Aspergillus oryzae NCH-42單寧酶之生產、純化及其特性分析

Abstract

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.