Production of high concentrations of fuel ethanol by fermentation of ligneous biomass

1)从丝状真菌柑橘青霉FERM P-15944培养滤液中纯化胞外内-1,4-β-木聚糖酶。纯化后的酶在SDS-PAGE上显示为单条带，表观M_r为31.6 kDa。木聚糖酶基因^<xynB的开放阅读框被9个内含子打断，编码25个氨基酸的推定前肽和302个氨基酸的成熟蛋白。10.2)从丝状真菌日本曲霉（Aspergillus japonicus）菌株MU-2培养滤液中纯化出一种具有β-木糖苷酶活性的胞外蛋白。纯化后的酶为糖蛋白，表观M_r为113.2 kDa，对对硝基苯基1-β- d -葡萄糖苷和对硝基苯基1-β- l -阿拉伯糖葡糖苷具有水解活性。β-xylo， sidase基因^<xyl>A的开放阅读框全长2412 bp，未被内含子打断，编码了17个氨基酸的s…More信号肽和787个氨基酸的成熟蛋白。推导出的氨基酸序列与黑曲霉β-木糖苷酶XlnD具有高度的同源性（69%），属于糖苷水解酶家族。3.3)从日本曲霉中纯化出一种胞外木聚糖酶，其产率为28.7%，比活性提高了21倍。木聚糖酶在SDS-PAGE上呈单条带，表观M_r为25.1 kDa。根据曲霉GH家族-11木聚糖酶的内部氨基酸序列（EDYGEYN）和高度保守序列（NHFNAWA）合成了一对简并PCR引物，以日本刺参基因组DNA为模板扩增出两个不同的内部序列。利用digg标记的扩增片段克隆了两个编码木聚糖酶的基因组DNA片段，推导出两个木聚糖酶基因^<xynG1和^<xynG2的氨基酸序列，结果表明纯化的木聚糖酶对应于^<xynG2基因产物。少

1)An extracellular endo-1,4-β-xylanase was purified from the culture filtrate of a filamentous fungus, Penicillium citrinum strain FERM P-15944. The purified enzyme showed a single band on SDS-PAGE with an apparent M_r of 31.6 kDa. An open reading frame of the xylanase gene ^<xynB was interrupted by nine introns and encoded a presumed prepropeptide of 25 amino acids and a mature protein of 302 amino acids. Sequence alignment showed that the P. citrinum enzyme belongs to glycoside hydrolase family 10.2)An extracellular protein exhibiting β-xylosidase activity was purified from the culture filtrate of a filamentous fungus Aspergillus japonicus strain MU-2. The purified enzyme was a glycoprotein with an apparent M_r of 113.2 kDa The enzyme also had hydrolytic activities toward p-nitropheny1-β-D-glucopyranoside and p-nitropheny1-β-L-arabinofuranoside. An open reading frame of the β-xylo, sidase gene ^<xyl>A, consisting of 2412 bp, was not interrupted by introns, and it encoded a presumed s … More ignal peptide of 17 amino acids and a mature protein of 787 amino acids. The deduced amino acid sequence showed a high degree of identity (69%)to Aspergillus nigerβ-xylosidase XlnD that belongs to glycoside hydrolase family 3.3)An extracellular xylanase from A. japonicus was purified with a yield of 28.7% of the activity and a 21-fold increase in specific activity. The xylanase showed a single band on SDS-PAGE with an apparent M_r of 25.1 kDa. A pair of degenerate PCR primers was synthesized according to the internal amino acid sequence (EDYGEYN)and a highly conserved sequence (NHFNAWA)among GH family-11 xylanases from Aspergillus spp. Two distinct internal sequences were amplified from genomic DNA of A. japonicus as a template by PCR with the primer pair. Two genomic DNA segments encoding xylanase were cloned using two DIG-labeled amplified fragments The deduced amino acid sequences of two xylanase genes, ^<xynG1 and ^<xynG2, revealed that purified xylanase corresponded to ^<xynG2 gene product. Less