Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan.
J Biosci Bioeng. 2010 Aug;110(2):152-7. doi: 10.1016/j.jbiosc.2010.02.008. Epub 2010 Mar 20.
A cell-associated enzyme exhibiting beta-xylosidase activity was purified from the cell extract of the dimorphic fungus, Aureobasidium pullulans strain ATCC 20524, grown on oat spelt xylan. The purified enzyme was homogeneous as judged by SDS-PAGE, which showed an apparent M(r) of 88.5 kDa. beta-Xylosidase activity was optimal at pH 3.5 and 70 degrees C. The enzyme exhibited apparent K(m) and V(max) values of 3.5 mM and 263 micromol/mg/min, respectively, for p-nitrophenyl-beta-d-xylopyranoside. The enzyme also showed some alpha-l-arabinofuranosidase activity. Southern blot analysis indicated that the beta-xylosidase gene (xylI) was present as a single copy in the genome. The genomic DNA and cDNA encoding this protein were cloned and sequenced. The open reading frame of 2415 bp was interrupted by two introns of 54 and 52 bp, and it encoded a presumed signal peptide of 20 residues and a mature protein of 785 residues with a calculated M(r) of 85,045 Da and a deduced isoelectric point of 4.57. The protein was N-glycosylated and possessed 16 potential N-glycosylation sites. Two distinct transcription start points of the xylI gene were present at nt -32 (A) and -26 (A) from the start codon. The xylI cDNA was functionally expressed in the yeast Pichia pastoris. The deduced amino acid sequence of the xylI gene product was 49% identical to the Talaromyces emersonii beta-xylosidase Bxl1, which belongs to the glycoside hydrolase family 3.
一株从生长在燕麦大麦芽半纤维素上的二相真菌 Aureobasidium pullulans 菌株 ATCC 20524 的细胞提取物中分离得到的具有β-木糖苷酶活性的细胞结合酶。该纯化酶通过 SDS-PAGE 显示出均一性，表观 M(r)为 88.5 kDa。β-木糖苷酶活性在 pH 3.5 和 70°C 时最佳。该酶对 p-硝基苯-β-d-木吡喃糖苷表现出明显的 K(m)和 V(max)值，分别为 3.5 mM 和 263 μmol/mg/min。该酶还表现出一些α-l-阿拉伯呋喃糖苷酶活性。Southern 印迹分析表明，β-木糖苷酶基因（xylI）在基因组中以单拷贝存在。该蛋白的基因组 DNA 和 cDNA 被克隆和测序。2415 bp 的开放阅读框被 54 和 52 bp 的两个内含子中断，它编码一个假定的 20 个残基的信号肽和一个 785 个残基的成熟蛋白，计算的 M(r)为 85045 Da，推测的等电点为 4.57。该蛋白被 N-糖基化，具有 16 个潜在的 N-糖基化位点。xylI 基因有两个不同的转录起始点，分别位于起始密码子前的 nt -32(A)和-26(A)。xylI cDNA 在酵母 Pichia pastoris 中具有功能性表达。xylI 基因产物的推导氨基酸序列与 Talaromyces emersonii 的β-木糖苷酶 Bxl1 有 49%的同一性，后者属于糖苷水解酶家族 3。
