Kotake Toshihisa, Kaneko Satoshi, Kubomoto Aya, Haque Md Ashraful, Kobayashi Hideyuki, Tsumuraya Yoichi
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
Biochem J. 2004 Feb 1;377(Pt 3):749-55. doi: 10.1042/BJ20031145.
The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no. AB104898. A gene encoding endo-beta-(1-->6)-galactanase from Trichoderma viride was cloned by reverse transcriptase-PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His(6) tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase. As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned.
图1所示的核苷酸序列已提交至DDBJ核苷酸序列数据库，登录号为AB104898。通过逆转录聚合酶链反应（reverse transcriptase-PCR）克隆了来自绿色木霉（Trichoderma viride）的编码内切β-(1→6)-半乳聚糖酶的基因，并在大肠杆菌（Escherichia coli）中进行表达。该基因包含一个由1437 bp（479个氨基酸）组成的开放阅读框。推导的该蛋白质氨基酸序列与其他已知糖苷水解酶的相似性很小。在该蛋白质的N端区域发现了一个信号序列（20个氨基酸），成熟形式的分子量经计算为50.488 kDa。在大肠杆菌中表达的与硫氧还蛋白和His(6)标签融合的重组蛋白形式的基因产物，其底物特异性和作用方式与从绿色木霉商业纤维素酶制剂中纯化的天然酶几乎相同，即重组酶内切水解聚合度（DP）高于3的β-(1→6)-半乳糖寡聚物，并且它还可以水解经α-L-阿拉伯呋喃糖苷酶处理的来自萝卜的阿拉伯半乳聚糖蛋白。在初始水解阶段，它产生聚合度从2到至少8的β-(1→6)-半乳糖寡聚物，在最终反应阶段，主要产物为半乳糖和β-(1→6)-半乳糖二糖。这些结果表明，克隆的基因编码一种内切β-(1→6)-半乳聚糖酶。据我们所知，这是首次克隆出内切β-(1→6)-半乳聚糖酶。
