X-ray diffraction study and reaction mechanism of bacterial alginate lyase

细菌海藻酸裂解酶的X射线衍射研究及反应机制

基本信息

批准号：
07660111
负责人：
MURATA Kousaku
金额：
$ 1.41万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660111/
关键词：
Bacterial infection Pseudomonas aeruginosa Alginate X-ray diffraction Alginate lyase

项目摘要

Alginate is a highly viscous heteropolysaccharide composed of manuronate and gluronate. Alginate formation by Pseudomonas aeruginosa is a leading cause of bacterial infectious disease, which is also called biofilm infection. The effective therapeutic methods for the disease have not been developed. For the treatment of bacterial infectious disease, the removal of diofilm is expected as a useful and effective procedures. The present study aimed to apply bacterial enzyme : alginate lyase that depolymerizes alginate for the removal of biofilm and to analyze three-dimensional structure. Following results were obtained. (1) Preliminary X-ray diffraction study was performed and crystal constants were determined. However, the enzyme crystal did not accept heavy metal ions, although the reason for this resistance is unclear. (2) The reaction mechanism of the lyase was analyzed and reaction constants were obtained with an estimation of the active site structure. (3) The alginate lyase was chemically modified with polyyethylenglycol. The modified enzyme showed sufficient lyase activity with extremely low lebel of antigenic activity. The results, together with others, strongly suggested that the enzyme will be applied to the treatment of bacterial infectious patients. (4) To further decrease antigenic activity, the alginate lyase was fragmented and low molecular size enzyme was generated. (5) The bacterium producing alginate lyase had a pit on the cell surface. This is the first finding in the history of microbiology. (6) The fine structural analysis of the pit structure by electron microscopy and pit-deficient mutant indicated that the pit is responsible for the uptake of macromolecules with energy and information. Our paper summarized these results was awarded from Japan Society of Fermentation and Bioengineering in 1996.

藻酸盐是一种高粘性的杂菌酸酯，由口腔素酸盐和胶质酸盐组成。铜绿假单胞菌形成藻酸盐是细菌传染病的主要原因，也称为生物膜感染。尚未开发出有效的疾病治疗方法。为了治疗细菌传染病，预期去除借叶膜是有用的有效程序。本研究旨在应用细菌酶：藻酸盐裂解酶，将藻酸盐解散以去除生物膜并分析三维结构。获得以下结果。（1）进行初步的X射线衍射研究，并确定晶体常数。然而，酶晶体不接受重金属离子，尽管这种抗性的原因尚不清楚。（2）分析裂解酶的反应机理，并通过估计活性位点结构获得反应常数。（3）藻酸盐裂解酸酯用聚乙基甘油化学修饰。修饰的酶显示出足够的裂解酶活性，具有极低的抗原活性。结果与其他结果一起强烈建议将酶应用于细菌感染性患者的治疗。（4）为了进一步降低抗原活性，藻酸盐裂解酶被碎裂并产生低分子大小酶。（5）产生藻酸盐裂解酶的细菌在细胞表面上有一个凹坑。这是微生物学史上的第一个发现。（6）通过电子显微镜和缺陷型突变体对坑结构的精细结构分析表明，该凹坑负责用能量和信息吸收大分子。我们的论文总结了1996年日本发酵和生物工程协会颁发的这些结果。