糸状菌Acremonium celluloyticus起源の3種エンドキシラナーゼの精製と基本性質 Purification and Properties of Three Endo-xylanases from Acremonium celluloyticus

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Abstract

糸状菌、4cremonzum cellulolyticus起源の市販酵素製剤から,各種カラムクロマトグラフィーを組み合わせ,電気泳動的に均一な3種のエンドキシラナーゼを高純度に精製し,キシラナーゼI,II,III と呼称した.精製酵素の分子量および等電点は,それぞれ30kDa,5.1,25.5kDa,5.2,および33.5kDa,5.7であった.N末端側第1一第25アミノ酸残基の配列は,3酵素ともNH2-Ala-Glu-Ala-lle-Asn-Tyr-Asn-Gln-Asn-Tyr-lle-Ala-Ser-Gly-Ala-Asn-Val-Gln-Tyr-Ser-Pro-Asn-lle-Ala-Ala-であった.キシラナーゼI,II,III は,可溶性キシランに高い反応性を示し,比活性値はそれぞれ112 .1,86.1,74.8U/mgであり,反応至適pHおよび至適温度は,それぞれ3.5,55℃,3.8,55℃ および3.5,50℃ であった.キシラナーゼI,II,III はpH3.5-9.5(25℃,2時間処理),55℃ 以下,pH3.0-9.5,55℃ 以下およびpH2.5-9.5,50℃ 以下において,それぞれ完全に安定であった.3酵素とも1mMのKMnO4によりほぼ完全に阻害され,1mMのSDS,PCMB,Zn2+,Ag+により部分阻害を受けた.当該酵素の基質特異性や反応速度パラメータなどが精査された.キシランおよびキシロオリゴ糖に対する作用パターンから,当該3酵素はいずれもエンドキシラナーゼであった.3種の精製酵素は,N末端アミノ酸配列,物理化学的・酵素学的性質,基質特異性などから,互いにアイソフォームであると推定された.

Three distinct endo-xylanase components derived from Acremonium cellulase, a commercial cellulase product from a filamentous fungus Acremonium cellulolyticus, were extensively purified by consecutive column chromatographies and designated as xylanaseI, xylanase II and xylanase III. XylanasesI, IIand III were each homogeneous on both Native- and SDS-PAGE . The molecular weight (SDS-PAGE) and p1 values of xylanases I, II and III were 30 kDa and 5.1, 25 .5 kDa and 5.2, and 33.5 kDa and 5.7, respectively. The N-terminal amino acid sequences from the 1st up to the 25th residue of three enzymes were identical as follows: NH<SUB>2</SUB>-Ala-Glu-Ala-Ile-Asn-Tyr-Asn-Gln-Asn-Tyr-Ile-Ala-Ser-Gly-Ala-Asn-Val-Gln-Tyr-Ser-Pro-Asn-Ile-Ala-Ala-. Xylanases I, II and III had high, specific soluble xylan saccharification activities of 112.1, 86.1 and 74.8 U/mg of protein, respectively. The optimum pH and temperature for xylanases I, II and III were pH 3.5 and 55°C, pH 3.8 and 55°C, and pH 3.5 and 50°C, respectively. Xylanases I, II and III were completely stable over the ranges of pH 3.5-9.5 at 25°C for 2 h and at temperatures below 55°C, pH 3.0-9.5 and below 55°C, and pH 2.5-9.5 and below 50°C, respectively. The enzymes were almost completely inhibited by 1 mM KMnO<SUB>4</SUB> and partially by 1 mM SDS, PCMB, Zn<SUP>2+</SUP>and Ag<SUP>+</SUP> Substrate specificities and kinetic parameters for these enzymes were precisely examined. The enzymes were characterized as endo-type xylanases on the basis of their action patterns on soluble xylan and xylooligosaccharides. Xylanases I, II and III seem to be isoforms judging from N-terminal amino acid sequences and basic physicochemical and enzymatic properties as well as substrate specificities.

Journal

  • Journal of Applied Glycoscience

    Journal of Applied Glycoscience 48(1), 45-54, 2001-01-01

    The Japanese Society of Applied Glycoscience

References:  17

Codes

  • NII Article ID (NAID)
    10008252367
  • NII NACSIS-CAT ID (NCID)
    AN10453916
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    13403494
  • NDL Article ID
    5648245
  • 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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