Luderer M E, Hofer F, Hagspiel K, Allmaier G, Blaas D, Kubicek C P
Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, Wien, Austria.
Biochim Biophys Acta. 1991 Feb 15;1076(3):427-34. doi: 10.1016/0167-4838(91)90487-k.
An endo beta-1,4-glucanase (EC 3.2.1.4, 1.4-(1,3;1,4)-beta-D-glucan 4 glucanhydrolase) was purified to apparent homogeneity from culture filtrates of Trichoderma reesei QM 9414. Identity of the protein with endoglucanase I (EG I) was examined by subjecting CNBr fragments of the protein to analysis by plasma desorption mass spectrometry. Seven non-glycosylated fragments, mapped on the eg1 gene sequence, could be identified, hence proving at least 39.4% identity of the amino acid sequence. No sign for microheterogeneity was observed. Purified EG I was used to prepare monoclonal antibodies. 17 stable clones were obtained, of which one--Mab EG 3--was used to analyze several commercial T. reesei cellulase preparations as well as culture filtrates from T. pseudokoningii and T. longibrachiatum for the presence of EG I. Most of them contained immunoreactive material migrating as a prominent 50-55 kDa band on SDS-PAGE, resembling EG I, but in some instances additional lower molecular weight bands were also observed. Cultivation of T. reesei at low pH led to an increase of these lower molecular weight bands. EG I was rather stable against proteolysis by papain in vitro, but after prolonged treatment, immunopositive products of 50 and 45 kDa were produced at the expense of the 55 kDa band. Our monoclonal antibodies failed to react with a low-molecular-weight endoglucanase, which was previously shown to be detectable with polyclonal antiserum against EG I. However, all monoclonals reacted with a 118 kDa protein which is most probably a dimer of EG I. These results are discussed with respect to the occurrence of multiple forms of EG I in T. reesei cellulase preparations.
从里氏木霉QM 9414的培养滤液中纯化出一种内切β-1,4-葡聚糖酶（EC 3.2.1.4, 1.4-(1,3;1,4)-β-D-葡聚糖4-葡聚糖水解酶），使其达到表观均一性。通过对该蛋白的溴化氰片段进行等离子体解吸质谱分析，检测该蛋白与内切葡聚糖酶I（EG I）的同一性。可以鉴定出7个非糖基化片段，它们定位在eg1基因序列上，从而证明氨基酸序列的同一性至少为39.4%。未观察到微异质性的迹象。纯化的EG I用于制备单克隆抗体。获得了17个稳定的克隆，其中一个——单克隆抗体EG 3——用于分析几种商业里氏木霉纤维素酶制剂以及拟康氏木霉和长枝木霉的培养滤液中EG I的存在情况。它们中的大多数含有免疫反应性物质，在SDS-PAGE上迁移为一条突出的50-55 kDa条带，类似于EG I，但在某些情况下也观察到了额外的低分子量条带。在低pH条件下培养里氏木霉会导致这些低分子量条带增加。EG I在体外对木瓜蛋白酶的蛋白水解作用相当稳定，但经过长时间处理后，会产生50 kDa和45 kDa的免疫阳性产物，而55 kDa条带减少。我们的单克隆抗体未能与一种低分子量内切葡聚糖酶发生反应，该酶先前已被证明可以用针对EG I的多克隆抗血清检测到。然而，所有单克隆抗体都与一种118 kDa的蛋白发生反应，该蛋白很可能是EG I的二聚体。针对里氏木霉纤维素酶制剂中EG I多种形式的出现情况对这些结果进行了讨论。
