This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da. The alpha-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid. The K(m) for MeGlcAX2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date. When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.

译文

这是第一份报告,描述了编码 α-葡萄糖醛酸苷酶的基因的分析,该酶是取代木聚糖完全分解所必需的酶。从嗜热细菌Thermotoga maritima msb8的染色体DNA中分离出携带 α-葡萄糖醛酸苷酶基因的DNA片段。借助核苷酸序列分析,缺失实验和在大肠杆菌中的表达研究,对 α-葡萄糖醛酸苷酶基因 (aguA) 进行了鉴定和表征,并通过纯化的重组酶的氨基末端测序来定义编码区的起始。aguA基因编码674氨基酸,主要是亲水性多肽,其计算分子量为78593 Da。maritima的 α-葡萄糖醛酸苷酶具有新颖的一级结构,与任何其他已知的氨基酸序列没有显着相似性。通过sdds-PAGE判断,将重组酶纯化至均一。在低盐浓度下的凝胶过滤分析显示重组酶的高表观分子量 (> 630 kDa),但是寡聚结构随着离子强度或pH的变化而改变,产生也具有酶活性的六聚和/或二聚体形式。该酶将2-O-(4-O-methyl-alpha-D-glucopyranosyluronic酸)-D-木二糖 (MeGlcAX2) 水解为木二糖和4-o-甲基葡萄糖醛酸。MeGlcAX2的K(m) 为0.95 mM。最适pH为6.3。在85 ℃ 、约25 ℃ 或高于迄今已知的所有其他 α-葡萄糖醛酸糖苷酶报告的值时测量最大活性。当在55-75摄氏度下孵育时,该酶发生部分失活,但此后几天内残留活性几乎保持恒定。

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