Escherichza coliで発現したThermotoga maritima由来のエンド-β-1,4-グルカナーゼの特性
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- マジブル ラーマン
- 独立行政法人食品総合研究所酵素機能研究室
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- シャカワットホッサン ブイヤン
- 独立行政法人食品総合研究所酵素機能研究室
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- 韮澤 悟
- 独立行政法人食品総合研究所酵素機能研究室
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- 北岡 本光
- 独立行政法人食品総合研究所酵素機能研究室
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- 林 清
- 独立行政法人食品総合研究所酵素機能研究室
書誌事項
- タイトル別名
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- Characterization of an Endo-.BETA.-1,4-glucanase of Thermotoga maritima Expressed in Escherichia coli.
- Characterization of an Endo-β-1, 4-glucanase of Thermotoga maritima Expressed in Escherichia coli
- Characterization of an Endo ベータ 1 4 glucanase of Thermotoga maritima Expressed in Escherichia coli
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抄録
A gene (TM1751, Swissprot Q9X273, GenBank AAD36816) of Thermotoga maritima encoding endo-β-1, 4-glucanase of family 5 was cloned and expressed in Escherichia coli. The recombinant protein was purified by combination of Ni-NTA and Q-Sepharose FF column chromatography. SDS-PAGE analysis of purified fractions showed a homogeneous protein band with an expected molecular mass of 38 kDa. The specific activity of the enzyme increased about 16-fold while the recovery was 21% after purification. The optimal temperature of the enzyme was found to be 90°C while its optimal pH was 6.6. The enzyme was active over a wide pH range (4-9.5) and stable up to 85°C. Kinetic studies showed that the PNP-β-D-cellotetaoside and PNP-β-D-cellopentaoside had similar Km values of 0.25 and 0.24 mM respectively. However, maximum catalytic efficiency (kcat/ Km) was observed with PNP-β-D-cellopentaoside. Further analysis of the enzyme hydrolyzate by HPLC suggests that the carbohydrate binding cleft of the enzyme may be composed of four sub-sites for glucopyranose units. At the initial stage, the hydrolysis of PNP-oligosaccharides (DP up to 5) yielded a range of products with cellobiose, cellotriose and cellotetraose being the major prod-ucts. CMC hydrolysis also predominantly produced cellobiose and cellotriose as end products. The enzyme hydrolyzed mixed linked β-1, 3/1, 4 soluble substrates such as barley glucan and lichenan more strongly than CMC. Transglycosylation activity was also found to be taking place with smaller soluble cellooligosaccharides.
収録刊行物
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- Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ
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Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 49 (4), 487-495, 2002
日本応用糖質科学会
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詳細情報 詳細情報について
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- CRID
- 1390282681270652544
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- NII論文ID
- 10009930208
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- NII書誌ID
- AN10453916
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- ISSN
- 18807291
- 13447882
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- NDL書誌ID
- 6335528
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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