Romaniec M P, Kobayashi T, Fauth U, Gerngross U T, Demain A L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Appl Biochem Biotechnol. 1991 Nov;31(2):119-34. doi: 10.1007/BF02921783.
Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C. thermocellum chromosomal DNA fragments constructed in the vector lambda gt11. A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated. No similarity with other previously cloned DNA fragments has been found. The anti-SL crossreacting polypeptide was isolated from recombinant Escherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity. Using CMC as the substrate, a temperature optimum of 55 degrees C and a pH optimum of 6.6 were observed. These properties were compared to those of C. thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities. In addition, the SL proteins produced in C. thermocellum and E. coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.
用针对嗜热栖热放线菌纤维小体的SL亚基产生的抗体筛选构建于λgt11载体中的嗜热栖热放线菌染色体DNA片段文库。分离出一个编码与抗SL抗体发生交叉反应的多肽的DNA片段，并阐明了其限制酶切图谱。未发现与其他先前克隆的DNA片段有相似性。从重组大肠杆菌中分离出抗SL交叉反应多肽，发现其分子量为37000Da，具有低水平的羧甲基纤维素酶（CMCase）和微晶纤维素酶（Avicelase）活性。以羧甲基纤维素（CMC）为底物时，观察到最适温度为55℃，最适pH为6.6。将这些特性与通过从SDS凝胶中电洗脱分离得到的嗜热栖热放线菌SL的特性进行比较，发现其也具有低水平的羧甲基纤维素酶和微晶纤维素酶活性。此外，嗜热栖热放线菌和大肠杆菌中产生的SL蛋白能够与从嗜热栖热放线菌粗纤维素酶制剂中纯化的内切葡聚糖酶（Ss）以及与抗Ss抗体发生交叉反应的重组蛋白对微晶纤维素产生正向相互作用。
