Nakashima K I, Watanabe H, Azuma J I
Cell Mol Life Sci. 2002 Sep;59(9):1554-60. doi: 10.1007/s00018-002-8528-1.
Cellulase genes of Pseudotrichonympha grassii (Hypermastigida: Eucomonymphidae), the symbiotic flagellate in the hindgut of the wood-feeding termite Coptotermes formosanus, were isolated and characterized. The nucleotide sequences of the major cellulase component in the hindgut of C. formosanus were determined based on its N-terminal amino acid sequence. The five isolated nucleotide sequences (PgCBH-homos) had an open reading frame of 1350 bp showing similarity to catalytic domains of glycoside hydrolase family (GHF) 7 members, and primary structure comparison with GHF7 members whose tertiary structures are well-characterized revealed the overall similarity between PgCBH-homo and the catalytic domain of a processive cellulase Cel7A (formerly CBHI) from the aerobic fungus Trichoderma reesei. Functional expression of PgCBH-homos in Escherichia coli, using the carboxymethylcellulose-Congo red assay, demonstrated the actual cellulolytic activity of PgCBH-homo. RT-PCR showed that PgCBH-homos were expressed, from the three flagellates in the hindgut, specifically in P. grassii.
从取食木材的台湾乳白蚁后肠中的共生鞭毛虫——绿草伪拟披发虫（超鞭毛虫纲：真拟披发虫科）中分离并鉴定了纤维素酶基因。基于其N端氨基酸序列测定了台湾乳白蚁后肠中主要纤维素酶组分的核苷酸序列。分离得到的5个核苷酸序列（PgCBH-homos）具有1350 bp的开放阅读框，与糖苷水解酶家族（GHF）7成员的催化结构域具有相似性，与三级结构已得到充分表征的GHF7成员进行一级结构比较后发现，PgCBH-homo与需氧真菌里氏木霉的连续性纤维素酶Cel7A（原CBHI）的催化结构域总体相似。利用羧甲基纤维素-刚果红测定法在大肠杆菌中对PgCBH-homos进行功能表达，证实了PgCBH-homo具有实际的纤维素分解活性。逆转录聚合酶链反应（RT-PCR）表明，PgCBH-homos在后肠中的三种鞭毛虫中表达，且在绿草伪拟披发虫中特异性表达。
