• Journal of Microbiology and Biotechnology
• /
• 제24권1호
• /
• pp.48-51
• /
• 2014

${\alpha}$-Neoagarooligosaccharide (${\alpha}$-NAOS) hydrolase was purified from Cellvibrio sp. OA-2007 by using chromatographic techniques after hydroxyapatite adsorption. The molecular masses of ${\alpha}$-NAOS hydrolase estimated using SDS-PAGE and gel filtration chromatography were 40 and 93 kDa, respectively, and the optimal temperature and pH for the enzyme activity were $32^{\circ}C$ and 7.0-7.2. ${\alpha}$-NAOS hydrolase lost 43% of its original activity when incubated at $35^{\circ}C$ for 30 min. The enzyme hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose to galactose, agarotriose, and agaropentaose, respectively, and produced 3,6-anhydro-L-galactose concomitantly; however, it did not degrade agarose.

• Journal of Microbiology and Biotechnology
• /
• 제20권10호
• /
• pp.1378-1385
• /
• 2010

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 Gram-staining 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 MonoQ 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 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were $35^{\circ}C$ 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 thin-layer 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 ${\beta}$-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.

• 생명과학회지
• /
• 제31권3호
• /
• pp.356-365
• /
• 2021

최근, 본 연구진은 담수 환경 유래 한천 분해 세균인 Cellvibrio sp KY-GH-1 (KCTC13629BP)의 전체 유전체 염기 서열을 분석하여 아가로오스를 L-AHG 및 D-갈락토오스로 가수분해시키는 아가레이즈들을 암호화하는 유전 정보를 탐색하였다. 그 결과, KY-GH-1 균주는 유전체 상의 약 77 kb 길이의 아가레이즈 유전자 클러스터 내에 9개의 β-아가레이즈 유전자들과 2개의 α-네오아가로비오스 가수분해효소(α-NABH) 유전자들을 지닌 것으로 나타났다. 이러한 유전자 정보를 바탕으로 KY-GH-1 균주가 한천을 탄소원으로 자화하기 위해 단량체인 L-AHG와 D-갈락토오스로 분해시키는 공정은, 엔도형 GH16 β-아가레이즈, 엔도형 GH86 β-아가레이즈 등에 의해 개시되어 NA4, NA6, NA8 등을 생성시킨 후, 이들에 대해 엑소형 GH50 β-아가레이즈가 추가로 작용하여 NA2를 생성시키고, 이어서 GH117 α-NABH가 작용하여 생성된 NA2를 단량체 L-AHG와 D-갈락토오스로 분해함으로써 종결되는 것으로 예측되었다. 대장균 발현 시스템과 PET-30a 벡터를 함께 사용하여, KY-GH-1 균주 유래의 GH50 패밀리 β-아가레이즈 유전자들(GH50A, GH50B, GH50C)과 GH117 패밀리 α-NABH 유전자들(GH117A α-NABH, GH117B α- NABH)을 발현시켜 His-태그 재조합 효소단백질들로 확보하여, 이들 효소단백질을 이용하여 효소 활성을 비교 분석한 결과, 재조합 GH50A β-아가레이즈가 세 개의 GH50A 패밀리 β-아가레이즈 동위효소들 중에서 가장 높은 엑소형 β-아가레이즈 활성을 나타내며, 또한 재조합 GH117A α-NABH가 NA2를 L-AHG와 D-갈락토오스로 강력하게 가수분해할 수 있으나 재조합 GH117B α-NABH는 NA2 가수분해 활성이 없음을 확인하였다. 연이어 GH50A β-아가레이즈 및 GH117A α-NABH의 효소 특성을 추가로 조사하였다. 아울러 이들 각 효소가 나타내는, 아가로오스를 분해하여 NA2를 생성시키는 효율성과 NA2를 분해하여 L-AHG 및 D-갈락토오스를 생성시키는 효율성을 평가하였다. 본 총설에서는, L-AHG 및 D-갈락토오스의 양산을 위한 아가로오스의 효소적 가수분해에 성공적으로 활용될 수 있을 것으로 기대되는, 담수 유래 한천 분해 세균 Cellvibrio sp. KY-GH-1 유래의 재조합 GH50A β-아가레이즈 및 GH117A α-NABH의 장점들에 대해 기술한다.

• Journal of Microbiology and Biotechnology
• /
• 제22권9호
• /
• pp.1237-1244
• /
• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of Microbiology
• /
• 제45권6호
• /
• pp.505-509
• /
• 2007

An obligately aerobic bacterium, strain KOPRI $20902^T$, was isolated from a marine sediment in Ny-${\AA}$lesund, Spitsbergen Islands, Norway. Cells were irregular rods and motile with polar monotrichous flagellum. The optimum growth temperature was $17-22^{\circ}C$. Cells grew best in pH 7.0-10.0 and 3-4% sea salts (corresponding to 2.3-3.1% NaCl). The novel strain required $Ca^{2+}$ or $Mg^{2+}$ in addition to NaCl for growth. Sequence analysis of 16S rRNA gene revealed that the Arctic isolate is distantly related with established species (<92.4% sequence similarity) and formed a monophyletic group with Cellvibrio, which formed a distinct phylogenetic lineage in the order Pseudomonadales. Predominant cellular fatty acids [$C_{16:1}\;{\omega}7c/15:0$ iso 2OH (45.3%), $C_{16:0}$ (18.4%), ECL 11.799 (11.2%), $C_{10:0}$ 3OH (10.4%)]; DNA G+C content (37.0 mol%); nitrate reduction to nitrogen; absence of aesculin hydrolysis, N-acetyl-${\beta}$-glucosaminidase and esterase; no assimilation of arabinose, galactose, glucose, lactose, maltose, and trehalose differentiated the strain from the genus Cellvibrio. Based on the phylogenetic and phenotypic characteristics, Dasania marina gen. nov., sp. nov. is proposed in the order Pseudomonadales. Strain KOPRI $20902^T$ (=KCTC $12566^T$=JCM $13441^T$) is the type strain of Dasania marina.

• Journal of Microbiology and Biotechnology
• /
• 제21권7호
• /
• pp.667-674
• /
• 2011

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to the class IV family. The purified enzyme worked optimally at $50^{\circ}C$ and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate ($C_4$), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.