Elucidation of cellulase gene expression mechanisim in cellulolytic bacteria

纤维素分解菌中纤维素酶基因表达机制的阐明

• 批准号: 12839005
• 负责人: KARITA Shuichi
• 金额: $ 2.37万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12839005/
• 关键词: Clostridium; Catabolite control protein; cellulase; xylanase; chitinase; ccpA gene; ccpA遺伝子; Clostrdium; カタボライト調節蛋白質; ヘリックスーターン-へリックスモチーフ; Bacillus subtilis; CcpA遺伝子

A gene encoding the catabolite control protein A (CcpA) having helix-turn-helix DNA binding motif was cloned from the cellulolytic anaerobe, Clostridium cellulovorans, using PCR and colony hybridization. A fusion ccpA gene with the promoter region of Bacillus subtilis ccpA gene was examined to complement test using ccpA defect mutant of B. subtilis. A gluconate kinase activity was measured to complement the mutant. As a result, the ccpA gene from C, cellulovorans was not able to complement the defect mutant. The CcpA protein expressed in Escherichia coli was purified using Ni-NTA and Resource Q column. The purified protein was examined for gel shift assay with the promoter region of C. cellulovorans scaffolding protein gene and B. subtilis amyE gene, being reglutated by catabolite. The DNA bands showed no shifting ; suggesting other factors need to bind the protein to DNA. And the protein sequence has similarity to B. subtilis CcpA, but the function is not same. In addition, cloning experiments of genes encoding phosphoenolpyruvate dependent phosphotransferase system (PTS) were done. PTS enzyme I and enzyme II Glc gene were cloned from C. cellulovorans, successfully. The presence of PTS genes suggest the catabolite control mechanism mediated PTS in C. cellulovorans. A ccpA gene from chitinolytic Clostridium paraputrificum was also cloned and sequenced. These have similar protein sequence.

利用PCR和菌落杂交技术从厌氧纤维素分解菌Clostridium cellulovorans中克隆了一个具有螺旋-转角-螺旋DNA结合基序的分解代谢物控制蛋白A（CcpA）基因。利用枯草芽孢杆菌ccpA基因启动子区与ccpA基因的融合基因，对B的ccpA缺陷突变株进行了互补试验。枯草芽孢杆菌测量葡萄糖酸激酶活性以补充突变体。结果，来自食纤维梭菌的ccpA基因不能补充缺陷突变体。利用Ni-NTA和ResourceQ柱对原核表达的CcpA蛋白进行纯化。纯化的蛋白用C.食纤维菌支架蛋白基因和B. subtilis amyE基因，经降解产物再突变。DNA条带没有显示出移动;这表明其他因素需要将蛋白质结合到DNA上。并且蛋白质序列与B具有相似性。subtilis CcpA，但功能不同。此外，还进行了编码磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）基因的克隆实验。从C.食纤维素菌，成功。PTS基因的存在提示了C.食纤维菌。此外，还克隆了一个来自几丁质分解梭菌的ccpA基因，并进行了序列测定。它们具有相似的蛋白质序列。

项目成果

J.-X.Feng: "Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product."Biosci.Biotechnol.Biochem.. 64(12). 2614-2624 (2000)

J.-X.Feng：“编码来自 Josui 梭菌的细胞结合多域木聚糖酶的基因的克隆、测序和表达，以及翻译产物的表征。”Biosci.Biotechnol.Biochem.. 64(12)。

J.-X.Feng, S.Kalita, et al.: "Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the tranlated product"Biosci.Biotechnol.Biochem.. 64. 2614-2624 (2000)

J.-X.Feng、S.Kalita 等人：“编码来自 Josui 的细胞结合多域木聚糖酶的基因的克隆、测序和表达，以及翻译产物的表征”Biosci.Biotechnol.Biochem

J.-X. Feng, S. Karita, et al.: "Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product"Biosci. Biotechnol. Biochem.. 64(12). 2614-2624 (2000)

J.-X。