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標題: Aspergillus niger NCH-3聚木糖之生產條件及酵素性質研究
Studies on the production and properties of xylanase from Aspergillus niger NCH-3
作者: 朱文銘
關鍵字: Aspergillus niger group;Aspergillus niger;xylanase;oat spelt xylan;potassium nitrate;sulfhydryl (-SH);聚木糖;燕麥聚木糖;硝酸鉀;硫氫基
出版社: 食品科學系
本研究由台灣中部地區的土壤中篩選出一株真菌NCH-3,能分泌高活性之聚木糖。 將菌株NCH-3培養於CZ及MEA培養基上,於顯微鏡下觀察其特徵並與標準菌株比對後,暫命名為Aspergillus niger NCH-3。
接著以Mandels-Reese培養基為基礎進行A. niger NCH-3生產聚木糖之較適培養條件之探討。結果發現碳源以0.75% 燕麥聚木糖,氮源0.38% 硝酸鉀最佳。培養溫度35℃,起始pH 8.0,孢子接種量105 spores/ml及振盪速率130rpm為最佳培養條件。在上述較適培養條件下A. niger NCH-3 於第五天時有最大酵素活性(26.17U/ml)。另外,培養液中β-xylosidase的最高活性為17.01U/ml。A. niger NCH-3聚木糖經超過濾濃縮、硫酸銨沈澱(飽和度40%-70%)、離子交換層析及膠體過濾層析等步驟純化後,分別以SDS-PAGE及IEF等電焦集電泳分析聚木糖之分子量及pI值,發現此酵素分子量約為35k Da ,而pI值為4.0。另外酵素性質如下:最適反應pH值為pH 5, 在pH 5-7 之間活性仍高,最適反應溫度50℃,溫度超過60℃活性明顯下降。基質特異性高,只對由木糖構成之多醣如樺木、櫸木及燕麥等來源之聚木糖有活性。推測硫氫基(-SH )應位於酵素之活性中心區域或其附近。而酵素之硫醇基應涉及催化反應。錳離子(Mn2+)對酵素活性有提高的作用。

A high xylanase producing mold, strain NCH-3 was isolated from Taiwan soil. According to morphologic characterization of strain NCH-3 grown on CZ and MEA media, it was identified to be the same as a type culture of Aspergillus niger and was named temporarily as Aspergillus niger NCH-3.
The optimal medium composition and cultivation conditions for A. niger NCH-3 was determined in shaking flasks (capacity 500 ml). The results showed that the Mandels-Reese medium with oat spelt xylan at 0.75% as carbon source, 0.38% KNO3 as nitrogen sources, initial pH at 8.0, inoculum size at 105 spores/ml, incubation temperature at 35 ℃, and shaking rate at 130 rpm gave the best result for the enzyme production. The maximum xylanase activity of 26.17 U/ml in the culture broth was obtained after 5 days under above conditions. The broth filtrate from A. niger NCH-3 was purified by ammonium sulfate precipitation (40-70%)、ion exchange chromatography and gel filtration chromatography. The purified enzyme appeared as single protein band on SDS-PAGE with molecular weight of 35 kDa. The isoelectric point of purified xylanase was 4.0. The optimum pH of activity was pH 5.0;howere, the enzyme remained quite active in pH ranging from pH 5.0 to 7.0. Both thermal stability and optimal temperature for purified xylanase were at 50℃. Enzyme showed high specificity on hydrolysis of xylans which came from beech wood, birch wood and oat spelt . The activity of xylanase was inhibited by Hg2+ ion, while Mn2+ and -mercaptoethanol at concentrations of 5mM stimulated the enzyme activity. It was suggested
that the sulfhydryl (-SH) group in the protein molecule was located at or near the active site, and probably involved in the hydrolysis reaction catalyzed by the enzyme.
Appears in Collections:食品暨應用生物科技學系

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