Wang Hui-Chang, Chen Yo-Chia, Huang Ching-Tsan, Hseu Ruey-Shyang
Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan.
Protein Expr Purif. 2013 Aug;90(2):153-9. doi: 10.1016/j.pep.2013.06.004. Epub 2013 Jun 13.
An 1888-bp cDNA designated celA, isolated from a cDNA library of Neocallimastix patriciarum J11 was cloned. The celA had an open reading frame of 1530 bp encoding J11 CelA of 510 amino acids. The primary structure analysis of J11 CelA revealed a complete cellulose-binding domain at the N-terminal, followed by an Asn, Ala, Gly, Gln and Pro-rich linker and ending with a C-terminal glycosyl hydrolase family 6 catalytic domain. The mature J11 CelA was overexpressed in Escherichia coli and purified to homogeneity. This enzyme had high specific activities towards barley β-glucan and lichenan, low toward carboxymethyl cellulose (CMC), Avicel, and H3PO4-swollen Avicel (PSA). The product of Avicel hydrolysis was cellobiose indicating that J11 CelA is a typical cellobiohydrolase. The recombinant J11 CelA had an optimal pH of 6.0 and was stable over a wide range of pH (5.2-11.3). The enzyme showed an optimal temperature of 50°C and was still maintained approximately 50% of the maximum activity in response to the treatment at 70°C for 1h. Cobalt and Fe(3+) at 1 mM greatly activated the enzyme activity. As a thermostable and pH stable enzyme with crystalline cellulose-degrading activity, J11 CelA is a potential candidate for the bioethanol industry.
从新美鞭菌属J11的cDNA文库中分离出一个1888 bp的cDNA，命名为celA，并进行了克隆。celA具有一个1530 bp的开放阅读框，编码510个氨基酸的J11 CelA。J11 CelA的一级结构分析表明，其N端有一个完整的纤维素结合结构域，接着是一个富含天冬酰胺、丙氨酸、甘氨酸、谷氨酰胺和脯氨酸的连接区，C端为糖基水解酶家族6催化结构域。成熟的J11 CelA在大肠杆菌中过量表达并纯化至均一。该酶对大麦β-葡聚糖和地衣多糖具有高比活性，对羧甲基纤维素（CMC）、微晶纤维素和磷酸膨胀微晶纤维素（PSA）的比活性较低。微晶纤维素水解产物为纤维二糖，表明J11 CelA是一种典型的纤维二糖水解酶。重组J11 CelA的最适pH为6.0，在较宽的pH范围（5.2 - 11.3）内稳定。该酶的最适温度为50°C，在70°C处理1小时后仍保持约50%的最大活性。1 mM的钴和Fe(3+)能极大地激活该酶的活性。作为一种具有结晶纤维素降解活性的热稳定和pH稳定酶，J11 CelA是生物乙醇工业的潜在候选酶。
