 90% 이상의 잔존활성을 보였다. 또한, $50^{\circ}C$에서 2시간 동안 노출된 후에도 56%의 잔존활성을 보였다. 60과 $70^{\circ}C$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 ${\beta}$-agarase로 확인되었다. 따라서 Agarivorans sp. BK-1가 생산하는 ${\beta}$-agarase는 보습효과, 세균성장 억제, 미백효과, 식품, 화장품 및 전분노화 방지 등의 기능을 가지는 네오한천올리고당의 생산에 유용할 것으로 판단된다.

• KSBB Journal
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• 제14권1호
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• pp.96-102
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• 1999

해양세균 Bacillus cereus ASK202는 특0]적으로 한천의 존재 하에서만 높은 한천분해효소 생산능을 가지는 것으로 확인되었다. 이 균주는 기본배지에서 배양하였올 경우, 그 배양상총액은 21 umts/l의 효소활성을 보였으며, 최적조건하의 발효조를 이용한 배양시에는 생산량은 160.8 umts/l로 기본배지에서보다 효소생산량이 약 7.7배 정도 증가한 결과를 보였다. 해양세균 Bacillus cereus ASK2027가 생산하는 한전분해효소(agarase)에 대하여 treeze drying, DEAE Sepharose CL-6B, Superose 6HR 10/30 column chromatography등에 의해 분리.정제한 결과 최종적으로 31.5 배의 정제도 27.8 %의 수율, 3,780 umt/mg의 specific actIvity을 지닌 정제된 효소를 얻을 수 있었다. 또한 HPLC상에서 정제된 효소가 90,000 daltons의 분자량을 지닌 단백질임을 확인하였다. 정제된 한천분해효소의 최적 pH 및 온도는 각각 6.0과 $40^{\circ}C$. 였으며, pH 5.0 ~ 10.0 및 $30^{\circ}C$에서 장기 보존하였을 경우 효소활성이 안정적으로 유지 됨을 확인하였다. 또한 정제된 한천분해효소 용액은 $Zn(NO_3)_2$의 첨가에 의해 약 16배정도 효소활성의 상승효과를 가져 왔으며, $CuSO_4,\; SnCl_2$에 대해서는 강한 저해효과를 나타내었다. 정제효소에 대한 기질특이성을 조사한 결과, agar와 agarose에 대해서만 특이적인 분해능을 나타내었으며, 그 밖의 polysaccharides에 대해서는 전혀 분해능을 보이지 않았다. 한편 정제된 한친분해효소의 Km 및 Vmax 값은 각각 $2.4mg/m\ell$와 $13.6 mg/m\ell$로 확인되 었다.