Oxacillinase-79抗藥性蛋白在鮑氏不動桿菌之酵素活性分析

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 博碩士論文 > Item 310902500/5742

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Title:	Oxacillinase-79抗藥性蛋白在鮑氏不動桿菌之酵素活性分析 Study of the enzyme activity of Oxacillinase-79 in Acinetobacter baumannii
Authors:	許荻靈 Hsu, Ti-Ling
Contributors:	中山醫學大學;醫學院;生化暨生物科技研究所;陳凌雲
Keywords:	Oxacillinase-79抗藥性蛋白;鮑氏不動桿菌;酵素活性分析 enzyme activity;Oxacillinase-79;Acinetobacter baumannii
Date:	2012
Issue Date:	2012-12-21T06:36:01Z (UTC)
Abstract:	鮑氏不動桿菌（Acinetobacter baumannii）是一種革蘭氏陰性桿菌，容易造成免疫力低下病患感染，屬於伺機性感染的菌種。Carbapenem 呈抗藥性的鮑氏不動桿菌於全球有急遽增加的趨勢，也因此增加了感染後治療的困難度，成為全球棘手的問題。而鮑氏不動桿菌Carbapenem 抗藥性增加主要是因帶了class D OXA-type β-Lactamase，又稱為Oxacillinase。OXA主要分為OXA-51-like、OXA-23-like、OXA-24-like、OXA-58-like四大類。過去實驗室探討OXA-51-like中OXA-66的129位置Ile、Val、Leu酵素活性的差異；也探討了OXA-66的222位置Trp、Leu酵素活性的差異，結果以蛋白結構模擬圖來解釋，發現當OXA-66的129位置由Ile突變為Val 及Leu，會使得抗藥性蛋白催化中心入口底部的平台更為寬闊；另當OXA-66的222位置由Trp 突變為Leu後，會使得催化中心上方疏水性入口屏障更為緊密，讓受質能更穩固的留在催化中心，因而酵素活性增加。根據過去的文獻發現，抗藥性蛋白OXA-79的突變點，也在222的位置，是由Trp突變為Gly，而此突變對酵素活性有何影響，目前並無人探討，因此本篇探討Oxacillinase-79對酵素活性的影響，以了解Trp 222 Gly的意義。利用SOE（Spliced overlapping extension）及基因選殖的方法，人工合成OXA-79及OXA-83G，並進行酵素活性分析。結果顯示，OXA-79酵素相對比活性是比OXA-66還降低。以蛋白結構模擬圖推測是因為催化中心上方入口雙側凸出屏障，當222位置由Trp突變為Gly，使得雙側凸出屏障僅剩單側屏障，造成受質不易穩固的停留在催化中心內，因而造成酵素活性下降。 Acinetobacter baumannii, opportunistic infections bacteria, is Gram-negative bacilli and can easily lead to immunocompromised patients with infections. There is a global trend of rapid increasing Carbapenem resistant Acinetobacter baumannii and this raise creates the difficulties of the treatment after infection, which has becomes an intractable problem around the world.Carbapenem resistance increases with the class D OXA type β-Lactamase, also known as Oxacillinase. Four types of OXA are identified: OXA-51-like, OXA-23-like, OXA-24-like and OXA-58-like enzymes. Previous laboratory research focuses on the OXA-51-like in the OXA and-66, 129 location, Ile, Val, Leu enzyme activity differences and the OXA-66, www position, Trp, Leu enzyme activity differences. The protein structure analysis indicated that resistance of OXA-66 129 position by Ile mutation for the Val nd Leu made the protein catalytic entrance at the bottom of the broader platform and OXA-66, 222 position by Trp mutations Leu made the top of the catalytic center the hydrophobic barrier entrance closer. The results suggest the substrate can be more stably stay in the catalytic center, and thus increase enzyme activity. We lack of understandings of Oxacillinase-79 mutation, located in 222 position by Trp mutations Gly of the resistance of protein, and its influneces on enzyme activity. This study focuses on the influences of Oxacillinase-79 on the enzyme activity in order to understand the role of Trp (222 position) and Gly. SOE (Spliced overlapping extension) and gene cloning synthetic are conducted to produce the synthetic OXA-79 and OXA-83G for enzyme activity analysis. Results indicate that enzyme activity is lower in OXA-79 then OXA-66. Protein structure simulation suggests the catalytic center of the entrance unilateral barriers disappeared at one side and made the substrate cannot stably stay at center, which explained the enzyme activity declined.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/5742
Appears in Collections:	[生化微生物免疫研究所] 博碩士論文

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