通过反转录PCR(RT-PCR)从黑曲霉(Aspergillus niger)DL08中提取内切葡聚糖酶基因(GeneBank No.KJ437592),PCR测序表明该基因全长999个核苷酸,编码332个氨基酸,预测相对分子质量为36.75 kDa,等电点(pI)为4.38,命名eg1。结构域分析表明,该蛋白包括18个氨基酸构成的信号肽和C末端1个糖基水解酶家族5的催化结构域。重组内切葡聚糖酶蛋白通过Ni-NTA亲和层析柱纯化,酶学性质研究表明,以羧甲基纤维素钠为底物时重组酶最适作用pH为5.0,最适作用温度为45℃。通过薄层层析法检测重组内切葡聚糖酶酶解1%羧甲基纤维素钠的产物,主要为连续寡糖。这些特性为纤维素酶酶解纤维素生产生物化学品和可再生生物燃料提供技术参考。

A gene encoding an endo-β-1,4-glucanase,which was isolated from Aspergillus niger DL08 by using an RT-PCR protocol and its nucleotide sequence was determined. The eg1 cDNA contained a 999 bp open reading frame encoding a 332 amino acid protein with an estimated molecular mass of 36. 75 kDa and isoelectronic point( pI)of 4. 38,named eg1. Based on domain analysis presumed the eg1 contains 18 amino acids signal peptide,a catalytic domain of glycosyl hydrolase family 5( GH5) C-terminal catalytic region. The recombinant endo-β-1,4-glucanse eg1was purified by Ni-affinity chromatography from the intracellular fraction of Escherichia coli. The optimal activity of the purified enzyme was displayed at a temperature of 45 ℃ and pH 5. 0 when sodium carboxymethyl cellulose was used as a substrate,respectively. The end products of the hydrolysis of 1% sodium carboxymethyl cellulose by eg1 were detected by thin layer chromatography,mainly continuously oligosaccharides. These properties would provide technical parameters for utilizing cellulose for the production of biochemicals and renewable biofuels.