Purification and Biochemical Characterization of β-agarase Produced by Marine Microorganism Cellulophga sp. J9-3

⧕᧲ၙᔾྜྷ Cellulophga sp. J9-3ᯕ ᔾᔑ⦹۵ ᄁ┡-ᦥa౩ᯕᷩ᮹ ᇥญ ၰ ᔾ⪵⦺ᱢ ✚ᖒ

ʡ݅ᗽ¹, ʡ᳦⯍², ḡᬱᰍ¹*

1ǎพᔾྜྷᯱᬱšၙᔾྜྷᯱᬱŝ

2ᕽᯝݡ⦺Ʊᩢ᧲⦺ŝ

Received: May 23, 2021 / Revised: July 5, 2021 / Accepted: July 7, 2021

ᕽ ು

⦽⃽^(agar)ᮡ⧕᳑ඹᖙ⡍ᄞ᮹ᵝ᫵ᖒᇥᯕ໑^, ᦥaಽᜅ

(agarose)᪡⡍⦝௡(porphyrin)᮹ࢱaḡ ^galactanᮥ⡍⧉⦽

݅^{[1, 2].} ✚⯩, Gelidiumŝ Gracilaria ᗮŝzᮡ⪮᳑ඹᮁ

௹⦽⃽ᮡᵝಽᦥaಽᜅಽǍᖒࡹᨕᯩ۵ߑ^, ᯕ۵ agarobiose (4-O-β-d-galactopyranosyl-3,6-anhydro-l-galactose) ݉᭥ℕa

α^-1,3 đ⧊ᮝಽᩑđࡽၹᅖࡽᯕݚඹ᮹ᖁ⩶ᔍᜍಽǍᖒࡽ

݅^[3].

⦽⃽ᮡ᪅ఌ࠺ᦩ᜾⣩ᯕӹ᜾⣩ᖒ⩶ᮥ᭥⦽℉aᱽಽᔍ

ᬊ⧕᪉ᦩᱥ⦽᜾⣩ᗭᰍᯕ໑^, ၙᔾྜྷ႑᧲ᬊ႑ḡ^, ⪵⧊ྜྷ

᮹ݕℕ^, Ⓧಽษ☁ə௹⦝ᬊݕℕ^, ၵᯕ᪅⥭௝ᜅ❒ᗭᰍ॒ᮝ

ಽəᮁᬊᖒᯕ᷾ݡ⦹Łᯩ݅^{[4, 5].} ↽ɝᨱ۵ᦥaಽᜅෝ

⬉ᗭᱢᮝಽᇥ⧕⦽᪍ญŁݚॅ᮹݅᧲⦽ᔾྜྷ⦺ᱢ⪽ᖒᯕ ᅕŁࡹŁᯩ݅^[6−^9]. ੱ⦽^, ᦥaಽᜅ᮹⬉ᗭᱢᇥ⧕↽᳦ᔑ

ྜྷᯙ D-galactoseӹ 3,6-anhydro-l-galactose ॒᮹⪽ᬊᨱš

⦽ᩑǍࠥ⪽ၽ⯩ᙹ⧪ࡹŁᯩ݅^{[10, 11].} ঑௝ᕽ^, ᦥaಽᜅ ᮹⬉ᗭᱢᇥ⧕Ŗᱶ}ၽᮡ⦽⃽ၵᯕ᪅ີᜅ᮹ᔑᨦᱢᯕᬊ ᨱaᰆᵲ᫵⦹݅^.

ᦥaಽᜅ᮹⬉ᗭᱢᇥ⧕Ğಽ۵ࢱaḡĞಽaݡ⢽ᱢᯕ

݅^[2]. α-agarolytic Ğಽ۵α^-1,3 đ⧊ᮥᖁ┾ᱢᮝಽᱩ݉⦹

۵α^-agaraseᨱ᮹⧕ᵝࠥࡹ໑^, ⪹ᬱั݉ᨱ 3,6-anhydro-l- galactoseෝw۵ᯝಉ᮹ agarooligosaccharidesෝอॅᨕԙ

݅^. β-agarolytic Ğಽ۵β^-1,4 đ⧊ᮥᖁ┾ᱢᮝಽᱩ݉⦹۵ Purification and Biochemical Characterization of β-agarase Produced by Marine Microorganism Cellulophga sp. J9-3

Da Som Kim¹, Jong-Hee Kim², and Won-Jae Chi¹*

1Microorganism Resources Division, National Institute of Biological Resource, Incheon 22689, Republic of Korea

2Department of Food and Nutrition, Seoil University, Seoul 02192, Republic of Korea

Cellulophga sp. J9-3, is a gram-negative, aerobic marine bacterium belonging to the family Flavobacteriaceae.

In addition to cellulose degradability, the J9-3 strain is also capable of hydrolyzing agar in the solid and liq- uid medium, and the production of agarase in the presence of agarose can be remarkably induced by the bacterium. From the cell culture broth of Cellulophga sp. J9-3, ammonium sulfate precipitation and three kinds of column chromatography were successively performed to purify a specific agarase protein, the AgaJ93. Purified AgaJ93 showed the strongest hydrolyzing activity towards agarose (approximately 22%), and even displayed activity towards starch. AgaJ93 hydrolyzed agarose into neoagarotetraose and neoaga- rohexaose via various oligosaccharide intermediates, indicating that AgaJ93 is an endo-type β-agarase.

AgaJ93 showed maximum activity at a pH of 7.0 and temperature of 35 °C. Its activity increased by more than six times in the presence of Co²⁺ ions. The N-terminal sequence of AgaJ93 showed 82% homology with the heat-resistant endo-type β-agarase Aga2 of Cellulophaga sp. W5C. However, the biochemical properties of the two enzymes were different. Therefore, AgaJ93 is expected to be a novel agarose, different from the previously reported β-agarases.

Keywords: Cellulophga sp. J9-3, neoagarotetraose, neoagarohexaose, agarose, agar

*Corresponding author

Tel.: +82-32-590-7371, Fax: +82-32-590-7230 E-mail: [email protected]

β^-agaraseᨱ᮹⧕ᵝࠥࡹ໑^, ⪹ᬱั݉ᨱ D-galactoseෝw۵ ᯝಉ᮹ neoagarooligosaccharidesෝอॅᨕԙ݅^. ḡɩʭḡ ᅕŁࡽ ^agarase᮹ݡᇡᇥᮡβ^-agaraseᨱᗮ⦹໑^, α^-agarase ۵ ⁵ÕอᯕᅕŁࡹᨕᯩ݅^[5]. ঑௝ᕽ^, ၙᔾྜྷᨱ᮹⦽⦽⃽

ݡᔍ۵ݡᇡᇥβ^-agaraseašᩍ⦹۵ β-agarolytic Ğಽᨱ᮹

⦽äᮝಽᩩᔢ⧁ᙹᯩ݅^.

ə࠺ᦩ݅᧲⦽ၙᔾྜྷಽᇡ░ฯᮡβ^-agaraseaᅕŁࡹᨩ

݅^. ᯕᵲᨱ۵ Ł᪉ᖒ^[12], ᱡ᪉ᖒ^[13], ԕᩝᖒ^[14], ⪙ᔑᖒ

[15], ⪙᦭⋝ญᖒ^[16] ॒⬉ᗭᱢ✚ḶᯕǍᇥࡹ۵⬉ᗭॅᯕ

݅ᙹᯩḡอ^, ᔑᨦ⪵ᨱᱢᬊࡽ⬉ᗭ᮹ᩩ۵ᦥḢʭḡᅕŁࡽ

ၵᨧ݅^. ঑௝ᕽ^, ⦽⃽᮹ᔑᨦᱢᯕᬊෝ᭥⧕ᕽ۵޵ᬒ݅᧲

⦽⬉ᗭ᮹ၽǕᯕ᫵ǍࡹŁᯩ݅^. ᅙᩑǍḥᮡᱽᵝࠥ⧕ᙹ ᨱᕽᇥญ⦽ Cellulophga sp. J9-3 Ɂᵝ᮹ᔾญ⦺ᱢ✚Ḷŝ

ᅙɁᯕᔾᔑ⦹۵ ^cellulase᮹⬉ᗭᱢ✚ᖒᨱš⦹ᩍᅕŁ⦽

ၵᯩ݅^[17]. ᯕ⬥᮹ĥᗮࡽ ᩑǍෝ☖⧕, Cellulophga sp.

J9-3 Ɂᵝaᔾᔑ⦹۵ ^agarase᮹✚ᖒᨱš⦹ᩍᅙםྙᨱ ᱶญ⦹ᩡ݅^. ⪮᳑ඹᖙ⡍ᄞᨱ۵ᖡ൑ಽ᪅ᜅa᧞ ^20% ⧉ᮁ

ࡹᨕᯩ݅۵ᱱᨱᕽ, cellulase᪡ ^agaraseෝ༉ࢱᔾᔑ⧁ᙹ

ᯩ۵ Cellulophga sp. J9-3 Ɂᵝ۵⪮᳑ඹၵᯕ᪅ີᜅᇥ⧕

ၰᯕᬊᨱᦥᵝᱢ⧊⦽Ɂᵝᯝäᮝಽᩩᔢࡽ݅^.

ᰍഭ ၰ ႊჶ

ၙᔾྜྷ ႑ḡ ၰ ႑᧲

Cellulophga sp. J9-3 (KACC 92201P)ᮡᱽᵝࠥ⧕ᙹᨱᕽ ᇥญ⦹ᩍ࠺ᱶ⦹ᩡŁ^, Ɂᵝ᮹ᇥඹ⦺ᱢ✚ᖒŝ cellulase ᔾ ᔑ܆ಆᨱš⦹ᩍ۵ᯕၙ ᅕŁ⦽ၵᯩ݅[17]. Cellulophga sp. J9-3 Ɂᵝ᮹ ^agarase ᔾᔑ܆ಆᮥ᳑ᔍ⦹ʑ᭥⧕^, Ɂᵝෝ

Marine agar 2216 (MA) (Difco, USA) ႑ḡᨱ ᱲ᳦⦹ᩍ 28Ⳅᨱᕽ ⁴⁸᜽e࠺ᦩ႑᧲⦽⬥, Lugol's Iodine ᬊᧂ^{(0.05 M} Iodine in 0.12 M KI)ᮝಽᩝᔪ⦹ᩍ⎽ಽܩᵝ᭥ᨱ⩶ᖒࡽ

ᇥ⧕⪹ᮥ šₑ⦹ᩡ݅^. Ɂᵝ᮹ ĥݡ ၰ ႑᧲ᨱ۵ ^MA᪡ Marine broth 2216 (MB) (Difco) ႑ḡෝᔍᬊ⦹ᩡ݅^.

Cellulophga sp. J9-3 Ɂᵝ᮹ ᔾᰆłᖁ ၰ ⬉ᗭ ᔾᔑ ᇥᕾ Cellulophga sp. J9-3 Ɂᵝෝ ^{50 ml MB} ႑ḡ^(250-ml flask)ᨱᱲ᳦⦹ᩍ ²⁸Ⳅᨱᕽ ²⁴᜽eḥ┶႑᧲^{(200 rpm)} ⦹

ᩡ݅^. ႑᧲ᧂ ^{1 ml}ෝᔩಽᬕ ^MB ႑ḡ ^{100 ml}ᨱᱲ᳦⦹Ł^,

࠺ᯝ᳑Õᨱᕽ ¹⁰⁴᜽e࠺ᦩ႑᧲⦹໕ᕽᯝᱶ⦽᜽eeĊᮝ

ಽ႑᧲ᧂᮥ ^{3 ml}ᦊᔹ⥭ย⦹ᩡ݅^. ႑᧲ᧂᔹ⥭ᮡᇥŲŲ

ࠥĥಽ ^{600 nm}ᨱᕽ᮹┢ࠥ^(OD600)ෝ⊂ᱶ⦹ᩍᖒᰆłᖁᮥ

᪥ᖒ⦹ᩡ݅^. ੱ⦽^, ႑᧲ᧂᮥ ^{10,000 ×g}ᨱᕽ ¹⁰ᇥeᬱᝍᇥ ญ⦽⬥ᔢ॒ᧂᮥ≉⦹ᩍ^{, DNS} ჶ^[18]ᮝಽᖙ⡍᫙ᇡಽᇥእ

ࡽ ^agarase᮹⪽ᖒᮥ⊂ᱶ⦹ᩡ݅^.

Agarase ⬉ᗭ᮹ ᱶᱽ

Cellulophga sp. J9-3ෝ ^{0.2% agar}a⡍⧉ࡽ ^MB ႑ḡ 1 Lᨱᱲ᳦⦹ᩍᔢʑ᪡࠺ᯝ᳑Õᨱᕽ ⁴⁸᜽e႑᧲⦹ᩡ݅^. ႑

᧲ᧂᮥ ^{10,000 ×g}ᨱᕽ ²⁰ᇥeᬱᝍᇥญ⦹ᩍ᨜ᮡᔢ॒ᧂᨱ ammonium sulfate (75%)ෝ℉a⦹ᩡ݅^{. 12}᜽e ࠺ᦩԪ

ᰆŁᨱᕽ ݉႒ḩᮥ⋉ᱥ᜽┉⬥^, ႑᧲ᧂᮥ ^{10,000 ×g}ᨱᕽ

60ᇥeᬱᝍᇥญ⦹ᩍ⋉ᱥྜྷอᮥ≉⦹ᩍ᪥∊ᧂ ^{A (50 mM} Tris-Cl, pH 7.5) 50 mlᨱ⩥┢⦹ᩡ݅^. ݉႒ḩ⩥┢ᧂᮥ࠺ ᯝ⦽᪥∊ᧂᮝಽ ¹²᜽e࠺ᦩ ⁴Ⳅᨱᕽ⚍ᕾ⦹ᩍ┩ᩝ⦽⬥^, 10,000 × gᨱᕽ ³⁰ᇥe ᬱᝍᇥญ⦹Ł^, ᔢ॒ᧂᮥ ^0.22 μ^m syringe filter (Millipore, USA)ಽᩍŝ⦹ᩍ݉႒ḩ׮⇶ᧂ (75 ml)ᮥᵡእ⦹ᩡ݅^.

݉႒ḩ׮⇶ᧂᮝಽᇡ░ ^agaraseෝᱶᱽ⦹ʑ᭥⧕ ^DEAE- sepharose FFᮭᯕ᪉Ʊ⪹Ⓧಽษ☁ə௹⦝(GE Healthcare, USA)ෝᙹ⧪⦹ᩡ݅^. ⍍ౝ(15 mm × 140 mm, Econo-Pac, BioRad, USA)ᨱ౩ḥᮥ ^{2 ml} ₥ᬑŁ^, ᪥∊ᧂ ^Aಽ⠪⩶⪵᜽

┉౩ḥᨱ݉႒ḩᮥ⯂₊᜽┉⬥^, ࠺ᯝ᪥∊ᧂᨱ ^{100, 200,} 300, 400, 500 mM NaClᮥ⧉ᮁ⦽ᬊᧂ⁽bᬊᧂ᮹^{volume =} 20 ml)ᮝಽ݉ĥᱢᮝಽ݉႒ḩᮥᬊ⇽᜽⎑݅⁽ᮁᗮ ^{= 1 ml/}

min). DNS ჶᮝಽᬊ⇽ᧂ᮹ ^agarase ⪽ᖒᮥ⊂ᱶ⦽đŝ^,

⪽ᖒᮡ౩ḥᨱđ⧊⦹ḡᦫŁ☖ŝ⦽ᔹ⥭ᨱᕽá⇽ࡹᨩᮝ ໑^, ঑௝ᕽ☖ŝᔹ⥭ಽᇡ░ ^agarase᮹ ²₉ᱶᱽෝᙹ⧪⦹

ᩡ݅^{. 2}₉ᱶᱽෝ ᭥⧕ Sulfopropyl (SP)-sepharose cation exchange chromatography (⍍ౝ volume = 2 ml, GE Healthcare)ෝᙹ⧪⦹ᩡŁ^, ᯕ࠺ᔢᮡ᪥∊ᧂ ^Aෝᔍᬊ⦹ᩡ

݅^. ⯂₊⦽݉႒ḩ᮹ᬊ⇽ᨱ۵ ¹⁰⁰−500 mM NaCl (100 mM eĊ⁾ᮥ⧉ᮁ⦽᪥∊ᧂ ^Aෝ݉ĥᱢᮝಽᔍᬊ⦹ᩡ݅⁽bᬊᧂ ᮹ volume = 20 ml, ᮁᗮ = 1 ml/min). DNS ჶᮝಽ ^agarase

⪽ᖒᮥ⊂ᱶ⦽đŝ, 100 mM NaCl ᬊ⇽ᇥ⫮ᨱᕽ⪽ᖒᯕá

⇽ࡹᨩ݅^. ⪽ᖒ ᇥ⫮ᮥ amicon ultracentrifugal filter (Millipore; 10 kDa cut off)ಽ ׮⇶ ⬥, Superdex 75 gel permeation chromatography (GE Healthcare)ಽ ^agarase ᮹ ³₉ᱶᱽෝᙹ⧪⦹ᩡ݅^. ᯕ࠺ᔢᮡ 100 mM NaClᯕ℉a

ࡽ ᪥∊ᧂ ^Aෝ ⁽ᮁᗮ = 0.5 ml/min)ෝᔍᬊ⦹ᩡŁ^, ⅾ ²⁴ fraction (1 ml/fraction)ᮥᇥ⫮⦹ᩡ݅^. bᇥ⫮ᨱݡ⧕ ^DNS

ჶᮝಽ ^agarase ⪽ᖒᮥ⊂ᱶ⦹ᩡŁ^, ⪽ᖒᯕ⪶ᯙࡽᇥ⫮ᮡ

0.1% sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE)ෝ☖⦹ᩍᙽࠥෝ⪶ᯙ⦹ᩡ݅

[19]. ᱶᱽࡽ ݉႒ḩᮡ ^PVDFส(Millipore)ᨱ ᱥʑᱢᮝಽ transfer ⦹ᩡ݅. Transfer ᪥∊ᧂ᳑ᖒ^{(1,000 ml)}ᮡ ^glycine 14.4 g, Tris base 3.03 g, MeOH 200 mlᯕ໑^{, transfer}۵ 48 mAᨱᕽ ⁴᜽e ᙹ⧪⦹ᩡ݅^. สᨱ ^transferࡽ݉႒ḩᮡ

Edman degradation ჶᨱ᮹⦽ ^N-ั݉ᦥၙיᔑᕽᩕᮥᇥ ᕾ⁽ᔍᬊʑʑ: ABI492 procise protein sequencer, USA)ᨱ

ᔍᬊ⦹ᩡ݅^. ᦥၙיᔑᕽᩕᇥᕾᮡᕽᬙݡ⦺Ʊ׮ᨦᔾ໦ŝ

⦺ݡ⦺׮ᔾ໦ŝ⦺Ŗ࠺ʑʑᬱ^(NICEM)ᨱ᮹഑⦹ᩍḥ⧪⦹

ᩡ݅^.

Agarase᮹ ᔾ⪵⦺ᱢ ✚ᖒ ᇥᕾ

Gel permeation chromatographyᨱᕽ᨜ᮡ ^agarare ⪽ᖒ

ᮥᅕᯕ۵ ⁹−¹¹ჩᇥ⫮ᮡ ^SDS-PAGEᨱᕽ݉ᯝ݉႒ḩ႕ ऽෝᅕᩡ݅. ঑௝ᕽ, ⪽ᖒᇥ⫮ᮥ amicon ultracentrifugal filter (10 kDa cut off)ෝᯕᬊ⦹ᩍ᪥∊ᧂ ^Aಽ⊹⪹⦹ᩍ⬉ ᗭ᮹✚ᖒᇥᕾᨱᔍᬊ⦹ᩡ݅^. ᔍᬊ⦽⬉ᗭᧂ᮹݉႒ḩ׮ࠥ

۵ 2.75 mg/mlᮡ ^agarase᮹ specific activity۵ ^283.1/mgᯕ

݅(Table 1).

⬉ᗭ᮹ʑḩ✚ᯕᖒᮥ⪶ᯙ⦹ʑ᭥⧕ᕽ ^0.3%᮹ ^agarose, starch (Sigma-Aldrich, USA), carboxylmethylcellulose (Sigma-Aldrich), brichwood xylan (Megazyme, USA), beechwood xylan (Megazyme)ᮥʑḩಽ℉a⦹ᩍ^{, DNS} ჶ ᮝಽbʑḩᨱݡ⦽⬉ᗭ᮹aᙹᇥ⧕⪽ᖒᮥ⊂ᱶ⦹ᩡ݅^.

⬉ᗭၹ᮲ᮡ⢽ᵡ᳑Õ⁽᪥∊ᧂ ^{A, 37}Ⳅ^{, 30}ᇥ⁾ᨱᕽᙹ⧪⦹ᩡ

݅^. əđŝ^, ᦥaಽᜅa↽ᱢ᮹ʑḩಽ❱݉ࡹᨕ^, ᯕ⬥᮹

ᝅ⨹ᮡ ^0.3% ᦥaಽᜅʑḩᨱݡ⧕ᕽอḥ⧪⦹ᩡ݅^.

⬉ᗭၹ᮲↽ᱢ᪉ࠥ᳑Õđᱶᮥ᭥⧕^, ⬉ᗭၹ᮲⁽᪥∊ᧂ A, 30ᇥ⁾ᮥ ³⁰−⁷⁰ⳄǍeᨱᕽ ⁵ⳄeĊᮝಽǍᇥ⦹ᩍḥ⧪

⦹ᩡ݅^. ⬉ᗭ᮹᪉ࠥᦩᱶᖒđᱶᮡ⬉ᗭᧂᮥ ³⁰−⁷⁰Ⳅ ⁽⁵Ⳅ eĊ⁾ᨱᕽ ⁶⁰ᇥ࠺ᦩႊ⊹⬥^, ⢽ᵡ᳑Õᨱᕽ⬉ᗭ⪽ᖒᮥ⊂ ᱶ⦹ᩡ݅^. ⬉ᗭ᮹↽ᱢ ^pH۵⬉ᗭၹ᮲ᧂᮥ ^{pH 6.0}−^{10.0 (pH} 0.5 eĊ⁾ ჵ᭥ᨱᕽᵡእ⦹Ł^{, 37}Ⳅᨱᕽ ³⁰ᇥeၹ᮲᜽⍽⬉ ᗭ⪽ᖒᮥ⊂ᱶ⦹ᩡ݅^. ᔍᬊࡽ᪥∊ᧂᮡ 50 mM MOPS (pH 6.0−7.0), 50 mM Tris-Cl (pH 7.0−9.0), 50 mM glycine- NaOH (pH 9.0−^10.0)ᯕ݅^. ⬉ᗭ᮹ᔢݡ⪽ᖒᮡ↽ݡsᮥӹ

┡ԕ۵᜽ഭ᮹⪽ᖒᮥ ^100%ಽᱶ⦹Ł^, ᯕᨱݡ⦽ᔢݡsᮝ

ಽ⪹ᔑ⦹ᩍĥᔑ⦹ᩡ݅^.

⬉ᗭၹ᮲ᨱᩢ⨆ᮥၙ⊹۵ɩᗮᯕ᪉ၰĥ໕⪽ᖒᱽ᮹⬉ ŝෝ᦭ᦥᅕʑ᭥⧕ᕽ↽᳦׮ࠥ ^{1 mM}᮹ ^{Ca2+, Co2+, Cu2+,} Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺, Na⁺, K⁺᪡ ^1%᮹ ^SDSෝbb⬉ᗭ ၹ᮲ᧂᨱ℉a⦹ᩍ^, ⢽ᵡ᳑Õᨱᕽ⬉ᗭ⪽ᖒᮥ⊂ᱶ⦹ᩡ݅^.

⬉ᗭ᮹ᔢݡ⪽ᖒᮡྕ℉aݡ᳑Ǒ᮹⪽ᖒᮥ ^100%ಽ⦹Ł^,

ᯕᨱݡ⦽ᔢݡsᮝಽ⪹ᔑ⦹ᩍĥᔑ⦹ᩡ݅^.

⬉ᗭၹ᮲ྜྷ᮹ ᇥᕾ

ᱶᱽࡽ݉႒ḩᨱ᮹⦽ ^agarsoe᮹aᙹᇥ⧕ᔑྜྷᮥᇥᕾ⦹

ʑ᭥⧕ᕽ^, ᪥∊ᧂ ^Aᨱךᯙ ^0.3% ᦥaಽᜅ(volume = 130 µl) ᨱ⬉ᗭᧂ ^{20 µl}ෝᕿᮡ⬥^{, 37}Ⳅᨱᕽ ²⁴᜽eၹ᮲ᮥᮁࠥ⦹

ᩡ݅^. ၹ᮲ྜྷ ^{5 µl}ෝ Silica gel 60 plate (Merck, USA)ᨱᜅ

⡠❦⦹Łᯕ࠺ᔢᬊๅ(n-ᇡ┥᪍:ᦥᖙ✙ᔑ:᷾ඹᙹ= 2:1:2)ಽ thin-layer chromatography (TLC)ෝᝅ᜽⦹ᩡ݅^. እƱෝ

᭥⦽ ⢽ᵡ⣩ᮡ 5 mM neoagaobiose, neoagarotetraose, neoagarohexaose (DyneBio, Korea)ෝ ᔍᬊ⦹ᩡ݅^. ᱥ}

⬥^, ၽᔪ᜽᧞⁽ᨱ┥᪍^:⫊ᔑ ^{= 9:1)}ᮥʼnŁ൉ᜅ⥥౩ᯕ⦹Ł^, 120Ⳅᨱᕽaᩕ⦹ᩍၹ᮲ᔑྜྷ᮹ၽᔪᮥ⪶ᯙ⦹ᩡ݅^[20].

TLC ᇥᕾđŝ^{, 2}}᮹↽᳦ᇥ⧕ᔑྜྷᯕá⇽ࡹᨩᮝ໑^, ᯕ

ॅᮥbb ^TLC ❱ᨱᕽ ɢᨕԕᨕ 100% methanolಽ⇵⇽

⦽ ⬥^, ᕽᬙݡ⦺Ʊ ^NICEMᨱᕽ Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Autoflex III; Bruker, USA)ಽᇥᕾᮥᙹ⧪⦹

ᩍᇥᯱపᮥ⪶ᯙ⦹ᩡ݅^.

đŝ ၰ Łₑ

Cellulophga sp. J9᮹ ⦽⃽ ᇥ⧕ ⪽ᖒ

ᅙᩑǍḥᮡ Cellulophga sp. J9-3a ^celluloseෝᇥ⧕⦹ᩍ cellobioseෝᔾᔑ⦹۵ cellulase ᔾᔑ܆ಆᮥw۵ၙᔾྜྷಽ ၽ⢽⦽ၵᯩ݅^[17]. ə్ӹ^, ݅᧲⦽݅ݚℕᨱݡ⦽ၙᔾྜྷ᮹ ᇥ⧕܆ಆ᜽⨹đŝ^{, J9-3} Ɂᵝav⦽ ^agarase ⪽ᖒࠥw ۵ᔍᝅᮥ⪶ᯙ⦹ᩡ݅(Fig. 1A). Ɂᵝ ^J9-3ᮡ ^agara⧉ᮁࡽ

Łℕ႑ḡᨱᕽ ^Lugol ᩝᔪ᜽ᨱ⎽ಽܩᵝᄡᨱv⦽ ^agar ᇥ

⧕⪽ᖒᮥᅕᩡᮝ໑^, ᯕಽᇡ░ ^J9-3 Ɂᵝa ^agaraseෝᖙ⡍

᫙ಽᇥእ⧁äᮝಽᩩᔢ⦹ᩡ݅^.

ᝅᱽ^{, MB} ᧂℕ႑ḡᨱ ^J9-3 Ɂᵝෝ႑᧲⧩ᮥভ^, Ɂℕෝᱽ Ñ⦽႑᧲ᧂᨱᕽv⦽ ^agarase ⪽ᖒᯕšₑࡹᨩᮝ໑^, ᯕ۵ Cellulophga sp. J9-3ᯕᖙ⡍᫙ᇡಽ ^agar ᇥ⧕⬉ᗭෝᇥእ⦽

݅۵ ᔍᝅᮥघၼ⋉⦽݅(Fig. 1B). Cellulophga sp. J9-3᮹ MB ႑ḡᨱᕽ᮹ᖒᰆᮡ ᱲ᳦ ²⁴᜽e ⬥ stationary phase Table 1. Summary of purification for agarase AgaJ93.

Purification step Volume (ml)

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Purification fold

Culture supernatant 880.0 3431.1 203083.1 59.2 1.0

Ammonium sulfate precipitation 75.0 279.7 46562.6 166.5 2.8

DEAE-sepharose FF chromatography 7.5 31.4 5457.1 173.7 2.9

SP-sepharose chromatography 7.5 23.4 5562.7 237.8 4.0

Superdex 75 chromatography 2.0 5.5 1567.9 283.1 4.8

(OD₆₀₀= 1.876)ᨱ ḥ᯦⦹ᩍ ³⁶᜽eᨱ ↽ݡ ᖒᰆ^(OD600= 1.956)ᮥᅕᩡŁ^, ᯕ⬥႑᧲⬥ʑʭḡ⃽⃽⯩qᗭ⦹ᩡ݅^. ၹ ໕ᨱ ^0.2% ᦥaಽᜅෝ℉a⦽ ^MB ႑ḡᨱᕽ۵^, ᱲ᳦⬥ ³⁶᜽ e⬥ᨱ stationary phase (OD₆₀₀= 2.516)ᨱḥ᯦⦹ᩍ ⁴⁸᜽ eᨱ↽ݡᖒᰆ^(OD600= 2.652)ᮥᅕᩡŁ^, ႑᧲⬥ʑʭḡ⃽

⃽⯩qᗭ⦹ᩡ݅^. ᷪ, Cellulophga sp. J9-3ᮡᦥaಽᜅ᮹℉ aᨱ᮹⧕ᕽɁ᮹ᖒᰆᯕ᧞ ^1.3႑᷾aࡹᨩᮝ໑^, ↽ݡᖒᰆ

᜽eࠥ ¹²᜽e܇ᨕḡ۵⩥ᔢᮥᅕᩡ݅(Fig. 1B).

✚⯩, Cellulophga sp. J9-3ᮡ ^MB ႑ḡᨱᕽ۵ ^agarase ⪽ ᖒᯕÑ᮹ӹ┡ӹḡᦫ۵ၹ໕ ⁽↽ݡ⪽ᖒ ^A540= 0.055), 0.2%

ᦥaಽᜅෝ℉aࡽ႑ḡ᮹႑᧲ᧂᨱᕽ᮹⬉ᗭ⪽ᖒᮡ^{, 24}᜽ eᇡ░ɪĊ⯩᷾a⦹ᩍ ⁸⁴᜽eʭḡ ^(A540= 0.685−^0.699) ᮁ ḡ⦽ ⬥^, ᕽᕽ⯩qᗭ⦹ᩡ݅^. ᯕ۵ Cellulophga sp. J9-3᮹

agarase ⬉ᗭᔾᔑᯕ݅ෙ⦽⃽ᇥ⧕ၙᔾྜྷᨱᕽᅕŁࡽä

⃹ౝᦥaಽᜅʑḩ᮹℉aಽᮁࠥࢁᙹᯩᮭᮥ᜽ᔍ⦹໑^, đŝᱢᮝಽ ᧞ ^12.7႑᮹ ⩥ᱡ⦽⬉ᗭᔾᔑ ᷾aෝᅕᩡ݅

(Fig. 1B).

Cellulophga sp. J9 ႑᧲ᧂᮝಽᇡ░ agarase AgaJ93᮹ ᱶᱽ

Cellulophga sp. J9-3᮹ᧂℕ႑᧲ᧂᮝಽᇡ░ ^agaraseෝᱶ ᱽ⦹ʑ᭥⧕^, ᖙ⡍ෝᱽÑ⦹Ł᨜ᮡ႑᧲ᧂอᮥ ^ammonium

sulfateಽ⋉ᱥ᜽⍽݉႒ḩᮥ׮⇶⦹ᩡ݅^. ᯕಽᇡ░ᮭᯕ᪉

Ʊ⪹ᙹḡᯙ DEAE-sepharose FFෝᯕᬊ⦹ᩍ ¹₉ᱶᱽෝ᜽

ࠥ⦽đŝ^{, agarase} ⪽ᖒᮡ ^resinᨱđ⧊⦹ḡᦫŁ☖ŝ⦽ᇥ

⫮ᨱᕽอšₑࡹᨩ݅^. ☖ŝ⦽ᇥ⫮ᮥ ^{7.5 ml}ಽ׮⇶⦹ᩍ᧲ ᯕ᪉Ʊ⪹ᙹḡᯙ SP-sepharose ⍍ౝᮥᯕᬊ⦹ᩍ ²₉ᱶᱽ

ෝ᜽ࠥ⦽đŝ, 100 mM NaCl ᇥ⫮ྜྷᨱᕽ׳ᮡ⬉ᗭ⪽ᖒ

Fig. 1. Production of extracellular agarase by Cellulophaga sp. J9-3. (A) Detection of agarase activity on Marine Agar plate.

The plate was stained with Lugol's Idoine solution. (B) Cell growth and agarase production depending on cultivation time in Marine broth (MB). Cell density (OD600) in MB (ø) and MB + 0.2% agarose (_ù), agarase activity (A540) in MB (_ý) and MB + 0.2%

agarose (þ).

Fig. 2. Purification of an extracellular agarase AgaJ93 from the culture broth of Cellulophaga sp. J9-3. (A) Gel permeation chromatogram in the final step of agarase purification process.

The protein concentration was measured at 280 nm (line) and agarase activity of each fraction was determined by DNS method at 540 nm (bar). (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein samples during purifica- tion. Lane M, protein molecular weight markers; 1, 75% ammo- nium sulfate precipitate; 2, Sample passing through DEAE- Sepharose Fast Flow column chromatography; 3, Sample after SP-Sepharose column chromatography; 4, Dialyzed sample after Superdex 75 column chromatography.

ᯕšₑࡹᨩ݅^. ᯕᇥ⫮ྜྷᮥ ^{1 ml}ಽ׮⇶⦹ᩍ Superdex-75 gel permeation chromatographyෝᝅ᜽⦽đŝ^{, 9}−¹²ჩᇥ

⫮ᨱᕽ׳ᮡ⬉ᗭ⪽ᖒᯕšₑࡹᨩ݅(Fig. 2A). ᯕॅᇥ⫮ྜྷ

ᮥ ^SDS-PAGEಽᇥᕾ⦹ᩍ݉ᯝ݉႒ḩ႕ऽෝᅕᯙᇥ⫮อ

ᮥᖁ┾⦹ᩡŁ^, ⧕ݚ݉႒ḩᮥ ^AgaJ93ᮝಽ໦໦⦹ᩍᝅ⨹ᨱ ᔍᬊ⦹ᩡ݅(Fig. 2B). AgaJ93᮹ b ᱶᱽ ݉ĥᄥ ᙹᮉᮥ

Table 1ᨱᱶญ⦹ᩡ݅^.

Cellulophga sp. J9 ᮁ௹ agarase AgaJ93᮹ ᱶᅕ⦺

ᙽᙹᱶᱽ⦽ ^AgaJ93 ݉႒ḩ᮹ ^N-ั݉ᦥၙיᔑᕽᩕᮥ⧕

ࠦ⦽đŝ, S-P-N-N-W-P-T-P-G-G-T-Vಽđᱶࡹᨩ݅^{. N-}ั

݉ᦥၙיᔑᕽᩕᮥ☁ݡಽ BlastP program [21]ᮝಽᔢ࠺

ᖒᯕᯩ۵݉႒ḩᮥáᔪ⦽đŝ^, ݅᧲⦽ʑ܆᮹݉႒ḩॅŝ ᔢ࠺ᖒᮥ ᅕᩡ݅^. ᯕ ᵲᨱᕽ aᙹᇥ⧕ šಉ ݉႒ḩᮡ

Cellulophaga sp. W5C᮹ β-agarase (82% identity with T-P-N-N-W-P-G-P-G-P-T-V), Xanthomonas campestris pv. campestris᮹ polyvinylalcohol dehydrogenase (90%

identity with S-P-N-N-W-P-T-A-G-G), Candidatus roseilinea sp. NK_OTU-006᮹ glycoside hydrolase family 31 protein

(75% identity with D-P-N-N-W-P-D-P-A-G-M-V) ॒ᯕ ᯩ

݅^. ᯕᵲ^, Ɂᵝ ^J9-3ŝzᮡᗮ᮹ Cellulophaga sp. W5C᮹ β-agarase (ATI14839)۵ ⁴⁸⁰}᮹ᦥၙיᔑᮝಽǍᖒࡹᨕᯩ ۵ glycoside hydrolase 16 familyᨱ ᗮ⦹۵ ^agaraseᯕ݅

[22]. ⦹ḡอᯕॅ݉႒ḩᨱᕽၽčࡽᔢ࠺ᕽᩕᮡ^{, N-}ั݉ᦥ

ၙיᔑᯕᦥܭ݉႒ḩԕᇡᦥၙיᔑᕽᩕᨱ⧕ݚ⦹ᩡᮝ໑^,

঑௝ᕽ ^AgaJ93 ݉႒ḩᮡáᔪࡽ݉႒ḩŝ۵݅ෙᦥၙיᔑ

ᕽᩕᮥwᮥäᮝಽ❱݉⦹ᩡ݅^.

Agarase AgaJ93 ⬉ᗭ᮹ ᔾ⪵⦺ᱢ ✚ᖒ

ʑḩ✚ᯕᖒ^, ↽ᱢ᪉ࠥ^, ᪉ࠥᦩᱶᖒ^, ↽ᱢ ^pH, ɩᗮ ^ion ၰ

detergent ⬉ŝ ॒⬉ᗭ᮹ ✚ᖒ ᇥᕾ ᝅ⨹ᮥ᭥⧕ ᱶᱽࡽ

AgaJ93ෝ 50 mM Tris-Cl buffer (pH 7.5)ಽ ⁵⁰႑⯍ᕾ⦹ᩍ ᔍᬊ⦹ᩡ݅^.

Agarose, birchwood xylan, beechwood xylan, starch,

CMC ॒᮹ʑḩᨱݡ⦽ ^AgaJ93᮹aᙹᇥ⧕ၹ᮲ᮥ⊂ᱶ⦽

đŝ^{, AgaJ93}ᮡᦥaಽᜅᨱ✚ᯕᱢᮝಽv⦽⪽ᖒᮥᅕᩡḡ

อ^{, starch}ᨱݡ⧕ᕽࠥᦥaಽᜅݡእ᧞ ^22% ᱶࠥ᮹⪽ᖒ

ᮥᅕᩡ݅(Fig. 3A). ə᫙ʑḩᨱݡ⧕ᕽ۵⪽ᖒᮥᅕᯕḡ

Fig. 3. Biochemical characterization of the purified extracellular agarase, AgaJ93.(A) Substrate specificity of AgaJ93 toward various polysaccharides. (B) Effect of temperature on AgaJ93 agarase activity toward agarose. _ù, optimum temperature; _þ, thermo- stability. (C) Effect of pH on AgaJ93 agarase activity toward agarose. þ, 50 mM MOPS (pH 6.0-7.0); ù, 50 mM Tris-Cl (pH 7.0-9.0); Ā, 50 mM glycine-NaOH (pH 9.0-10.0). (D) Effect of metal ions and SDS on AgaJ93 agarase activity toward agarose. The final concentration of each chemical used is indicated under the figure. The maximum enzyme activity in (A)-(C) and the enzyme activity without chemicals in (D) was set to 100% when calculating relative activities. All data shown are mean values from at least three replicate experiments.

ᦫᦹ݅^{. AgaJ93}۵ ³⁵Ⳅᨱᕽaᰆ׳ᮡ ^agarase ⬉ᗭ⪽ᖒᮥ

ᅕᩡ݅^{. 30}Ⳅ^{, 40}Ⳅ^{, 45}Ⳅᨱᕽ۵bb↽ݡ⪽ᖒݡእ᧞ ^75%, 69%, 74%᮹እƱᱢ׳ᮡ⪽ᖒᮥᅕᯙၹ໕^{, 50}Ⳅᯕᔢ᮹᪉

ࠥᨱᕽ۵ ^52% ᯕ⦹᮹ԏᮡ⪽ᖒᮥᅕᩡ݅(Fig. 3B). AgaJ93 ᮹᪉ࠥᦩᱶᖒእƱđŝ^{, 35}Ⳅʭḡ۵ᦩᱶ⦹í⪽ᖒᮥᮁ ḡ⦹ḡอ^{, 40}Ⳅᯕᔢᨱᕽ۵ݡᇡᇥ᮹⪽ᖒᮥᯤŁ᧞ ^27%

ᱶࠥ᮹ ⪽ᖒอᯕ ᮁḡࡹᨩ݅(Fig. 3B). ✚⯩^{, 50}Ⳅᨱᕽ۵ 5ᇥĞŝ⬥⬉ᗭ⪽ᖒᮥÑ᮹ᔢᝅ⦹ᩡ݅(data not shown).

AgaJ93 ⬉ᗭ۵ ^{pH 7.0}−^7.5 ᇡɝᨱᕽ↽ݡ⪽ᖒᮥᅕᩡŁ^, ᵲ ᖒ ^pH ᯕ᫙᮹Ǎeᨱᕽ۵⪽ᖒᯕqᗭ⦹ᩍ ^{pH 6.0}ŝ ^10.0

ᨱᕽ۵↽ݡ⪽ᖒݡእ ^27% ᱶࠥ᮹⪽ᖒอᮥӹ┡ԩ݅^(Fig.

3C). ᯕ్⦽đŝಽᇡ░^{, AgaJ93}۵ᵲᖒ ^pH ᳑Õᨱᕽ⪽ᖒ

ᮥw۵ᵲ᪉ᖒ⬉ᗭಽ❱݉ࡽ݅^.

AgaJ93 ⬉ᗭၹ᮲ᨱᩢ⨆ᮥၙ⊹۵ɩᗮᯕ᪉ၰĥ໕⪽ᖒ

ᱽ᮹⬉ŝෝ᳑ᔍ⦽đŝ^, ⬉ᗭ⪽ᖒᮡ ^Co2+ෝ℉a⦽ၹ᮲ᨱ ᕽၙ⃹ญǍݡእ ⁶႑׳ᮡ⪽ᖒ᷾aෝᅕᩡ݅^. ၹ໕^{, Cu2+,} Fe²⁺, Zn²⁺ŝ ^SDSෝ℉a⦽ၹ᮲ᨱᕽ۵⬉ᗭ⪽ᖒᯕ᪥ᱥ⯩

qᗭ⦹ᩡ݅^{. Mn2+}ᮥ℉a⦽ၹ᮲ᨱᕽ۵⬉ᗭ⪽ᖒᯕ ^45%᮹ qᗭ⦹ᩡŁ^{, Ca2+, Mg2+}ᮡၙၙ⦽᷾a⬉ŝෝ^{, Na+, K+}ᮡ

᧞e᮹qᗭ⬉ŝෝӹ┡ԩ݅^{(Fig. 3D).}

Fig. 4. Analysis of the agarose hydrolysates by agarase AgaJ93. (A) Thin layer Chromatogram of the agarose hydrolysate by agarase AgaJ93 depending on reaction time. The purified AgaJ93 was used 55 μg (left panel) or 1.1 μg (right panel) in 150 μl of total reaction volume. When a low concentration of enzyme is used (right panel), oligosaccharides larger than neoagarotetraose or neoagarohexaose appear as intermediates at the beginning of the reaction. However, if the reaction time is prolonged or if a high concentration of enzyme is used as shown in the left panel, neoagarotetroaose and neoagarohexaose are produced as final products. G, D-glucose; NA2, neoagarobiose; NA4, neoagarotetrasoe; NA6, neoagarohexaose; S, NA2 + NA4 + NA6. (B) MALDI-TOF mass spectrum of the agarose hydrolysate by agarase AgaJ93. The peaks for molecular ions at m/z of 665.8 (M + Na)⁺ and 971.8 (M + Na)⁺ corresponding to neoag- arotetraose and neoagarohexaose, respectively, are indicated by arrows.

AgaJ93 ⬉ᗭᨱ ᮹⦽ ᦥaಽᜅ ᇥ⧕ᔑྜྷ ᇥᕾ

AgaJ93 ⬉ᗭ᪡ ᦥaಽᜅෝ ʑḩಽ ⦹۵ ⬉ᗭၹ᮲ྜྷᮥ

TLCಽ ᇥᕾ⦽ đŝ^{, AgaJ93}ᮡ ᦥaಽᜅෝ ᇥ⧕⦹ᩍ

neoagarotetraose᪡ neoagarohexaoseෝ↽᳦ᔑྜྷಽᔾᔑ⦹

۵äᮝಽᩩᔢࡹᨩ݅(Fig. 4A, left panel). ੱ⦽^{, 50}႑ಽ⯍

ᕾ⦽ ^AgaJ93 ⬉ᗭᧂᮥᔍᬊ⦹ᩍ᜽eᨱ঑ෙᦥaಽᜅ᮹⬉

ᗭၹ᮲ ᔑྜྷᮥ ^TLCಽᇥᕾ⦹ᩡᮥ ভ, neoagarooctaoseӹ neoagarodecaose᪡zᯕ޵ⓑⓍʑ᮹᪍ญŁݚᯕၹ᮲Ⅹ ʑᨱ ᔾᔑࡹŁ^, ၹ᮲᜽eᨱ ঑௝ neoagarotetraose᪡ neoagarohexaose ಽaᙹᇥ⧕aḥ⧪ࢉᮥ⪶ᯙ⦹ᩡ݅^(Fig.

4A, right panel). ᯕ᪡zᮡ đŝ۵ ^AgaJ93 ⬉ᗭa ^endo- type᮹ᄁ┡^-ᦥa౩ᯕᷩᯥᮥ᮹ၙ⦽݅^.

⬉ᗭၹ᮲ྜྷᮥ MALDI-TOF mass spectrometerಽᇥᕾ⦽

đŝ^, ࢱ}᮹ᵝ᫵⦽⦝Ⓧa m/z 665.8 ŝ ^{m/z 971.8}ᨱᕽá

⇽ࡹᨩᮝ໑^, ᯕ۵bb neoagarotetraose᪡ neoagarohexaose ᮹ sodium-adducted form (M+Na)⁺᮹ᇥᯱపᨱᱶ⪶⯩ᯝ

⊹⦽݅(Fig. 4B). ᯕ్⦽đŝෝ᳦⧊⦹໕^{, AgaJ93} ⬉ᗭ۵ᦥ aಽᜅ᮹β-1,4 glycosidic đ⧊ᮥᇥ⧕⦹ᩍݡఖ ⁵⁰ݡ ⁵⁰᮹ እᮉಽ neoagarotetraose᪡ neoagarohexaoseෝ ᔾᔑ⦹۵ endo-type β^-agaraseᯙäᮝಽ❱݉ࡽ݅^.

✚ᯕ⦹íࠥ^{, AgaJ93}۵ᦥaಽᜅ⪽ᖒ᜽⨹ᨱᔍᬊ⦹۵ᯙ Ŗʑḩᯙ р-nitrophenyl-α-d-galactopyranoside᪡ ^р- nitrophenyl-β-d-galactopyranoside ʑḩ ༉ࢱᨱ⪽ᖒᮥᱥ

⩡ᅕᯕḡᦫᦹ݅(data not shown). ᯕ᪡zᮡĞᬑ۵ᦥᵝ ऽྙᩩಽ⧕᧲ၙᔾྜྷ Gayadomonas joobiniege G7aᔾᔑ

⦹۵ ^agaraseᨱᕽᅕŁࡽၵᯩ݅^[23]. ঑௝ᕽ^{, AgaJ93}۵ᦥ ᵝ✚ᯕ⦽⬉ᗭ⦺ᱢᖒĊᮥwᮥäᮝಽᩩᔢࡽ݅^.

ḡɩʭḡ, Cellulophaga ᗮᮝಽᇡ░ ³ }᮹ ^agarasea࠺ ᱶࡹᨩ݅[14, 22, 24]. AgaJ93᮹ N-terminal sequenceෝᯕ ᬊ⦽ ^homology እƱ đŝ^{, AgaJ93}ᮡ Cellulophaga sp.

W5C᮹ԕᩕᖒ endo-type β-agarase Aga2᪡ ^82%᮹ᔢ࠺ᖒ

ᮥᅕᩡ݅^{[22]. Aga2}۵ ^AgaJ93ŝzᯕᦥaಽᜅෝᇥ⧕⦹

ᩍ neoagarotetraose᪡ neoagarohexaoseෝᔾᔑ⦹۵ ^endo- type β^-agarase ᯕḡอ^, ⬉ᗭ⪽ᖒ↽ᱢ᳑Õᯕ ⁴⁵Ⳅ^{, pH 8.0} ᯕ໑^{, 45}Ⳅᯕᔢᨱᕽࠥᩕᦩᱶᖒᮥᅕᯕ۵ԕᩕᖒ⬉ᗭ௝۵ ᱱᨱᕽ Ⓧí ݅෕݅^. ᯕ᫙ᨱ^{, N-}ั݉ ᦥၙיᔑ ᕽᩕᯕ

AgaJ93ŝᯝ⊹⦹۵ᦥa౩ᯕᷩ۵ᅕŁࡽᱢᯕᨧᮝ໑^, ᨕ۱

ᱶࠥᔢ࠺ᖒᯕᯩ۵äᮝಽᩕÑ⦽݉႒ḩ᮹Ğᬑࠥ⧕ݚᕽ

ᩕᯕ ^N-ั݉ᯕᦥܭԕᇡᨱ᭥⊹⦹۵ᦥၙיᔑᕽᩕಽᇥᕾ

ࡹᨩ݅^. ঑௝ᕽ^{, AgaJ93}ᮡ Cellulophaga ᗮᯕӹʑ┡⦽⃽

ᇥ⧕ၙᔾྜྷᨱᕽᅕŁࡽβ^-agaraseॅŝ۵݅ෙᦥၙיᔑᕽ

ᩕŝ⬉ᗭᱢ✚ᖒᮥw۵ᝁȽ᮹ ^agaraseᯝäᮝಽᩩᔢࡹ

໑^, ᮁᱥℕᇥᕾᮥ☖⧕ᮁᱥᯱෝ⪶ᅕ⦹ᩍ⬉ᗭ✚ᖒᨱš

⦽ǍℕᱢᯙᩑǍaᯕ൉ᨕḡʙʑݡ⦽݅^.

᫵ ᧞

Cellulophga sp. J9-3ᮡ ᖡ൑ಽᜅ ᇥ⧕܆ಆᮥ wᮝ໑^, Flavobacteriaceae ŝᨱᗮ⦹۵ə௭^-ᮭᖒ⪙ʑᖒ⧕᧲ᖙɁ ᯕ݅^. ੱ⦽^{, J9-3} Ɂᵝ۵Łℕၰᧂℕ႑ḡᨱᕽ⦽⃽ᮥa ᙹᇥ⧕⧁ᙹᯩᮝ໑^, ႑ḡᨱ℉a⦽ᦥaಽᜅ^(agarose)ᨱ ᮹⧕ᦥa౩ᯕᷩ^(agarase)᮹ᔾᔑᯕ⩥ᱡ⦹íᮁࠥࡹ۵✚ ᖒᮥᅕᩡ݅. Cellulophga sp. J9-3᮹ᖙ⡍႑᧲ᧂᮝಽᇡ░^, ⫊ ᔑᦵ༉ۥ⋉ᱥၰ ³ ݉ĥ᮹⍍ౝⓍಽษ☁ə௹⦝ෝᩑᗮᱢ ᮝಽᙹ⧪⦹ᩍ^, ⦽}᮹ ^agarase ݉႒ḩ^{, AgaJ93}ᮥᙽᙹ⦹

íᱶᱽ⦹ᩡ݅^. ᱶᱽࡽ ^AgaJ93ᮡᦥaಽᜅᨱݡ⦽ᇥ⧕⪽ ᖒᯕaᰆv⦹ᩡᮝ໑, starchᨱݡ⧕ᕽࠥᦥaಽᜅݡእ᧞ 22% ᱶࠥ᮹ᇥ⧕⪽ᖒᮥӹ┡ԩ݅^{. AgaJ93}ᮡᦥaಽᜅෝᇥ

⧕⦹ᩍᔍᯕᷩaⓑ᪍ญŁݚᵲeℕෝĞᮁ⦹ᩍ^, ↽᳦ᔑྜྷ

ಽօ᪅ᦥaಽ▭✙௝᪅ᜅ᪡օ᪅ᦥaಽ⩆ᔍ᪅ᜅʭḡᇥ⧕⧉

ᮥ⪶ᯙ⦹ᩡᮝ໑^, ᯕ۵ ^AgaJ93ᯕ endo-type β^-ᦥa౩ᯕᷩᯥ

ᮥ᮹ၙ⦽݅^{. AgaJ93}ᮡ pH 7.0, 35Ⳅᨱᕽ↽ݡ⪽ᖒᮥӹ┡

ԩŁ^{, Co2+} ᯕ᪉ᨱ ᮹⧕ ⁶႑ ᯕᔢ᮹ ⪽ᖒ᷾aෝ ᅕᩡ݅^. AgaJ93᮹ N-terminal sequence ᇥᕾ đŝ^{, AgaJ93}ᮡ Cellulophaga sp. W5C᮹ ԕᩕᖒ endo-type β^-ᦥa౩ᯕᷩ

Aga2᪡ ^82%᮹ᔢ࠺ᖒᮥᅕᩡᮝӹ^, ࢱ⬉ᗭ᮹ᔾ⪵⦺ᱢ✚ ᖒᯕݍ௱݅^. ঑௝ᕽ^{, AgaJ93}ᮡʑ᳕ᨱᅕŁࡽβ^-ᦥa౩ᯕ

ᷩॅŝ۵݅ෙᝁȽ᮹ᦥa౩ᯕᷩᯝäᮝಽᩩᔢࡽ݅^.

Acknowlegments

This research was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202102109).

Conflict of Interest

The authors have no financial conflicts of interest to declare.

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