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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||摘要:||
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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