Purification and properties of mimosine hydrolyzing enzyme of Leucaena Psyllids (Jumping Plant Lice)

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  • キジラミのミモシン分解酵素の精製と諸性質
  • キジラミ ノ ミモシン ブンカイ コウソ ノ セイセイ ト ショ セイシツ

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Abstract

1)ミモシンをピルビン酸,アンモニアおよび3,4-DHPに分解するミモシン分解粗酵素液がキジラミ30gに30mlの200mMリン酸緩衝液(pH7.2)をいれてホモゲナイズしえた。その酵素液の総タンパク質1639.44mg,総活性3072.90Units,比活性1.87であった。2)その粗酵素液を60%飽和硫安分画法,熱処理法,DEAE=TOYOPEARL650M、DEAE-TOYOPEARL650M, Buthyl Sepharose 4B、RESOUCE-Q、Mono-Q、Superose-12カラムクロマトグラフィーにより精製した。比活性は1071.71μ/mgで,活性回収率は0.14%で,約570倍になった。3)精製した酵素は電気永動的にほど均一性を示し,分子量は約50,000であった。4)本酵素の至適pHは7.5-8.5で,至適温度は55-65℃であった。5)本酵素のN末端アミノ酸配列はELEDDXKKFXNPVIEAであった。

A crude enzyme of psyllid hydrolyzed mimosine, a strongly toxic substance for livestock, into 3-hydroxy-4(1H) pyridine, pyruvic acid, and ammonia with 1639.44mg of total protein, 307290 units of total activity and 1.87 of specific activity was obtained by homogenizing 3g of psyllid body with 200mM phosphate buffer(30ml) and centrifuging. Purification of the crude enzyme was carried out in order of 60% saturated ammonium sulfate fractionation, heat treatment at 60℃ for 1hour, DEAE-TOYO Pearl L650, Buthyl Sepharose 4B columns chromatography and FPLC with RESOUCE Q, MONO Q and Superose 12 columns. Purified enzyme had 1071.71U/mg of specific activity, 0.14% of recovery, and 570 times of purification. The enzyme with molecular weight of about 50,000, maximum pH ranges of 7.5-8.5 and maximum temperature ranges of 5.5-6.5 showed homogeneity by electrophoresis. N terminal Amino acid sequence of the enzyme was found to be ELEDDXKKFXNPVIEA

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