• 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.

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

• 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.

• 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.

• Fisheries and Aquatic Sciences
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• 제13권1호
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• pp.36-48
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• 2010

An agar-degrading Agarivorans sp. AG17 strain was isolated from the red seaweed Grateloupia filicina collected from Jeju Island. A beta-agarase gene from Agarivorans sp. AG17 was cloned and designated as agrA. agrA has a 2,985 bp coding region encoding 995 amino acids and was classified into the glycoside hydrolase family (GHF)-50. Predicted molecular mass of the mature protein was 105 kDa. His-tagged agrA was overexpressed in Escherichia coli and purified as a fusion protein. The enzyme showed 158.8 unit/mg specific activity (optimum temperature at $65^{\circ}C$ and pH 5.5 in acetate buffer) with unique biochemical properties (high thermal and pH stabilities). Enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose by degrading agar, and hydrolyzed neoagaro-oligosaccharides were biologically active. Hence the purified enzyme has potential for use in industrial applications such as the development of cosmetics and pharmaceuticals.

• 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.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.591-597
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• 2017

Maribacter dokdonensis DSW-8 was isolated from the seawater off Dokdo in Korea. To investigate the genomic features of this marine bacterium, we sequenced its genome and analyzed the genomic features. After de novo assembly and gene prediction, 16 contigs totaling 4,434,543 bp (35.95% G+C content) in size were generated and 3,835 protein-coding sequences, 36 transfer RNAs, and 6 ribosomal RNAs were detected. In the genome of DSW-8, genes encoding the proteins associated with gliding motility, molybdenum cofactor biosynthesis, and utilization of several kinds of carbohydrates were identified. To analyze the genomic relationships among Maribacter species, we compared publically available Maribacter genomes, including that of M. dokdonensis DSW-8. A phylogenomic tree based on 1,772 genes conserved among the eight Maribacter strains showed that Maribacter speices isolated from seawater are distinguishable from species originating from algal blooms. Comparison of the gene contents using COG and subsystem databases demonstrated that the relative abundance of genes involved in carbohydrate metabolism are higher in seawater-originating strains than those of algal blooms. These results indicate that the genomic information of Maribacter species reflects the characteristics of their habitats and provides useful information for carbon utilization of marine flavobacteria.

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