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|標題:||Aspergillus oryzae NCH-42單寧酶之生產、純化及其特性分析|
Production, purification and characterization of tannase from Aspergillus oryzae NCH-42
|摘要:||本研究由台灣南投之果園篩選出一株可分泌高單寧酶活性之菌株。經鏡檢觀察特徵及比對其5.8S rDNA序列後，暫命名為Aspergillus oryzae NCH-42，並進行酵素生產、純化及特性分析。A. oryzae NCH-42較適培養基與培養條件:碳源為4%單寧酸及1.5%葡萄糖，氮源0.485%酵母萃取物及無機鹽類0.1% KHSO4。培養溫度40℃，起始pH 5.0，接種量106 spores/ml及震盪速率 150 rpm培養三天後，有最高酵素活性，達48.32 U/ml。A. oryzae NCH-42單寧酶經硫酸銨沉澱(飽和度60%-90%)、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等電焦集電泳分析單寧酶之分子量及pI值，發現此酵素分子量約為52 kDa，而pI值為4.14。分析本菌株單寧酶之N-端胺基酸序列，發現與其他真菌單寧酶同源性最高可達83.3%。另外，酵素特性如下: 最適反應pH值為5.0，在pH 4.0~7.0時酵素活性仍高，最適反應溫度為30℃，溫度超過60℃活性明顯下降。Fe2+、Fe3+、Hg2+有明顯的抑制作用；另外化學修飾劑PMSF則對酵素有顯著抑制效果，推測絲胺酸(Serine)應是酵素活性中心所必需之胺基酸分子。單寧酶以單寧酸為基質之Km值與Vmax值分別為0.06 mM、15.3 μmol/min/mg，而本菌株單寧酶降解水解型單寧確實可獲得沒食子酸之產物。|
An extracellularly tannase-producing fungal strain NCH-42 was isolated from orchards in Nantou, according to morphological observation of strain by microscope, and comparison between the partial sequecne of 5.8S rDNA by BLAST, it was identified to be the same as a type culture of Aspergillus oryzae, and was therefore named temporarily as A. oryzae NCH-42. The optimal medium composition and cultivation conditions for A. oryzae NCH-42 was determined in shaking flasks (capacity 250 ml). The results showed that combination of 4% tannic acid with 1.5 % glucose as carbon source, 0.485% yeast extract as nitrogen source, and 0.1% KHSO4 as mineral salt, incubation temperature at 40℃, initial pH at 5.0, inoculum size at 106 spores/ml and shaking rate at 150 rpm gave the best result for the enzyme production. The maximum tannase activity of 48.32 U/ml in the culture broth was obtained after 3 days under above conditions. The broth filtrate from A. oryzae NCH-42 was purified by ammonium sulfate precipitation (60-90%), ion exchange chromatography and gel filtration chromatography. The purified enzyme appeared as single protein band on SDS-PAGE with molecular weight of 52 kDa. The isoelectric point of purified tannase was 4.14. The result of comparison of the N-terminal amino acid sequence of purified tannase with another tannase from A. oryzae showed a high degree of homology with each other (83.3%).The optimum pH of activity was pH 5.0, while the enzyme remained quite stable in pH ranging from pH 4.0 to 7.0. The optimum temperature for the purified tannase was 30℃, and good thermal stability ranging from 30℃ to 60℃ was found. The activity of tannase was inhibited by Fe2+、Fe3+、Hg2+. The serine residues in tannase was inactivated by PMSF, this indicates serine was probably involved in the active site or substrate binding site of the enzyme.The Km and Vmax of purified tannase for tannic acid were 0.06 mM and 15.3 μmol/min/mg, respectively.The action of purified tannase on various gallate ester substrates was examined, the purified tannase indeed catalyzes the hydrolysis of ester bonds of hydrolysable tannins that contain esterified phenolic OH groups and thereby release gallic acid.
|Appears in Collections:||食品暨應用生物科技學系|
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