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

1）从丝状真菌桔青霉（Penicillium citrinum）菌株FERM P-15944的培养滤液中纯化胞外内切-1，4-β-木聚糖酶。纯化后的酶在SDS-PAGE上呈单一条带，表观分子量为31.6kDa。木聚糖酶基因^<xynB的开放阅读框被9个内含子打断，编码一个25个氨基酸的前肽和一个302个氨基酸的成熟蛋白。序列比对显示桔黄青霉酶属于糖苷水解酶家族10。2）从丝状真菌Aspergillusglycerin菌株MU-2的培养物滤液中纯化了表现出β-木糖苷酶活性的胞外蛋白。该酶为糖蛋白，表观分子量为113.2kDa，对对硝基苯基-β-D-吡喃葡萄糖苷和对硝基苯基-β-L-阿拉伯呋喃糖苷具有水解活性。β-木糖苷酶基因^A的开放阅读框由2412 bp组成，不被内含子打断，它编码一个推测的S<xyl> ...更多信息 17个氨基酸的信号肽和787个氨基酸的成熟蛋白。推导的氨基酸序列与糖苷水解酶家族3的黑曲霉β-木糖苷酶XlnD具有较高的同源性（69%）。纯化后的酶活收率为28.7%，比活提高了21倍。SDS-PAGE显示木聚糖酶为单一条带，表观分子量为25.1kDa。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。从A.以该引物对为模板进行PCR。用地高辛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，推导了两个木聚糖酶基因^&lt;xynG 1和^&lt;xynG 2的氨基酸序列，表明纯化的木聚糖酶对应于^&lt;xynG 2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