金清，肖明

上海師范大學(xué)生命與環(huán)境科學(xué)學(xué)院，上海 200234

抗菌肽(antimicrobial peptide)具有抗菌譜廣、作用迅速強(qiáng)大、不易產(chǎn)生耐藥性等優(yōu)點(diǎn)，在醫(yī)藥、化妝品、食品工業(yè)中具有廣闊的應(yīng)用前景[1-3]。表面活性素(surfactin)、伊枯草菌素(iturin)和豐原素(fengycin)是芽胞桿菌產(chǎn)生的主要活性物質(zhì)，可抑制農(nóng)作物病害[4-6]。近年來發(fā)現(xiàn)這3類物質(zhì)在醫(yī)藥領(lǐng)域有重要應(yīng)用前景，具有抗病毒[5,7]、抗腫瘤[8-12]、抗細(xì)菌[13-15]、抗真菌[16-18]、抗支原體[19]、抗炎[20]作用，展示出極大的臨床應(yīng)用潛力[21]，是一種新型抗菌肽[22-26]，但人們對它們在醫(yī)藥領(lǐng)域中的研究進(jìn)展所知甚少。因此，本文就表面活性素、伊枯草菌素和豐原素的發(fā)現(xiàn)歷史、結(jié)構(gòu)特點(diǎn)、作用機(jī)制、生物合成及應(yīng)用價(jià)值進(jìn)行綜述。

1 發(fā)現(xiàn)歷史

表面活性素于1968年首次在枯草芽胞桿菌(Bacillussubtilis)的培養(yǎng)液中發(fā)現(xiàn)，由4種異構(gòu)體(枯草菌素A～D)組成，表現(xiàn)出各種生理活性，包括作為纖維蛋白凝固抑制劑和細(xì)胞裂解物[27]。伊枯草菌素于1957年首次在從土壤中分離到的枯草芽胞桿菌中發(fā)現(xiàn)[28]，豐原素則首次在枯草芽胞桿菌F29-3中發(fā)現(xiàn)[29]。

表面活性素、伊枯草菌素和豐原素具有廣泛的工業(yè)應(yīng)用價(jià)值，如制造洗滌劑[30-31]、抑制植物病害[32-34]、提高油采收率[31]等。近年來，這3種新型抗菌肽的醫(yī)療應(yīng)用研究也取得重大突破，在醫(yī)藥領(lǐng)域中扮演著越來越重要的角色。

與其他菌肽的生產(chǎn)類似，這3種抗菌肽也是通過發(fā)酵生產(chǎn)。目前研究者從各方面努力，包括優(yōu)良菌種選育[5]、發(fā)酵過程優(yōu)化[35-36]、高效分離純化方式的探索等來提高產(chǎn)率[37-38]，取得了一系列成果。

2 結(jié)構(gòu)特點(diǎn)及作用機(jī)制

表面活性素、伊枯草菌素和豐原素是一類主要由革蘭陽性芽胞桿菌產(chǎn)生的抗菌肽，一般由1個(gè)疏水的脂肪烴鏈以羧基、羰基或氨基與親水的由7～10個(gè)氨基酸構(gòu)成的肽鏈以酰胺鍵或內(nèi)酯的形式連接構(gòu)成環(huán)肽，其結(jié)構(gòu)上的差異主要在于脂肪鏈中碳原子的個(gè)數(shù)、氨基酸的種類及脂肪酸鏈與肽鏈連接鍵的不同(圖1)。

A: Surfactin is formed by β-hydroxy fatty acids (12-17 carbon atoms) with peptide chain through lactone bonds.The molecular peptide chain is composed of seven α-amino acids.B: Iturin is a ring formed by β-amino fatty acids (14-17 carbon atoms) with peptide chain through amide bond.The molecular peptide chain is composed of seven α-amino acids.C: Fengycin is composed of β-hydroxy fatty acids (14-17 carbon atoms) and peptide chain through lactone bond.Fengycin is different from surfactin and iturin.The macrocyclic ring is composed of peptide chain of ten α-amino acids.The Arabic numerals represent the position of the amino acid.

圖1表面活性素、伊枯草菌素和豐原素的分子結(jié)構(gòu)
Fig.1Molecularstructuresofsurfactin,iturinandfengycin

表面活性素由β-羥基脂肪酸與肽鏈以內(nèi)酯鍵結(jié)合而成[39-41]。多數(shù)細(xì)菌代謝產(chǎn)生的表面活性素的肽鏈為七元肽，即分子中的肽鏈由7個(gè)α-氨基酸組成，再與帶有12～17個(gè)碳原子的β-羥基脂肪酸構(gòu)成一個(gè)大內(nèi)酯環(huán)，相對分子質(zhì)量(Mr)為 1 000 左右。多肽中典型的氨基酸順序?yàn)閇27,42-43]：L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu(圖1A)。表面活性素家族常見的有surfactin A[44]、surfactin C[44]、lichenysin[44-46]、pumilacidin等[46]。

伊枯草菌素由β-氨基脂肪酸與肽鏈以酰胺鍵成環(huán)[47]。多數(shù)細(xì)菌代謝產(chǎn)生的伊枯草菌素的肽鏈也為七元肽，與帶有14～17個(gè)碳原子的β-氨基脂肪酸構(gòu)成一個(gè)大環(huán)，Mr 為 1 000 左右。多肽中典型的氨基酸順序?yàn)閇47-48]：L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser(圖1B)。伊枯草菌素家族種類較多，如iturin A[49-54]、iturin C[49,51]、iturin D[51]、iturin E[51-53]、iturin F[53]、桿菌霉素(bacillomycin)D[49-52]、bacillomycin F[49-51]、bacillomycin L[49-51]、mixirins[9,51-52]、mojavensin[52]、抗霉枯草菌素(mycosubtilin)[49-51,53-54]和subtulene[51]等。

豐原素由β-羥基脂肪酸與肽鏈以內(nèi)酯鍵結(jié)合而成。與表面活性素不同的是，豐原素的大環(huán)由肽鏈自行構(gòu)成，β-羥基脂肪酸并不參與[54-55]。多數(shù)細(xì)菌代謝產(chǎn)生的豐原素的肽環(huán)為十元肽，第三位的D-Tyr和最后一位的L-Ile以內(nèi)酯鍵結(jié)合成環(huán)，再與帶有14～17個(gè)碳原子的β-羥基脂肪酸以酯鍵結(jié)合，Mr 為 1 500 左右[54-56]。多肽中典型的氨基酸順序?yàn)閇54-58]：L-Glu-D-Orn-D-Tyr-D-allo-Thr-L-Glu-D-Ala-L-Pro-L-Gln-L-Tyr-L-Ile(圖1C)。豐原素家族常見的有fengycin A[54-58]、fengycin B[54-55,57-58]、fengycin C[54-55,58]、fengycin S[55]和制磷脂菌素(plipastatin)[54-55,57-59]。

表面活性素可通過溶解和破壞細(xì)胞膜發(fā)揮抗菌作用，其與膜中的極性頭部和疏水?；溇上嗷プ饔?，高濃度時(shí)引起磷脂雙分子層高度不穩(wěn)定，中等濃度時(shí)在細(xì)胞膜中形成離子傳導(dǎo)孔與Ca2+結(jié)合，有助于膜滲透；其還可通過有機(jī)屏障驅(qū)動(dòng)其他單價(jià)和二價(jià)陽離子，導(dǎo)致cAMP磷酸二酯酶活性被抑制[60-62]。伊枯草菌素能迅速引起細(xì)胞膜損傷，細(xì)胞通透性改變，細(xì)胞內(nèi)物質(zhì)外泄，從而達(dá)到抑制真菌孢子萌發(fā)、菌絲體生長的效果；其還可與細(xì)胞內(nèi)靶點(diǎn)(如細(xì)胞DNA)相互作用，破壞細(xì)胞內(nèi)鈣穩(wěn)態(tài)，導(dǎo)致細(xì)胞死亡[63-65]。豐原素對細(xì)胞膜有顯著擾亂作用，通過基于豐原素濃度的兩態(tài)躍遷過程使磷脂雙分子層損傷，從而導(dǎo)致細(xì)胞死亡。高濃度時(shí)，豐原素作為洗滌劑促進(jìn)細(xì)胞膜溶解，該過程主要由其對脂質(zhì)雙分子層的吸引力等物理化學(xué)性質(zhì)所驅(qū)動(dòng)；低濃度時(shí)，豐原素聚合形成孔洞，導(dǎo)致膜的滲透率發(fā)生變化[61,66-68]。

3 生物合成

芽胞桿菌能分泌多種肽類及由肽類衍生的抗菌活性物質(zhì)，按合成途徑分為核糖體肽和非核糖體肽[4]。非核糖體肽Mr較小，一般為 3 000 以下，通過非核糖體肽鏈合成酶(non-ribosomal peptide synthetase，NRPS)來合成，多發(fā)生于菌體生長停止之后；而核糖體肽Mr較大，大多于菌體快速生長時(shí)期合成[4,69]。

非核糖體途徑合成的脂肽類抗菌活性物質(zhì)合成于菌體生長停止之后，屬于微生物次級代謝產(chǎn)物，能繞開核糖體，不以mRNA 為模板，也不需tRNA 作為運(yùn)載工具，而是通過NRPS識別特定的氨基酸并連接成多肽鏈[4,70]。表面活性素、伊枯草菌素和豐原素就是NRPS合成的次級代謝產(chǎn)物，由胞內(nèi)游離氨基酸經(jīng)活化后結(jié)合到合成酶系特定的結(jié)構(gòu)域，從而實(shí)現(xiàn)肽鏈的延長和環(huán)化[71-72](圖2)。

NRPS是由多個(gè)功能模塊組成的復(fù)合酶體系，按功能分為必不可少模塊和可供選擇模塊，各模塊負(fù)責(zé)活化不同的氨基酸使肽鏈延長。必不可少模塊有氨基酸激活結(jié)構(gòu)域(amino acid activating domain)、氨酰載體結(jié)構(gòu)域(acyl carrier)、縮合結(jié)構(gòu)域(condensation domain)和硫酯酶結(jié)構(gòu)域(thioesterase domain)。氨基酸激活結(jié)構(gòu)域由550個(gè)氨基酸殘基構(gòu)成，負(fù)責(zé)識別和腺苷?；囟ǖ陌被幔址Q為腺苷?；Y(jié)構(gòu)域(adenylation domain)；氨酰載體結(jié)構(gòu)域負(fù)責(zé)運(yùn)載氨基酸，又稱為巰基化結(jié)構(gòu)域T或肽酰載體蛋白( peptidyl carrier protein，PCP)；縮合結(jié)構(gòu)域負(fù)責(zé)肽鍵形成；硫酯酶結(jié)構(gòu)域負(fù)責(zé)釋放多肽和肽的環(huán)化?？晒┻x擇模塊包括環(huán)化結(jié)構(gòu)域(cyclization domain)、甲基轉(zhuǎn)移酶結(jié)構(gòu)域(methyltransferase domain)、差向異構(gòu)酶結(jié)構(gòu)域(epimerization domain)等。差向異構(gòu)酶結(jié)構(gòu)域負(fù)責(zé)將被激活的L-氨基酸轉(zhuǎn)化為D-氨基酸。全酶由多個(gè)模塊按特定的空間順序排列而成，模塊的數(shù)量、種類及排列次序決定了氨基酸種類、順序和最終產(chǎn)物肽鏈的長短[73-74](圖2)。

A: amino acid activating domain；PCP: peptidyl carrier protein；C: condensation domain；E: epimerization domain；TE: thioesterase domain；MCT: monocarboxylate transporter.

圖2表面活性素、伊枯草菌素和豐原素的代謝通路
Fig.2Metabolicpathwaysofsurfactin,iturinandfengycin

NRPS合成多肽的一般過程為：首先，氨基酸激活結(jié)構(gòu)域選擇并結(jié)合特定的氨基酸，在ATP 作用下激活氨基酸(腺苷化)，形成氨酰腺苷酸。氨酰腺苷酸與氨酰載體結(jié)構(gòu)域上的4-磷酸泛酰巰基輔基以共價(jià)鍵形式結(jié)合，形成氨酰載體復(fù)合體。然后，攜帶有活化氨基酸的氨酰載體與縮合結(jié)構(gòu)域特定部位結(jié)合，在其合成酶的作用下，按相鄰合成酶各組成模塊的順序依次向前形成肽鍵。肽鍵形成后，進(jìn)入下一循環(huán)，即肽鏈延伸過程。最后，硫酯酶結(jié)構(gòu)域終止肽鏈合成，將肽鏈從磷酸泛酰巰基輔基釋放下來，并進(jìn)行環(huán)化[75-77]。

3.1 表面活性素

表面活性素合成酶包括3 個(gè)亞單位：SrfA、ComA(SrfB)和SrfC[4,26,78-86]。編碼表面活性素合成酶的基因srfA-A、srfA-B、srfA-C共同構(gòu)成srfA操縱子(長約25 kb)，分別負(fù)責(zé)編碼Mr為 401 000、402 000 和 144 000 的3個(gè)合成單體酶E1A、E1B和E2。srfA-C編碼的第一個(gè)硫酯酶結(jié)構(gòu)域負(fù)責(zé)終止肽鏈延伸并釋放多肽產(chǎn)物(圖2A)。

sfp基因(約4.5 kb)是參與表面活性素合成的第二調(diào)控元件，位于srfA操縱子下游30.5 kb處，與srfA-D末端相距約4 kb。sfp基因編碼的SFP 酶具有編碼磷酸泛酰巰基乙胺基轉(zhuǎn)移酶(phosphopantetheinyl transferase，PPTase)的功能，可催化非核糖體肽和載鐵蛋白前體的形成，并借此將表面活性素合成酶激活，屬PPTase超家族。有研究認(rèn)為sfp和srfA-C-TE基因共同發(fā)揮主導(dǎo)作用，還有研究認(rèn)為sfp基因在表面活性素合成中有更直接的調(diào)節(jié)作用[85]。在基因圖譜中，srfA與sfp相鄰，而與srfB相隔，srfB功能等同于comA基因，即激活srfA啟動(dòng)子的轉(zhuǎn)錄。

SrfA-A負(fù)責(zé)組裝前3位氨基酸；SrfA-B負(fù)責(zé)組裝第4～6位氨基酸；SrfA-C負(fù)責(zé)組裝第7位氨基酸，并將羥基脂肪酸轉(zhuǎn)移到蛋白SrfA-A上。

3.2 伊枯草菌素

伊枯草菌素通過聚酮合酶(polyketide synthase，PKS)-NRPS雜合體系合成[78,87-88]。

編碼伊枯草菌素合成酶的基因ituD、ituA、ituB和ituC共同構(gòu)成itu操縱子(長約38 kb)。ituD負(fù)責(zé)編碼丙二酰輔酶A轉(zhuǎn)酰酶，對伊枯草菌素的形成起重要作用，被破壞可導(dǎo)致iturin A產(chǎn)生特異性缺陷；其還可調(diào)控伊枯草菌素產(chǎn)量，可能與脂肪酸合成有關(guān)。ituA編碼Mr為 449 000 的蛋白，與脂肪酸合成酶、氨基酸轉(zhuǎn)移酶和肽合成酶具有同源性，可能與β-氨基脂肪酸形成有關(guān)；部分基因與聚酮合酶相關(guān)，其編碼的聚酮合酶參與脂肽類分子碳鏈合成的最終步驟，并為肽段部分氨基酸分子的組裝做好準(zhǔn)備。ituB編碼Mr為 609 000 的具有4個(gè)氨基酸腺苷酸化結(jié)構(gòu)域的肽合成酶。ituC編碼Mr為 297 000 的另一種肽合成酶，具有2個(gè)腺苷酸化結(jié)構(gòu)域、1個(gè)差向異構(gòu)酶結(jié)構(gòu)域和硫酯酶結(jié)構(gòu)域，這可能有助于肽環(huán)化。ItuA負(fù)責(zé)組裝第1位氨基酸，ItuB負(fù)責(zé)組裝第2～5位氨基酸，ItuC負(fù)責(zé)組裝第6和7位氨基酸(圖2B)。

3.3 豐原素

編碼豐原素合成酶的基因fenC、fenD、fenE、fenA、fenB共同構(gòu)成fen操縱子(長約37 kb)，分別編碼5個(gè)亞基，即5個(gè)單體酶——FenC、FenD、FenE、FenA和FenB。每個(gè)單體酶一般含1～3個(gè)氨基酸激活模塊，且每個(gè)模塊具有接受特定氨基酸及形成相應(yīng)肽鍵的功能。豐原素合成從肽單體酶FenC開始，途經(jīng)FenD、FenE和FenA，終止于肽單體酶FenB[60,89-91]。

FenC的Mr為 287 000，負(fù)責(zé)活化并組裝第1和2位氨基酸；FenD的Mr為 290 000，負(fù)責(zé)活化第3和4位氨基酸；FenE的Mr為 286 000，活化第5和6位氨基酸；FenA的Mr為 406 000，負(fù)責(zé)活化第7～9位氨基酸；FenB的Mr為 146 000，負(fù)責(zé)組裝最后一位氨基酸。FenB含有能中斷肽鏈合成的硫酯酶結(jié)構(gòu)域，具有釋放肽鏈的功能，在其下游也發(fā)現(xiàn)了與脂肪酸代謝有關(guān)的基因(圖2C)。

4 應(yīng)用價(jià)值

研究表明，表面活性素對新城疫病毒(Newcastle disease virus，NDV)不僅具有直接滅活作用，還可阻斷其對細(xì)胞的吸附；隨著濃度升高還可抑制NDV的生物合成，具有明顯的量-效關(guān)系。表面活性素的治療指數(shù)為 12.16，高于病毒唑的 9.70，有望開發(fā)成為一種有效的抗病毒藥物，這對養(yǎng)殖業(yè)生產(chǎn)及防治NDV感染所致人類疾病具有重要意義[7]。另有研究表明，表面活性素對人乳腺癌細(xì)胞Michigan Cancer Foundation-7(MCF-7)表現(xiàn)出較強(qiáng)的抑制作用。噻唑藍(lán)(methylthiazolyldiphenyl-tetrazolium bromide，MTT)法顯示，表面活性素能抑制MCF-7增殖，呈現(xiàn)濃度與時(shí)間依賴關(guān)系，細(xì)胞處理24 h后的半抑制濃度(half maximal inhibitory concentration，IC50)為10 μg/mL。隨著發(fā)酵期間表面活性素含量升高(0.3～48.2 mg/kg)，SEC(surfactin extractions of cheonggukjang)(100 μg/mL)的抗癌活性逐漸升高(20.3%～54.7%)[8]。表面活性素除具有抗病毒、抗腫瘤作用，還可抗細(xì)菌[13-15]、抗真菌[16-17]、抗支原體[19]、抗炎[20]，抗菌譜較廣，同時(shí)具有不易產(chǎn)生耐藥性、可被動(dòng)物消化酶降解、無殘留等優(yōu)點(diǎn)，均顯示其應(yīng)用于醫(yī)藥業(yè)的潛力。

伊枯草菌素具有強(qiáng)烈的抗真菌、抗腫瘤作用，還具有低毒、低殘留、低過敏性和抗菌譜廣的特點(diǎn)，是一種潛在的具有極大開發(fā)應(yīng)用價(jià)值的醫(yī)藥產(chǎn)品。研究表明，伊枯草菌素對紅色毛癬菌具有較強(qiáng)抑制作用[18]。Mixirins A、B和C具有抗腫瘤作用，可抑制人結(jié)腸癌細(xì)胞HCT-116的生長，其IC50分別為 0.68、1.6 和 1.3 μg/mL[9]。

豐原素具有顯著的抗腫瘤效果，對腫瘤細(xì)胞及腫瘤組織具有較好的選擇性，對腫瘤凋亡相關(guān)蛋白有明顯影響，而對正常造血系統(tǒng)和白細(xì)胞無影響，為尋找新型抗腫瘤藥物提供了方向。研究表明，與對照組相比，豐原素濃度達(dá)20 μg/mL時(shí)即可抑制人結(jié)腸癌細(xì)胞HT-29的生長，并呈濃度與時(shí)間依賴關(guān)系。蛋白免疫印跡分析發(fā)現(xiàn)，加入豐原素后，HT-29細(xì)胞中的Bax、Caspase-3和Caspase-6表達(dá)明顯增加，而Bcl-2和CDK4/cyclin D1表達(dá)降低，表明豐原素可通過影響人HT-29細(xì)胞周期的G1期停滯和誘導(dǎo)細(xì)胞凋亡而對其產(chǎn)生抑制作用[10]。此外，大量研究也表明豐原素可抑制人結(jié)腸癌HCT-15細(xì)胞增殖[11]，調(diào)節(jié)人肺癌95D細(xì)胞G0/G1期引起細(xì)胞周期停滯和促進(jìn)細(xì)胞凋亡來抑制癌細(xì)胞生長[12]，顯示其具有極大的抗腫瘤潛力。

綜上所述，抗菌肽是極具價(jià)值的新一代抗菌藥物，能作為抗病原體藥物，并可能發(fā)展成為抗腫瘤藥物，在免疫調(diào)節(jié)、促進(jìn)傷口愈合等方面有應(yīng)用價(jià)值。多數(shù)文獻(xiàn)表明，表面活性素、伊枯草菌素和豐原素在醫(yī)療領(lǐng)域有著巨大價(jià)值與廣闊前景，具備低毒、抗菌譜廣、不易產(chǎn)生耐藥性等優(yōu)勢及工業(yè)化生產(chǎn)潛力，但臨床應(yīng)用還不廣泛。隨著對抗菌肽研究的不斷深入及技術(shù)的不斷改進(jìn)，如何將其應(yīng)用于臨床、應(yīng)用于人類醫(yī)療事業(yè)將成為研究的主要方向。

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