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No.36 (1989/12) >

Title :成熟サトウキビ茎梢頭部のα-アミラーゼに関する研究(農芸化学科)
Title alternative :Studies on α-Amylase from Top Part of Mature Sugar Cane Stalks (Department of Agricultural Chemistry)
Authors :知念, 功
宮城, 大
伊藤, 敬史
隈元, 俊和
屋, 宏典
Authors alternative :Chinen, Isao
Miyagi, Masaru
Ito, Takafumi
Kumamoto, Tosikazu
Oku, Hirosuke
Issue Date :5-Dec-1989
Abstract :サトウキビ茎梢頭部には二種類のアミラーゼ,α-アミラーゼとβ-アミラーゼが存在した。本研究では前者のα-アミラーゼを高度に精製し諸性質を調べた。精製はα-シクロデキストリンセファロース6Bゲルを用いたアフィニィティーカラムクロマトグラフィーとセファデックスG-100カラムクロマトグラフィーにかけて行なった。その結果,さらに二種類のα-アミラーゼが存在する事がわかった。その両酵素のうち活性の高い酵素はポリアクリルアミドデスク電気泳動的に均一に精製された。同酵素はこの精製法で66,476.8倍に純化され,回収率は75.01%であった。精製酵素をSDS-ポリアクリルアミドゲル電気泳動法により分子量を求めた結果,約44,000であった。至適温度と至適pHはそれぞれ,65℃とpH5.0であった。また,可溶性澱粉に対するKm値とVmax値はそれぞれ,2.15mg/mlと146units/mgであった。本酵素は塩化カルシウムを1mM濃度で添加すると著しい熱安定性を示し,55℃で1時間加熱しても安定であった。基質特異性を調べた結果,いずれの基質でも主にマルトヘプタオース(G_7)を生成した。また澱粉分解物の変旋光を測定した結果,その生成物はα型である事がわかった。また,本酵素はα-アミラーゼ測定用着色基質に作用した。以上の事から本酵素の作用型式はエンド型で,主としてマルトヘプタオースを生成するα-アミラーゼであると推測された。精製を行なう際,セファデックスG-100カラムクロマトグラフィーで2つの活性ピークが見られた。今回は活性が高かったピークについて諸性質を調べた。しかし活性が低かったピークについても試験的に可溶性澱粉に作用させ,その生成糖の組成を調べてみた。その結果,本酵素は澱粉に作用させた場合マルトースのみを特異的に生成する事がわかった。
In the first place two types of starch-hydrolyzing enzyme were found to be in top part of mature sugar cane stalk. Because one of them was adsorbed to an α-cyclodextrin-Sepharose column, the other was passed through it. In this paper the former was purified and various properties of it were studied. Purification was carried out by affinity chromatography on epoxy activited Sepharose 6B substituted with α-cyclodextrin and then by gel filtration on Sephadex G-100. As a result, two kinds of amylase showing affinity to α-cyclodextrin-Sepharose existed in the fraction. One of them had high activity and showed homogeneity by disk gel electrophoresis. But the other had low activity and was obtained in very small amounts. So further studies of the former were achieved. The purity was 66,477 times and the recovery was 75%. The molecular weight was estimated to be about 69,000 by disk gel electrophoresis. The optimum temperature and pH volues were 65℃ and 5.0,respectively. The Km and Vmax values for soluble starch were 2.15mg/ml and 146 units/mg/min. The amylase indicated remarkable thermostability when 1mM calcium chloride was present. In other words, it was stable at 55℃ for one hour. The enzyme cleaved considerably soluble starch, amylopectin, amylose, dextrin, and glycogen, but wheat starch, rice starch, cassava starch, sweet potato starch, potato starch, in less extent than the formers. Oligosaccharide composition of products from theses starches was determined by high performance liquid chromatography. No difference in tendency of the composition was detected among the starches. But maltoheptaose (G_7) was mainly produced from each starch. Maltooctaose (G_8), maltohexaose (G_6) maltopentaose (G_5), maltotetraose (G_4), maltotriose (G_3) and maltose (G_2) were also formed in less content than the former. Optical rotation of decomposition products from soluble starch was determined, but the result was not clear. But the enzyme was adsorbed to α-cylodextrin Sepharose, hydrolyzed synthetic substrate (amylose azure), which has been used as a substrate of α-amylase, and was inhibited by α-amylase inhibitor from Triticum aestivlm, therefore it was decided to be α-amylase producing α-type oligosaccharides from various starches. Two kinds of α-amylase was found to be in this fraction in the purification as stated above. One of them showing minor activity was found to produce only maltose (G_2) from soluble starch by HPLC.
Type Local :紀要論文
ISSN :0370-4246
Publisher :琉球大学農学部
URI :http://hdl.handle.net/20.500.12000/3874
Citation :琉球大学農学部学術報告 = The Science Bulletin of the Faculty of Agriculture. University of the Ryukyus no.36 p.33 -49
Appears in Collections:No.36 (1989/12)

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