篩選乳酸桿菌Lactobacillus casei ATCC 334基因組中具潛力的第二型抑菌素及其活性分析

Characterization of Putative Bacteriocin Identified from Lactobacillus casei ATCC 334

郭楊正(Yang-Cheng Kuo)

指導教授 ： 林志侯

國立清華大學/生命科學院/分子醫學研究所/博士(2013年)

https://doi.org/10.6843/NTHU.2013.00090

摘要

抑菌素(bacteriocin )是指細菌所生產的抗菌胜肽，它通常為帶正電及熱穩定的小分子胜肽。本文研究的為第二型抑菌素，常以原型胜肽(propeptide)的形式生產，其N-端常帶有雙甘胺酸(double-glycine) 的前導胜肽，可被特定蛋白辨認、修飾並運輸至細胞膜外，而形成具有活性的抑菌素。根據這些特性，我們利用PFAM與APD2等資料庫來搜尋相關的序列，發現在Lactobacillus casei ATCC 334這株基因全定序的乳酸菌中，帶有八個可能為抑菌素的序列，而此菌株本身未曾被發現會產生任何抑菌素。因此，我們利用異體表現系統，在大腸桿菌中表現、純化其中六種較具潛力的抑菌素，並使用膠體擴散測試法測試其活性。實驗結果顯示m2163 可以抑制部分乳酸菌(lactobacilli)以及李斯特菌(Listeria)； 而m2386則可抑制李斯特菌生長。這兩個胜肽皆具有熱穩定以及容易被蛋白脢所分解的特性。經由質譜分析，m2163會透過在序列4與24的半胱胺酸(Cysteine)形成雙硫鍵。而利用定點突變法，以絲胺酸(serine)取代半胱胺酸的實驗顯示，雙硫鍵的形成對於m2163的抑菌活性相當重要。以合成的m2163與m2386進行膜通透性實驗及二級結構分析顯示，合成m2163 在緩衝液即可呈現α-螺旋結構，且依然有抑菌活性，並可以增加膜的通透性; 恒溫滴定熱卡計實驗顯示，m2163對於帶負電的POPG微脂粒之親和力並不強。綜合以上實驗推測m2163可能會針對細菌表面上的特定蛋白或其他物質做結合，而進一步造成膜的通透性改變。另一方面，合成的m2386在緩衝液中依然呈現無結構狀態，必須在30 % TFE環境中才能呈現α-螺旋結構，合成的m2386可能因此喪失其抑菌活性，亦無法改變膜的通透性。

關鍵字

第二型抑菌素 ； 乳酸桿菌

並列摘要

Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334 and all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activities. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121◦C for 30 min, but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced. Both synthetic m2163 and m2386 behave as a random coil in aqueous solution whereas anα-helix is formed by both in the membrane mimic environment. However, the synthetic m2163 can also behave as anα-helix in a sodium phosphate buffer which would allow it to permeate into the cell membrane of sensitive bacteria and hence a corresponding bactericidal activity is detected. The ITC results indicated that the interaction between m2163 and POPG was weak. Therefore, we suspected that m2163 might have a specific target on the surface of sensitive bacteria. On the contrary, due to the presence of random coil conformation in sodium phosphate buffer, the bactericidal activity of synthetic m2386 is absent and no permeattion into the bacterial membrane is assumed.

並列關鍵字

Class II bacteriocin ； Lactobacillus casei ； Double-glycine leader peptide ； Heterologous expression

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