• Title/Summary/Keyword: -Agarase

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Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7

  • Lee, Yeong Rim;Jung, Subin;Chi, Won-Jae;Bae, Chang-Hwan;Jeong, Byeong-Chul;Hong, Soon-Kwang;Lee, Chang-Ro
    • Journal of Microbiology and Biotechnology
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    • v.28 no.2
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    • pp.284-292
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    • 2018
  • A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

Characterization of α-agarase from Alteromonas sp. SH-1 (Alteromonas sp. SH-1균 유래의 α-agarase의 특성조사)

  • Lee, Sol-Ji;Shin, Da-Young;Kim, Jae-Deog;Lee, Dong-Geun;Lee, Sang-Hyeon
    • KSBB Journal
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    • v.31 no.2
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    • pp.113-119
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    • 2016
  • A novel agar-degrading marine bacterium, SH-1 strain, was isolated from seashore of Namhae at Gyeongnam province, Korea. The SH-1 strain exhibited 98% similarity with Alteromonas species based on 16S rDNA sequencing and named as Alteromonas sp. SH-1. Alteromonas sp. SH-1 showed agarase activity of 348.3 U/L (1.67 U/mg protein). The molecular masses of the enzymes were predicted as about 85 kDa and 110 kDa by SDS-PAGE and zymogram. The enzymatic activity was optimal at $30^{\circ}C$ and the relative agarase activity was decreased as temperature increase from $30^{\circ}C$ and thus about 90% and 70% activities were shown at $40^{\circ}C$ and $50^{\circ}C$, respectively. The optimum pH was 6.0 for agarase activity in 20 mM Tris-HCl buffer and activities were less than 70% and 85% activity at pH 5.0 and pH 7.0, respectively, compared with that at pH 6. Agarase activity has remained over 90% at $20^{\circ}C$ after 1.5 hour exposure at this temperature. However, its activity was less than 60% at $30^{\circ}C$ after 0.5 h exposure at this temperature. The enzymes produced agarooligosaccharides such as agaropentaose and agarotriose from agarose, indicating that the agarases are ${\alpha}$-agarases. Thus, Alteromonas sp. SH-1 and its agarases would be useful for the industrial production of agarooligosaccharides which are known as having anticancer and antioxidation activities.

Molecular Cloning, Overexpression, and Enzymatic Characterization of Glycosyl Hydrolase Family 16 ${\beta}$-Agarase from Marine Bacterium Saccharophagus sp. AG21 in Escherichia coli

  • Lee, Youngdeuk;Oh, Chulhong;Zoysa, Mahanama De;Kim, Hyowon;Wickramaarachchi, Wickramaarachchige Don Niroshana;Whang, Ilson;Kang, Do-Hyung;Lee, Jehee
    • Journal of Microbiology and Biotechnology
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    • v.23 no.7
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    • pp.913-922
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    • 2013
  • An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The ${\beta}$-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) ${\beta}$-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to ${\beta}$-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant ${\beta}$-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at $55^{\circ}C$ and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by $FeSO_4$ (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a ${\beta}$-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

Isolation of an Agarolytic Bacteria, Cellvibrio mixtus SC-22 and The Enzymatic Properties (한천분해세균 Cellvibrio mixtus SC-22의 분리 및 효소적 특성)

  • Cha, Jeong-Ah;Kim, Yoo-Jin;Seo, Yung-Bum;Yoon, Min-Ho
    • Journal of Applied Biological Chemistry
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    • v.52 no.4
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    • pp.157-162
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    • 2009
  • An agar-liquefying bacteria (SC-22), which produces a diffusible agarase that caused agar softening around the colony was isolated from Daecheong lake in Korea. Chemotaxanomic and phylogenetic analyses based on 16S rRNA gene sequences revealed the strain was classified as Cellvibrio mixtus SC-22. The isolate SC-22 showed maximal extracellular agarase activity with 58.5 U/mL after 48 h cultivation in the presence of 0.2% agar. It was observed that the isolate produced two kinds of extracellular and three kinds of intracellular isoenzymes. The major agarase was purified from the culture filtrate of agarolytic bacteria by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The molecular mass of the purified enzyme was estimated to be 25 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 7.0 and $50^{\circ}C$, respectively. The agarase activity was activated by $Fe^{2+}$, $Na^+$ and $Ca^{2+}$ ions while it was inhibited by $Hg^{2+}$, $Mn^{2+}$ and $Cu^{2+}$ at 1 mM concentration. The predominant hydrolysis product of agarose by the enzyme was galactose and disaccharide on TLC, indicating the cleavage of $\beta$-1,4 linkage in a random manner. The enzyme showed high substrate specificity for only agar and agarose among various polysaccharides.

Cloning, Expression, and Characterization of a Novel GH-16 β-Agarase from Agarivorans sp. JA-1 (Agarivorans sp. JA-1 유래 신규 GH-16 β-agarase의 클로닝, 발현 및 특성)

  • Jeon, Myong Je;Kim, A-Ram;Lee, Dong-Geun;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.22 no.11
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    • pp.1545-1551
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    • 2012
  • Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

Isolation of a Marine-derived Flammeovirga sp. mbrc-1 Strain and Characterization of Its Agarase (해양성 Flammeovirga sp. mbrc-1 균주의 분리 및 한천분해기능의 특성조사)

  • Jang, Hye-Ji;Lee, Dong-Geun;Lee, Seung-Woo;Jeon, Myong-Je;Chun, Won-Ju;Kwon, Kae-Kyoung;Lee, Hee-Soon;Lee, Sang-Hyeon
    • KSBB Journal
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    • v.26 no.6
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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.

Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 ${\beta}$-Agarase from Agarivorans sp. JA-1

  • Lee, Dong-Geun;Jeon, Myong Je;Lee, Sang-Hyeon
    • Journal of Microbiology and Biotechnology
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    • v.22 no.12
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    • pp.1692-1697
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    • 2012
  • We report a glycoside hydrolase (GH)-118 ${\beta}$-agarase from a strain of Agarivorans, in which we previously reported recombinant expression and characterization of the GH-50 ${\beta}$-agarase. The GH comprised an open reading frame of 1,437 base pairs, which encoded a protein of 52,580 daltons consisting of 478 amino acid residues. Assessment of the entire sequence showed that the enzyme had 97% nucleotide and 99% amino acid sequence similarities to those of GH-118 ${\beta}$-agarase from Pseudoalteromonas sp. CY24, which belongs to a different order within the same class. The gene corresponding to a mature protein of 440 amino acids was inserted, recombinantly expressed in Escherichia coli, and purified to homogeneity with affinity chromatography. It had maximal activity at $35^{\circ}C$ and pH 7.0 and had 208.1 units/mg in the presence of 300 mM NaCl and 1 mM $CaCl_2$. More than 80% activity was maintained after 2 h exposure to $35^{\circ}C$; however, < 40% activity remained at $45^{\circ}C$. The enzyme hydrolyzed agarose to yield neoagarooctaose as the main product. This enzyme could be useful for industrial production of functional neoagarooligosaccharides.

Isolation of Agarivorans sp. KC-1 and Characterization of Its Thermotolerant β-Agarase (한천분해세균 Agarivorans sp. KC-1의 분리 및 내열성 β-아가라제의 특성 규명)

  • Min, Kyung-Cheol;Lee, Chang-Eun;Lee, Dong-Geun;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.28 no.9
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    • pp.1056-1061
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    • 2018
  • 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 $70^{\circ}C$ 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 $50^{\circ}C$ in 20 mM of Tris-HCl buffer. The agarase activity was maintained at 90% or more until 2 hr exposure at $20^{\circ}C$, $30^{\circ}C$ and $40^{\circ}C$, but it was found that the activity decreased sharply from $60^{\circ}C$. 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 ${\beta}$-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 ${\beta}$-agarase would be useful for the production of neoagarooligosaccharides that inhibit bacterial growth and delay starch degradation.

Improvement of a Unified Saccharification and Fermentation System for Agaro-bioethanol Production in Yeast

  • Lee, So-Eun;Kim, Yeon-Hee
    • Microbiology and Biotechnology Letters
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    • v.48 no.1
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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.

Characteristics of a Marine Agarolytic Pseudomonas sp. from Porphyra dentata(Bangiales, Rhodophyta) and Some Properties of its Extracellular Agarase (김(Porphyra dentata) 병반조직에서 분리한 해양미생물의 특성과 생산된 체외 한천분해효소 특성)

  • 박상렬;조수정;김민근;임우진;류성기;안창룡;홍수영;이영한;김범규
    • Journal of Life Science
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    • v.11 no.4
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    • pp.291-297
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    • 2001
  • The marine bacterium isolated from Porphyra dentata showing green spot rot disease was identified as Pseudomonas sp. the strain have CNCase activity, xylanase activity and protease activity as well as agarase activity. But the strain has no pectate lyase activity. Porphyra dentata tissue inoculated this isolate was macerated after 1 week incubation. The characteristics of extracellular crude agarase of this isolate were examined, the optimal pH and temperature were pH7 and 3$0^{\circ}C$, respectively.

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