新型抗菌肽——表面活性素、伊枯草菌素和丰原素

金清，肖明

上海师范大学生命与环境科学学院，上海 200234

抗菌肽(antimicrobial peptide)具有抗菌谱广、作用迅速强大、不易产生耐药性等优点，在医药、化妆品、食品工业中具有广阔的应用前景[1-3]。表面活性素(surfactin)、伊枯草菌素(iturin)和丰原素(fengycin)是芽胞杆菌产生的主要活性物质，可抑制农作物病害[4-6]。近年来发现这3类物质在医药领域有重要应用前景，具有抗病毒[5,7]、抗肿瘤[8-12]、抗细菌[13-15]、抗真菌[16-18]、抗支原体[19]、抗炎[20]作用，展示出极大的临床应用潜力[21]，是一种新型抗菌肽[22-26]，但人们对它们在医药领域中的研究进展所知甚少。因此，本文就表面活性素、伊枯草菌素和丰原素的发现历史、结构特点、作用机制、生物合成及应用价值进行综述。

1 发现历史

表面活性素于1968年首次在枯草芽胞杆菌(Bacillussubtilis)的培养液中发现，由4种异构体(枯草菌素A～D)组成，表现出各种生理活性，包括作为纤维蛋白凝固抑制剂和细胞裂解物[27]。伊枯草菌素于1957年首次在从土壤中分离到的枯草芽胞杆菌中发现[28]，丰原素则首次在枯草芽胞杆菌F29-3中发现[29]。

表面活性素、伊枯草菌素和丰原素具有广泛的工业应用价值，如制造洗涤剂[30-31]、抑制植物病害[32-34]、提高油采收率[31]等。近年来，这3种新型抗菌肽的医疗应用研究也取得重大突破，在医药领域中扮演着越来越重要的角色。

与其他菌肽的生产类似，这3种抗菌肽也是通过发酵生产。目前研究者从各方面努力，包括优良菌种选育[5]、发酵过程优化[35-36]、高效分离纯化方式的探索等来提高产率[37-38]，取得了一系列成果。

2 结构特点及作用机制

表面活性素、伊枯草菌素和丰原素是一类主要由革兰阳性芽胞杆菌产生的抗菌肽，一般由1个疏水的脂肪烃链以羧基、羰基或氨基与亲水的由7～10个氨基酸构成的肽链以酰胺键或内酯的形式连接构成环肽，其结构上的差异主要在于脂肪链中碳原子的个数、氨基酸的种类及脂肪酸链与肽链连接键的不同(图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.

图1表面活性素、伊枯草菌素和丰原素的分子结构
Fig.1Molecularstructuresofsurfactin,iturinandfengycin

表面活性素由β-羟基脂肪酸与肽链以内酯键结合而成[39-41]。多数细菌代谢产生的表面活性素的肽链为七元肽，即分子中的肽链由7个α-氨基酸组成，再与带有12～17个碳原子的β-羟基脂肪酸构成一个大内酯环，相对分子质量(Mr)为 1 000 左右。多肽中典型的氨基酸顺序为[27,42-43]：L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu(图1A)。表面活性素家族常见的有surfactin A[44]、surfactin C[44]、lichenysin[44-46]、pumilacidin等[46]。

伊枯草菌素由β-氨基脂肪酸与肽链以酰胺键成环[47]。多数细菌代谢产生的伊枯草菌素的肽链也为七元肽，与带有14～17个碳原子的β-氨基脂肪酸构成一个大环，Mr 为 1 000 左右。多肽中典型的氨基酸顺序为[47-48]：L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser(图1B)。伊枯草菌素家族种类较多，如iturin A[49-54]、iturin C[49,51]、iturin D[51]、iturin E[51-53]、iturin F[53]、杆菌霉素(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。

丰原素由β-羟基脂肪酸与肽链以内酯键结合而成。与表面活性素不同的是，丰原素的大环由肽链自行构成，β-羟基脂肪酸并不参与[54-55]。多数细菌代谢产生的丰原素的肽环为十元肽，第三位的D-Tyr和最后一位的L-Ile以内酯键结合成环，再与带有14～17个碳原子的β-羟基脂肪酸以酯键结合，Mr 为 1 500 左右[54-56]。多肽中典型的氨基酸顺序为[54-58]：L-Glu-D-Orn-D-Tyr-D-allo-Thr-L-Glu-D-Ala-L-Pro-L-Gln-L-Tyr-L-Ile(图1C)。丰原素家族常见的有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]。

表面活性素可通过溶解和破坏细胞膜发挥抗菌作用，其与膜中的极性头部和疏水酰基链均可相互作用，高浓度时引起磷脂双分子层高度不稳定，中等浓度时在细胞膜中形成离子传导孔与Ca2+结合，有助于膜渗透；其还可通过有机屏障驱动其他单价和二价阳离子，导致cAMP磷酸二酯酶活性被抑制[60-62]。伊枯草菌素能迅速引起细胞膜损伤，细胞通透性改变，细胞内物质外泄，从而达到抑制真菌孢子萌发、菌丝体生长的效果；其还可与细胞内靶点(如细胞DNA)相互作用，破坏细胞内钙稳态，导致细胞死亡[63-65]。丰原素对细胞膜有显著扰乱作用，通过基于丰原素浓度的两态跃迁过程使磷脂双分子层损伤，从而导致细胞死亡。高浓度时，丰原素作为洗涤剂促进细胞膜溶解，该过程主要由其对脂质双分子层的吸引力等物理化学性质所驱动；低浓度时，丰原素聚合形成孔洞，导致膜的渗透率发生变化[61,66-68]。

3 生物合成

芽胞杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质，按合成途径分为核糖体肽和非核糖体肽[4]。非核糖体肽Mr较小，一般为 3 000 以下，通过非核糖体肽链合成酶(non-ribosomal peptide synthetase，NRPS)来合成，多发生于菌体生长停止之后；而核糖体肽Mr较大，大多于菌体快速生长时期合成[4,69]。

非核糖体途径合成的脂肽类抗菌活性物质合成于菌体生长停止之后，属于微生物次级代谢产物，能绕开核糖体，不以mRNA 为模板，也不需tRNA 作为运载工具，而是通过NRPS识别特定的氨基酸并连接成多肽链[4,70]。表面活性素、伊枯草菌素和丰原素就是NRPS合成的次级代谢产物，由胞内游离氨基酸经活化后结合到合成酶系特定的结构域，从而实现肽链的延长和环化[71-72](图2)。

NRPS是由多个功能模块组成的复合酶体系，按功能分为必不可少模块和可供选择模块，各模块负责活化不同的氨基酸使肽链延长。必不可少模块有氨基酸激活结构域(amino acid activating domain)、氨酰载体结构域(acyl carrier)、缩合结构域(condensation domain)和硫酯酶结构域(thioesterase domain)。氨基酸激活结构域由550个氨基酸残基构成，负责识别和腺苷酰化特定的氨基酸，又称为腺苷酰化结构域(adenylation domain)；氨酰载体结构域负责运载氨基酸，又称为巯基化结构域T或肽酰载体蛋白( peptidyl carrier protein，PCP)；缩合结构域负责肽键形成；硫酯酶结构域负责释放多肽和肽的环化。可供选择模块包括环化结构域(cyclization domain)、甲基转移酶结构域(methyltransferase domain)、差向异构酶结构域(epimerization domain)等。差向异构酶结构域负责将被激活的L-氨基酸转化为D-氨基酸。全酶由多个模块按特定的空间顺序排列而成，模块的数量、种类及排列次序决定了氨基酸种类、顺序和最终产物肽链的长短[73-74](图2)。

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

图2表面活性素、伊枯草菌素和丰原素的代谢通路
Fig.2Metabolicpathwaysofsurfactin,iturinandfengycin

NRPS合成多肽的一般过程为：首先，氨基酸激活结构域选择并结合特定的氨基酸，在ATP 作用下激活氨基酸(腺苷化)，形成氨酰腺苷酸。氨酰腺苷酸与氨酰载体结构域上的4-磷酸泛酰巯基辅基以共价键形式结合，形成氨酰载体复合体。然后，携带有活化氨基酸的氨酰载体与缩合结构域特定部位结合，在其合成酶的作用下，按相邻合成酶各组成模块的顺序依次向前形成肽键。肽键形成后，进入下一循环，即肽链延伸过程。最后，硫酯酶结构域终止肽链合成，将肽链从磷酸泛酰巯基辅基释放下来，并进行环化[75-77]。

3.1 表面活性素

表面活性素合成酶包括3 个亚单位：SrfA、ComA(SrfB)和SrfC[4,26,78-86]。编码表面活性素合成酶的基因srfA-A、srfA-B、srfA-C共同构成srfA操纵子(长约25 kb)，分别负责编码Mr为 401 000、402 000 和 144 000 的3个合成单体酶E1A、E1B和E2。srfA-C编码的第一个硫酯酶结构域负责终止肽链延伸并释放多肽产物(图2A)。

sfp基因(约4.5 kb)是参与表面活性素合成的第二调控元件，位于srfA操纵子下游30.5 kb处，与srfA-D末端相距约4 kb。sfp基因编码的SFP 酶具有编码磷酸泛酰巯基乙胺基转移酶(phosphopantetheinyl transferase，PPTase)的功能，可催化非核糖体肽和载铁蛋白前体的形成，并借此将表面活性素合成酶激活，属PPTase超家族。有研究认为sfp和srfA-C-TE基因共同发挥主导作用，还有研究认为sfp基因在表面活性素合成中有更直接的调节作用[85]。在基因图谱中，srfA与sfp相邻，而与srfB相隔，srfB功能等同于comA基因，即激活srfA启动子的转录。

SrfA-A负责组装前3位氨基酸；SrfA-B负责组装第4～6位氨基酸；SrfA-C负责组装第7位氨基酸，并将羟基脂肪酸转移到蛋白SrfA-A上。

3.2 伊枯草菌素

伊枯草菌素通过聚酮合酶(polyketide synthase，PKS)-NRPS杂合体系合成[78,87-88]。

编码伊枯草菌素合成酶的基因ituD、ituA、ituB和ituC共同构成itu操纵子(长约38 kb)。ituD负责编码丙二酰辅酶A转酰酶，对伊枯草菌素的形成起重要作用，被破坏可导致iturin A产生特异性缺陷；其还可调控伊枯草菌素产量，可能与脂肪酸合成有关。ituA编码Mr为 449 000 的蛋白，与脂肪酸合成酶、氨基酸转移酶和肽合成酶具有同源性，可能与β-氨基脂肪酸形成有关；部分基因与聚酮合酶相关，其编码的聚酮合酶参与脂肽类分子碳链合成的最终步骤，并为肽段部分氨基酸分子的组装做好准备。ituB编码Mr为 609 000 的具有4个氨基酸腺苷酸化结构域的肽合成酶。ituC编码Mr为 297 000 的另一种肽合成酶，具有2个腺苷酸化结构域、1个差向异构酶结构域和硫酯酶结构域，这可能有助于肽环化。ItuA负责组装第1位氨基酸，ItuB负责组装第2～5位氨基酸，ItuC负责组装第6和7位氨基酸(图2B)。

3.3 丰原素

编码丰原素合成酶的基因fenC、fenD、fenE、fenA、fenB共同构成fen操纵子(长约37 kb)，分别编码5个亚基，即5个单体酶——FenC、FenD、FenE、FenA和FenB。每个单体酶一般含1～3个氨基酸激活模块，且每个模块具有接受特定氨基酸及形成相应肽键的功能。丰原素合成从肽单体酶FenC开始，途经FenD、FenE和FenA，终止于肽单体酶FenB[60,89-91]。

FenC的Mr为 287 000，负责活化并组装第1和2位氨基酸；FenD的Mr为 290 000，负责活化第3和4位氨基酸；FenE的Mr为 286 000，活化第5和6位氨基酸；FenA的Mr为 406 000，负责活化第7～9位氨基酸；FenB的Mr为 146 000，负责组装最后一位氨基酸。FenB含有能中断肽链合成的硫酯酶结构域，具有释放肽链的功能，在其下游也发现了与脂肪酸代谢有关的基因(图2C)。

4 应用价值

研究表明，表面活性素对新城疫病毒(Newcastle disease virus，NDV)不仅具有直接灭活作用，还可阻断其对细胞的吸附；随着浓度升高还可抑制NDV的生物合成，具有明显的量-效关系。表面活性素的治疗指数为 12.16，高于病毒唑的 9.70，有望开发成为一种有效的抗病毒药物，这对养殖业生产及防治NDV感染所致人类疾病具有重要意义[7]。另有研究表明，表面活性素对人乳腺癌细胞Michigan Cancer Foundation-7(MCF-7)表现出较强的抑制作用。噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide，MTT)法显示，表面活性素能抑制MCF-7增殖，呈现浓度与时间依赖关系，细胞处理24 h后的半抑制浓度(half maximal inhibitory concentration，IC50)为10 μg/mL。随着发酵期间表面活性素含量升高(0.3～48.2 mg/kg)，SEC(surfactin extractions of cheonggukjang)(100 μg/mL)的抗癌活性逐渐升高(20.3%～54.7%)[8]。表面活性素除具有抗病毒、抗肿瘤作用，还可抗细菌[13-15]、抗真菌[16-17]、抗支原体[19]、抗炎[20]，抗菌谱较广，同时具有不易产生耐药性、可被动物消化酶降解、无残留等优点，均显示其应用于医药业的潜力。

伊枯草菌素具有强烈的抗真菌、抗肿瘤作用，还具有低毒、低残留、低过敏性和抗菌谱广的特点，是一种潜在的具有极大开发应用价值的医药产品。研究表明，伊枯草菌素对红色毛癣菌具有较强抑制作用[18]。Mixirins A、B和C具有抗肿瘤作用，可抑制人结肠癌细胞HCT-116的生长，其IC50分别为 0.68、1.6 和 1.3 μg/mL[9]。

丰原素具有显著的抗肿瘤效果，对肿瘤细胞及肿瘤组织具有较好的选择性，对肿瘤凋亡相关蛋白有明显影响，而对正常造血系统和白细胞无影响，为寻找新型抗肿瘤药物提供了方向。研究表明，与对照组相比，丰原素浓度达20 μg/mL时即可抑制人结肠癌细胞HT-29的生长，并呈浓度与时间依赖关系。蛋白免疫印迹分析发现，加入丰原素后，HT-29细胞中的Bax、Caspase-3和Caspase-6表达明显增加，而Bcl-2和CDK4/cyclin D1表达降低，表明丰原素可通过影响人HT-29细胞周期的G1期停滞和诱导细胞凋亡而对其产生抑制作用[10]。此外，大量研究也表明丰原素可抑制人结肠癌HCT-15细胞增殖[11]，调节人肺癌95D细胞G0/G1期引起细胞周期停滞和促进细胞凋亡来抑制癌细胞生长[12]，显示其具有极大的抗肿瘤潜力。

综上所述，抗菌肽是极具价值的新一代抗菌药物，能作为抗病原体药物，并可能发展成为抗肿瘤药物，在免疫调节、促进伤口愈合等方面有应用价值。多数文献表明，表面活性素、伊枯草菌素和丰原素在医疗领域有着巨大价值与广阔前景，具备低毒、抗菌谱广、不易产生耐药性等优势及工业化生产潜力，但临床应用还不广泛。随着对抗菌肽研究的不断深入及技术的不断改进，如何将其应用于临床、应用于人类医疗事业将成为研究的主要方向。

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