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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.

海藻酸盐是一种高粘度的杂多糖，由肉桂酸和谷氨酸组成。铜绿假单胞菌形成藻酸盐是引起细菌感染性疾病的主要原因，细菌感染性疾病又称生物膜感染。这种疾病的有效治疗方法还没有开发出来。对于细菌感染性疾病的治疗，去除双膜有望成为一种有用和有效的方法。本研究旨在应用细菌酶：海藻酸裂解酶来去除生物膜，并对其三维结构进行分析。获得了以下结果。(1)进行了初步的X射线衍射研究，测定了晶体常数。然而，该酶晶体不接受重金属离子，尽管产生这种抗性的原因尚不清楚。(2)对裂解酶的反应机理进行了分析，通过对活性中心结构的估算，得到了反应常数。(3)用聚乙二醇对海藻酸裂解酶进行化学修饰。修饰后的酶具有较高的裂解酶活力和极低的抗原性。这些结果与其他结果一起，强烈表明该酶将用于治疗细菌感染患者。(4)为进一步降低海藻酸裂解酶的抗原性，对海藻酸裂解酶进行了裂解，产生了低分子量的酶。(5)藻酸盐裂解酶产生菌在细胞表面有一个凹坑。这是微生物学历史上的第一次发现。(6)电子显微镜和缺陷型突变体对凹坑结构的精细结构分析表明，凹坑负责摄取含有能量和信息的大分子。本文总结了1996年日本发酵与生物工程学会授予的研究成果。