糸状菌Acremonium cellulolyticus起源の2種エンドセルラーゼの精製と基本性質 Purification and Properties of Two Endo-cellulases from Acremonium cellulolyticus

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Abstract

糸状菌Acremonium cellulolyticusの市販酵素製剤から,エンドセルラーゼの二成分を各種カラムクロマトグラフィーを組み合わせ,高純度に精製し,それぞれをセルラーゼIII-AおよびIII-Bと呼称した.精製酵素は,いずれも電気泳動的に単一なタンパク質であり,両者はβ-グルコシダーゼ活性を全く示さない標品であった.精製セルラーゼIII-AおよびIII.Bの分子量(SDS-PAGE法)および等電点は,それぞれ58kDa,4.6および49kDa,4.2であった.両酵素はいずれも14-16%の糖含量(グルコース換算)を示した.また,これら2種の精製酵素のN末端側第2一第20番目までのアミノ酸配列をエドマン分解法により決定した.セルラーゼIII-AおよびIII-Bの反応至適pHおよび温度は,それぞれ5.5,55。Cおよび5.5,65。Cであり,セルラーゼIII-AおよIII-BのpHおよび温度に対する安定領域はそれぞれpH4.2-8.0,55。C 以下およびpH3.3-7.8,60。C 以下であった.また,セルラーゼIII-AおよびIII-Bは,70。C,10分間の加熱処理後,それぞれ25および88%の残存CMC糖化活性を示した.セルラーゼIII-BによるCMCの加水分解は,セルラーゼIII-Aに比し,よりエンド水解度の高いものであった.両精製酵素を種々のセルロース性基質に作用させると,いずれの基質からも多量のセロビオースおよび少量のグルコースが最終生成糖として得られた.

Two distinct endo-cellulase components derived from Acremonium cellulase, a commercial cellulase preparation from Acremonium cellulolyticus, were extensively purified by consecutive column chromatography and designated as cellulase III-A and cellulase III-B. Cellulases III-A and III-B were each homogeneous on both Native- and SDS-PAGE, and were completely free from β-glucosidase. The molecular mass (SDS-PAGE) and pI Tvalues of cellulases IIIV-A and III-B were 58 kDa and 4.6, and 49 kDa and 4.2, respectively. Both enzymes contained 14-16% carbohydrates (as glucose). The N-terminal amino acid sequences from the 2nd up to the 20th residue of bothenzymes were determined by Edman degradation. Some enzymatic properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases III-A and III-B were pH 5.5 and 55°C, and pH 5.5 and 65°C, respectively. Cellulases III-A and III-B were completely stable over the range of pH 4.2-8.0 at 4°C for 24 h and at temperatures below 55°C, and pH 3.3-7.8 and below 60°C, respectively. Cellulases III-A and III-B retained 25 and 88% of the original CMC-saccharification activities, respectively, after heating at 70°C for 10 min. The hydrolysis of CMC by cellulase III-B was more endo-lytic than that by cellulase III-A. Both enzymes splitvarious soluble and insoluble substrates to produce predominant cellobiose and a small amount of glucose as the final hydrolysis products.

Journal

  • Journal of Applied Glycoscience

    Journal of Applied Glycoscience 47(3), 293-302, 2000-08-31

    The Japanese Society of Applied Glycoscience

References:  24

Codes

  • NII Article ID (NAID)
    10008252079
  • NII NACSIS-CAT ID (NCID)
    AN10453916
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    13403494
  • NDL Article ID
    5521380
  • NDL Source Classification
    ZP24(科学技術--化学・化学工業--糖・澱粉)
  • NDL Call No.
    Z17-15
  • Data Source
    CJP  NDL  J-STAGE  JASI 
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