School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China.
J Ind Microbiol Biotechnol. 2012 Feb;39(2):347-57. doi: 10.1007/s10295-011-1039-z. Epub 2011 Sep 21.
Using 3' and 5' rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A, named as Aucel12A, is 1,027 bp in length harboring 5' and 3' non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence is 1,576 bp in length, consisting of a 5' flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp. The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5'- and 3'-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W. of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5-7.0, and at a temperature of 55°C or below. Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag⁺, Hg²⁺ and Fe²⁺. The K(m) and V(max) of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively.
利用 3' 和 5' 快速扩增 cDNA 末端方法，从 Aspergillus usamii E001 的总 RNA 中扩增了编码内切 1,4-β-葡聚糖酶的全长 cDNA 序列（缩写为 AuCel12A）。该基因编码 AuCel12A 的克隆 cDNA 序列，命名为 Aucel12A，长度为 1027 bp，包含 5' 和 3' 非编码区，以及 720 bp 的开放阅读框，编码 16 个氨基酸的信号肽和 223 个氨基酸的成熟 AuCel12A，理论分子量为 24294 Da，理论等电点为 4.15，有一个潜在的 N-糖基化位点。使用我们实验室最初开发的常规 PCR 和 pUCm-T 载体介导的 PCR 从 A. usamii E001 的基因组 DNA 中扩增了基因 Aucel12A 的完整 DNA 序列。克隆 DNA 序列长 1576 bp，由 5' 侧翼调控区、三个外显子和两个内含子组成，大小分别为 50 和 66 bp。编码成熟 AuCel12A 的 cDNA 片段在毕赤酵母中以完全活性形式表达。选择表达最高重组 AuCel12A(rAuCel12A)活性的一个毕赤酵母转化子，标记为 P. pastoris GSCel2-1，用于后续研究。使用 5'- 和 3'-AOX1 引物的 PCR 分析证实了 Aucel12A 整合到毕赤酵母基因组中。SDS-PAGE 和酶活性分析表明，rAuCel12A 是一种糖基化蛋白，表观分子量为 27.0 kDa，碳水化合物含量为 4.82%，分泌到培养基中。纯化的 rAuCel12A 在 pH5.0 和 60°C 时表现出最高的活性。它在 pH 3.5-7.0 范围内非常稳定，在 55°C 或以下的温度下也非常稳定。它的活性不受一系列金属离子和 EDTA 的显著影响，但被 Ag⁺、Hg²⁺ 和 Fe²⁺抑制。rAuCel12A 对羧甲基纤维素-Na(CMC-Na)的 K(m)和 V(max)，在 pH5.0 和 50°C 时分别为 4.85 mg/ml 和 160.5 U/mg。
