【摘要】：為提高豬溶菌酶(Sus scrofa lysozyme,SSL)的抗革蘭氏陰性菌活性,將其進行了不同蛋白酶的水解,選擇抗革蘭氏陰性菌效果最好的水解產(chǎn)物,利用凝膠過濾色譜和反相制備色譜進行分離,對其功能成分進行液質(zhì)聯(lián)用鑒定。對分離得到的物質(zhì)進行抗菌活性驗證和生物信息學(xué)的分析,并在此基礎(chǔ)上對抗菌物質(zhì)的殺菌機理進行了探討。結(jié)果表明,胰蛋白酶的水解產(chǎn)物具有較高的殺滅革蘭氏陰性菌的活性,進一步分離純化得到了具有抗革蘭氏陰性菌活性的六肽A-W-V-A-W-K。經(jīng)化學(xué)合成驗證,該六肽既保留了SSL的部分抗菌活性,也具備殺滅多種革蘭氏陰性菌的能力。進一步分析發(fā)現(xiàn)其位于SSL分子C端的一個螺旋-回環(huán)-螺旋的結(jié)構(gòu)中,并由此推測其殺菌機理是通過改變細(xì)胞膜的滲透性,進而使細(xì)胞內(nèi)溶物流出而造成細(xì)胞死亡,而抗菌實驗也驗證了這一推測。該抗菌肽的發(fā)現(xiàn)為后續(xù)提高SSL的抗菌活性提供了理論依據(jù)。
[Abstract]:In order to improve the activity of porcine lysozyme (Sus scrofa lysozyme,SSL against Gram-negative bacteria, different proteases were hydrolyzed. The functional components were separated by gel filtration chromatography and reversed phase preparation chromatography. The antimicrobial activity and bioinformatics of the separated substances were verified and the bactericidal mechanism of the antibacterial substances was discussed. The results showed that the hydrolysate of trypsin had higher activity to kill Gram-negative bacteria, and the six peptide A-W-V-A-W-Kwith the activity of anti-Gram-negative bacteria was obtained by further isolation and purification. It was proved by chemical synthesis that the six peptides not only retained some of the antibacterial activity of SSL, but also had the ability to kill many Gram-negative bacteria. Further analysis revealed that it was located in a helix-loop-helix structure at the C-terminal of SSL molecule, and its bactericidal mechanism was presumed to result in cell death by changing the permeability of the cell membrane and resulting in the release of intracellular solutes. Antimicrobial experiments also confirmed the hypothesis. The discovery of the antimicrobial peptide provides a theoretical basis for improving the antibacterial activity of SSL.
【作者單位】： 江南大學(xué)工業(yè)生物技術(shù)教育部重點實驗室;江南大學(xué)糧食發(fā)酵工藝與技術(shù)國家工程實驗室;江南大學(xué)生物工程學(xué)院;
【基金】：國家重點基礎(chǔ)研究發(fā)展計劃(973計劃)(No.2013CB733602) 中央高�；究蒲袠I(yè)務(wù)費專項資金資助(No.JUSRP51302A) 江蘇高校優(yōu)勢學(xué)科建設(shè)工程項目資助~~
【分類號】：Q51
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本文編號：2386266