Multi-cellulase components produced by Sporotrichum cellulophilum

从嗜纤维链霉菌粗提液中分离纯化出CII1、CII2、CIII1、CIII2、CIII3、CIV等纤维素酶，并去除CIII2和CIV中的糖分。尝试制备了抗所有纤维素酶的单抗，但仅获得了4个克隆。用酶联免疫吸附试验检测抗纤维素酶的单抗的反应性。纤维素酶CIV与CIII2关系最密切，与CIII1关系较近，与CIII3关系较近，与CII2关系不大。用5C_4.300柱对两个组分进行了纯化，测定了22个和31个氨基酸的氨基酸序列。从这些序列中合成了8个DNA探针，由14~24个碱基组成。提取嗜纤维链霉菌的RNA，构建其cDNA文库。用探针T(20mer)从文库中筛选出阳性克隆，重新克隆后与探针(23mer)杂交，没有一条不是阳性克隆。然后从细胞中提取DNA，用SamIIIA1对其进行部分酶切，得到DNA文库。用探针B(23mer)筛选，得到4.9kbp的片段。将其克隆到400bpAluI-AluII片段上，测得其DNA序列，但有部分序列与氨基酸序列不符。然后，用D探针(29mer)分离出500bpSMALKpnI和1050EcoT22I-Xba1片段，测定了它们的DNA序列，发现了与7和6个氨基酸序列对应的区域，但在这些区域附近没有找到与其他连接氨基酸序列相对应的DNA序列。因此，纤维素酶基因还没有被克隆。

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.