Multi-cellulase components produced by Sporotrichum cellulophilum

纤维素酶CII 1、CII 2、CIII 1、CIII 2、CIII 3、CIV从S.除去CIII 2和CIV的糖部分。尝试制备抗所有纤维素酶的单克隆抗体，但仅获得4个克隆。用ELISA法分析了单克隆抗体的反应性。纤维素酶CIV与CIII 2最接近，与CIII 1更接近，与CII 1和CIII 3接近，与CII 2几乎不相关。通过HPLC在5C_4.300上纯化两个级分，并测定22和31个残基的氨基酸序列。从这些序列中，合成了由14至24个碱基组成的8个DNA探针。S.提取嗜纤维素菌，制备cDNA文库。用探针T（20 mer）从文库中筛选出阳性克隆，再克隆后与探针（23 mer）杂交，无一不杂交。然后从细胞中提取DNA，用SamIIIA 1部分水解后，制备DNA文库。用探针B筛选。(23 mer），得到4.9kbp的片段。对400 bp的cDNA片段进行再克隆后，测定其DNA序列，但序列的任何部分都与氨基酸序列不一致。接着，使用探针D（29 mer），分离500 bp Smal-kpn I和1050 bp EcoT 22 I-Xba 1片段，测定这些DNA序列，发现对应于7和6个残基的氨基酸序列的区域，但在该区域附近未发现对应于其它连接氨基酸序列的DNA序列。因此纤维素酶基因尚未克隆。

Cellulases CII1, CII2, CIII1, CIII2, CIII3, CIV were purified to homogeneity from the crude extract of S. cellulophilum, and sugar moieties of CIII2 and CIV were removed. Monoclonal antibodies against all the cellulases were tried to prepare, but only 4 clones were obtained. The reactivities of monoclonal antibodies against all the cellulases were analyzed by ELISA method. Cellulase CIV was most close to CIII2, more close to CIII1, close to CII1 and CIII3, and hardly related to CII2.The amino terminus of cellulase CIV was blocked and CIV was hydrolyzed with lysyl endopeptidase. Two fractions were purified by HPLC on 5C_4.300 and amino acid sequences of 22 and 31 residues were determined. From those sequences, 8 DNA probes composing of 14 to 24 bases were synthesized. The RNA of S. cellulophilum was extracted, the cDNA library was prepared. By using probe T (20 mer), positive clones were screened from the library, those were recloned, and hybridized with probe (23 mer), but none of them was not.In the above method the cDNA was not prepared with good yield. Then we extracted DNA from the cell, and after hydrolyzing it partially with SamIIIA1, the DNA library was prepared. It was screened with probe B.(23 mer), and 4.9 kbp fragment was obtained. After recloning to 400 bp AluI-AluII fragment, its DNA sequence was determined, but any part of the sequence did not correspond with the amino acid sequence. Next by using probe D (29 mer), 500 bp Smal-kpnI and 1050 EcoT22I-Xba1 fragments were isolated, those DNA sequences were determined, regions corresponding to amino acid sequence of 7 and 6 residues were found but the DNA sequences corresponding to other connecting amino acid sequence were not found near the regions. So cellulase gene is not cloned yet.