Stenotrophomonas maltophilia 乙內醯胺酶之特性分析; Characterization of b-lactamases of Stenotrophomonas maltophilia

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题名:	Stenotrophomonas maltophilia 乙內醯胺酶之特性分析;Characterization of b-lactamases of Stenotrophomonas maltophilia
作者:	蔣凱弘;Kai-Hung Chiang
贡献者:	中國醫藥大學：醫學檢驗生物技術學系
关键词:	乙內醯胺酶;酵素動力學分析;抑制劑;b-lactamase;enzymatic kinetic assay;inhibitor
日期:	2008-07-09
上传时间:	2009-08-12 17:09:53 (UTC+8)
摘要:	Stenotrophomonas maltophilia具有L1和L2兩型b-lactamases，而此兩型b-lactamases是屬於共同被誘導表現。本研究分別包含定性和定量兩部分，探討S. maltophilia的L1和L2兩型b-lactamases之特性。在定性的部分：b-lactamases在IEF電泳上之酵素活性圖騰型式可作為b-lactamases定性分析之依據。本研究發現20株S. maltophilia分離菌株在IEF電泳上有兩型的酵素活性圖騰表現。除了酸性b-lactamase和鹼性b-lactamase各有一條條紋(band)這種典型的酵素活性圖騰之外；另外還有一種特殊的IEF酵素活性圖騰，其中有一條酸性b-lactamase的條紋，和數條同屬於鹼性b-lactamase的階梯狀條紋(ladder-shapped bands)，以S. maltophilia KH菌株為代表。利用基因替換策略構築取得KH菌株的L2同源突變株。從KH和KHL2-菌株的IEF和native-PAGE上之酵素活性表現，我們證實鹼性階梯條紋狀的b-lactamases是由L2基因所表現的。在定量的部份，我們提出CENTA-clavulanic acid方法，來單獨定量S. maltophilia 的cell extracts中，被誘發的L1或L2?涀-lactamase。分析L1和L2酵素，對於nitrocefin和CENTA的酵素動力學分析，以及對clavulanic acid抑制劑的影響。CENTA對於L1是一種穩定的受質，但較難被L2酵素水解。L2酵素活性容易被10 mM 的clavulanic acid抑制；相反的，L1酵素活性則不受clavulanic acid影響。因此，我們根據上述L1和L2對CENTA的水解能力和被clavulanic acid的抑制效果，發展一種定量方法，能單獨定量破菌後的extracts中，L1酵素活性。把破菌收集好的cell extracts預先加入clavulanic acid作用10分鐘，接著利用分光光度計測量L1酵素活性，監測對受質CENTA的降解情形。 Stenotrophomonas maltophilia has two types of b-lactamase, L1 and L2. Both b-lactamases are co-inducibly expressed. To characterize the L1 and L2 b-lactamases of S. maltophilia, two parts are included in this study, in respect of quality and quantity. Firstly, to qualitatively characterize these b-lactamases, the zymogram pattern of isoelectric focusing electrophoresis (IEF) is generally referred. The zymogram of twenty S. maltophilia isolates were determined by IEF and two types of zymogram were revealed. In addition to the typical zymogram type of an acid b-lactamase and a basic one, a peculiar IEF zymogram pattern with an acid?涀-lactamase and several basic (ladder-shapped) b-lactamases was identified, like S. maltophilia KH did. The isogenic L2 mutant of strain KH was constructed by gene replacement strategy. From IEF and native-PAGE zymograms of strains KH and KHL2-, we demonstrate that the basic b-lactamases were encoded by the same L2 gene. Secondly, to differentially quantify the induced L1 and L2 b-lactamases in the cell extracts of S. maltophilia, a CENTA-clavulanic acid method is proposed. The L1 and L2 enzymes were characterized, including the enzyme kinetic assay against nitrocefin and CENTA, as well as the inhibitory effect toward clavulanic acid. CENTA was shown to be a susceptible substrate for L1, although it was hardly hydrolyzed by L2 enzyme. The L2 activity was sensitive to 10 mM clavulanic acid. In contrast, the L1 activity was unaffected by clavulanic acid. A differential quantification method for L1 enzyme activity in the induced sonicated extracts was developed based on their discrepant characteristics of L1 and L2 in the aspects of the hydrolysis ability against CENTA and the inhibitory effect by clavulanic acid. The prepared sonicated extracts were pre-incubated with clavulanic acid for 10 min, and then the activity of L1 enzyme was determined by spectrophotometrically monitoring the degradation of substrate CENTA.
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