Denman S, Xue G P, Patel B
CSIRO Division of Tropical Crops and Pastures, St. Lucia, Queensland, Australia.
Appl Environ Microbiol. 1996 Jun;62(6):1889-96. doi: 10.1128/aem.62.6.1889-1896.1996.
The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II.
对瘤胃真菌梨形新丽鞭毛虫（Neocallimastix patriciarum）的一种纤维素酶cDNA（celA）的核苷酸序列及其编码的蛋白质一级结构进行了表征。celA cDNA长1.95 kb，具有1284 bp的开放阅读框，编码一个含有428个氨基酸残基的多肽。序列比对表明，纤维素酶A（CELA）与B族纤维素酶（6族糖基水解酶）具有显著同源性，特别是与需氧真菌里氏木霉（Trichoderma reesei）的纤维二糖水解酶II。与先前表征的梨形新丽鞭毛虫糖基水解酶不同，CELA与先前描述的任何其他瘤胃微生物纤维素酶均无同源性。使用缺失分析和与其他特征明确的纤维素酶进行序列比较的一级结构和功能研究表明，CELA在N端由一个纤维素结合结构域和C端的一个催化结构域组成。这两个结构域由一个富含天冬酰胺的连接子隔开。纤维素结合结构域的缺失导致纤维素结合能力和对结晶纤维素的活性显著降低。当CELA在大肠杆菌中表达时，它主要位于周质空间，表明CELA的信号序列在大肠杆菌中具有功能。酶学研究表明，CELA的最适pH为5.0，最适温度为40℃。免疫亲和纯化的CELA对微晶纤维素的比活性为9.7 U/mg蛋白质，与报道的其他纤维二糖水解酶（如里氏木霉纤维二糖水解酶I和II）的比活性相比，CELA似乎是一种相对活跃的纤维二糖水解酶。
