• 한국미생물·생명공학회지
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• 제47권4호
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• pp.662-666
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• 2019

본 연구에서는 Saccharomyces cerevisiae를 이용해서 neoagarobiose hydrolase (NABH)를 효율적으로 생산하기 위한 NABH558 유전자 발현시스템을 구축하였다. ADH1 promoter와 GAL10 promoter 하류에 NABH558 유전자를 가진 pAMFα-NABH plasmid와 pGMFα-NABH plasmid는 S. cerevisiae 2805 균주에 형질전환되었다. 2805/pAMFα-NABH 균주는 YPD (2% dextrose) 배지에서 가장 높은 NABH 효소 활성(0.069 unit/ml/DCW)을 보였고, 2805/pGMFα-NABH 균주의 경우는 배지의 조성과 상관없이 비슷한 수준의 NABH 활성(0.02-0.027 unit/ml/DCW)을 보였다. RT-PCR을 통한 NABH558 유전자의 transcription level은 NABH 활성 증가에 따라 비슷한 수준으로 증가되었음을 확인할 수 있었다. 또한 재조합균주에서 생산된 NABH는 agarose를 galactose와 AHG로 분해하였다. 따라서 NABH558 유전자의 발현에는 ADH1 promoter를 사용하는 것이 더 효율적이며 GAL10 promoter와 비교해서 최대 3배정도 높은 활성의 재조합 NABH를 생산할 수 있음을 알 수 있었다.

• 한국미생물·생명공학회지
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• 제48권1호
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• pp.32-37
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• 2020

We improved on a unified saccharification and fermentation (USF) system for the direct production of ethanol from agarose by increasing total agarase activity. The pGMFα-NGH plasmid harboring the NABH558 gene encoding neoagarobiose hydrolase and the AGAG1 and AGAH71 genes encoding β-agarase was constructed and used to transform Saccharomyces cerevisiae 2805. NABH558 gene transcription level was increased and total agarase activity was increased by 25 to 40% by placing the NABH558 gene expression cassette upstream of the other gene expression cassettes. In the 2805/pGMFα-NGH transformant, three secretory agarases were produced that efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexaose. During the united cultivation process, a maximum of 2.36 g/l ethanol from 10 g/l agarose was produced over 120 h.

• KSBB Journal
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• 제26권6호
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• pp.552-556
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• 2011

A novel agar-degrading bacterium mbrc-1 was isolated from seashore of Kyungpo at Gangwon province and cultured in marine broth 2216 medium. Isolated bacterium mbrc-1 was named as Flammeovirga sp. mbrc-1 based on the 16S rDNA sequence. Its agarase showed maximum activity of 923 units/L at pH 7.0 and $45^{\circ}C$ and sustained 90% remaining activity after exposed to $45^{\circ}C$ for 2 hours. The enzyme hydrolyzed agarose to yield neoagarohexaose (18.5%), neoagarotetraose (38%) and neoagarobiose (43.5%), indicating that the enzyme is ${\beta}$-agarase. Thus, isolated bacterium and its ${\beta}$-agarase would be useful for the industrial production of neoagarotetraose and neoagarobiose.

• KSBB Journal
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• 제13권5호
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• pp.524-529
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• 1998

An agarase was partially purified from the culture broth of marine bacterium Bacillus cereus ASK202. Optimal pH and temperature of this agarase were found to be 7.0 and 40$^{\circ}C$, respectively. The maximum productivity of agarooligosaccharides was obtained from 0.3 %(w/v) agar by using of 1 unit agarase. As the results of TLC and HPLC analysis, these oilgosaccharides consisted of neoagarobiose, neoagarotetraose and neoagarohexaose. Under the optimal reaction conditions, 77.5 %(w/v) neoagarobiose and 6.2 %(w/v) neoagarotetraose were produced from agar and the conversion yield of total agarooligosaccharides was 83.7 %(w/v) after for 2 h reaction at 40$^{\circ}C$.

• 생명과학회지
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• 제18권1호
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• pp.58-62
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• 2008

기능성 한천올리고당 생산에 사용할 수 있는 유전자원을 확보하기 위하여 동해안 기장 해수에서 한천분해활성을 보이는 해양유래 세균 SL-12를 분리하였으며 165 rDNA염기서열 분석으로 해양기원의 Glaciecola 속과 가장 유사한 균주임을 확인하였다. SL-12 균주가 생성하는 한천분해효소(agarase)의 최적 pH는 pH 7.0 (20 mM sodium phosphate 완충용액)이고 최적 온도는 $30^{\circ}C$로 나타났다. 분리 된 Glaciecola sp. SL-12 균주가 생산하는 한천분해효소의 분해산물에 대한 TLC 분석결과, 기능성 한천올리고당을 생산하는 ${\beta}$-agarase로 판명되어 산업적 활용 가능성이 높은 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제29권4호
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• pp.625-632
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• 2019

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The $pGMF{\alpha}-HGN$ plasmid harboring AGAH71 and AGAG1 genes encoding ${\beta}-agarase$ and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae $2805/pGMF{\alpha}-HGN$ strain was cultivated into YP-containing agarose medium at $40^{\circ}C$ for 48 h (for saccharification) and then $30^{\circ}C$ for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

• 생명과학회지
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• 제25권11호
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• pp.1273-1279
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• 2015

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.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1183-1189
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• 2021

Autodisplay of a multimeric protein complex on a cell surface is limited by intrinsic factors such as the types and orientations of anchor modules. Moreover, improper folding of proteins to be displayed often hinders functional cell surface display. While overcoming these drawbacks, we ultimately extended the applicability of the autodisplay platform to the display of a protein complex. We designed and constructed a cell surface attachment (CSA) system that uses a non-covalent protein-protein interaction. We employed the high-affinity interaction mediated by an orthogonal cohesin-dockerin (Coh-Doc) pair from Archaeoglobus fulgidus to build the CSA system. Then, we validated the orthogonal Coh-Doc binding by attaching a monomeric red fluorescent protein to the cell surface. In addition, we evaluated the functional anchoring of proteins fused with the Doc module to the autodisplayed Coh module on the surface of Escherichia coli. The designed CSA system was applied to create a functional attachment of dimeric α-neoagarobiose hydrolase to the surface of E. coli cells.

• 생명과학회지
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• 제26권4호
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• pp.453-459
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• 2016

경상남도 남해군 미조면 연안으로부터 채취한 해수를 Marine agar 2216 배지에 도말하여 한천분해활성을 보이는 SH-4 균주를 분리하였다. 선택된 SH-4 균주는 16S rDNA 염기서열분석을 통해 Simiduia sp. SH-4로 명명하였다. Simiduia sp. SH-4 균주의 배양액으로부터 한천분해효소를 획득하여 한천분해활성을 측정하였다. 한천분해활성의 강도에 있어서 Simiduia sp. SH-4 유래 한천분해효소의 최고활성은 120.4 U/l로 나타났다. 한천분해활성은 30℃에서 최고치를 나타내었으며, 30℃에서의 활성을 100%로 하였을 때 20℃에서 30%, 40℃에서 75%의 상대활성을 나타내었다. 최적 pH는 pH 6.0으로 pH 6.0의 활성을 100%로 하였을 때 pH 5.0과 pH 7.0에서 각각 91%와 59%의 상대활성을 나타내었다. TLC 분석 결과, Simiduia sp. SH-4는 neoagarotetraose 및 neoagarobiose 등의 neoagarooligosaccharides를 생성하는 것으로 보아 β-agarase를 생산하는 균주로 확인되었다. 따라서 Simiduia sp. SH-4균주와 이 균주가 생산하는 β-agarase는 식품, 화장품, 의약품 산업에서 기능성소재 생산자로서 유용하게 활용할 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1659-1669
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• 2020

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20℃, respectively. In particular, rAhg943 could maintain enzyme activity at 10℃ (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca²⁺ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K_m and V_max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.