生物表面活性剂——表面活性素

生物表面活性剂是由微生物在一定条件下合成的具有表面活性的物质,具有对环境无毒、生物降解性能好等特性,广泛应用于洗涤剂、化妆品、食品、医药、石油等工业领域及农业和环境保护方面。该文对表面活性素的生产、结构、性质及应用方面进行了综述。     

脂肽类生物表面活性剂的研究进展

脂肽是由微生物代谢产生的一类具有很强表面活性的生物表面活性剂 ,在医药、食品、化妆品和微生物采油等方面有良好的应用潜力。本文对脂肽的生产、分离、鉴定及应用方面进行了综述     

生物表面活性剂Surfactin生产菌株的定向改造策略

表面活性素(Surfactin)是芽胞杆菌属(Bacillussp.)代谢产生的脂肽类生物表面活性剂,是由非核糖体肽合成酶(NRPS)催化而得的一种次级代谢产物。由于surfactin具有稳定性好、可被降解、表面活性好等理化性质以及抑菌、抗肿瘤等生物活性,在医药、农业、食品、化妆品、石油开采等方面都具有很大的应用潜力。但是,天然菌株产率低、生产成本高等特点限制了surfactin的规模化应用。本文对surfactin的合成机理进行了简要阐述,并针对目前提升surfactin产量和改变结构组分的4种定向改造策略(启动子工程、强化外排分泌、改造NRPS结构域和脂肪酸链合成酶系)进行了综述,最后对surfactin的研究方向进行了展望。     

解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用

对一株解淀粉芽孢杆菌TF28产生的抗菌脂肽进行分离鉴定及抑菌活性研究,采用酸沉淀、乙酸乙酯和甲醇萃取技术制备了抗菌脂肽粗提物,经过2次HPLC分离纯化,在保留时间32～42min内获得8个抗菌脂肽纯品,经MALDI-TOF-MS鉴定为芬芥素(fengycins),对尖孢镰刀菌和禾谷镰刀菌显示出较强的抑菌活性,该研究为提高菌株TF28抗菌脂肽产量的定向遗传改造奠定基础。     

一种脂肽类生物表面活性剂产生菌的筛选

从油田地层水中筛选分离得到1株能够产生表面活性剂的细菌,经鉴定为枯草芽孢杆菌。分析了该菌株的生理形态和生长特性,以及该菌株代谢产生的生物表面活性剂的性质。薄层色谱与原位水解显色和红外光谱分析表明,培养后菌株代谢产生的生物表面活性为脂肽。它能使水的表面张力降低到26mN/m,其临界胶束浓度为0.025mg/mL。     

环脂肽类生物表面活性剂结构、功能及生物合成*

环脂肽是由微生物产生的一类生物表面活性剂,由亲水的肽环和亲油的脂肪烃链两部分组成,具有独特的化学结构和生理功能。除具表面活性外,环脂肽还具有抗真菌、抗细菌、抗病毒、抗肿瘤等生物活性,具有广阔的应用前景,但我国对这一领域的研究却刚刚起步。本文介绍了目前已知环脂肽的结构类型、结构与功能的关系、相关功能特点等,着重介绍了环脂肽生物合成的非核糖体多酶体系及合成酶基因操纵子,并对环脂肽的应用前景进行了展望。     

生物表面活性剂的合成与提取研究进展*

生物表面活性剂（Biosurfactant）是由微生物产生的具有高表面活性的生物分子。相对于化学合成的表面活性剂,生物表面活性剂对生态系统的毒性较低,且可生物降解。因此,生物表面活性剂开始应用于环境污染治理的各个方面。中从生物表面活性剂生产菌的筛选、培养基的优化及生物表面活性剂的提取等方面对近年来生物表面活性剂的研究进展进行了总结,并对未来的发展方向作了展望。     

生物表面活性剂脂肽的发酵生产及抑菌应用研究进展*

表面活性素(surfactin)是一种环脂肽型生物表面活性剂,具有卓越的表/界面活性,能够显著降低水的表面张力,表现出良好的抗真菌、抗病毒、抗肿瘤、杀虫和抗支原体等生物活性,在医药、农业、食品、日化、石油开采等领域具有很大的应用潜力,但高昂的成本和缺乏竞争力的应用领域使其难以真正地实际应用起来。多年来,大量的研究工作在于促进其工业化应用。综述了surfactin的结构、特性及发酵生产,同时系统的比较和总结了surfactin在抑菌方面的应用研究。     

糖脂生物表面活性剂——甘露糖赤藓糖醇脂的研究进展

甘露糖赤藓糖醇脂是一种糖脂类生物表面活性剂,主要由霉菌和酵母菌等微生物发酵生产,具有良好表面活性和特殊生物活性,其在食品、医药、化妆品等领域有着潜在的应用前景。近年来,其研究在国外备受关注,而国内却鲜有报道。文中综述了甘露糖赤藓糖醇脂的发酵生产、多样性结构及活性、构效关系、生物合成途径等方面的相关研究,对存在问题进行了分析,并探讨了今后的研究重点。     

一株产脂肽类表面活性剂的碱性Dietzia菌及特性研究

【目的】筛选降解性能良好的产生物表面活性剂的菌株,对其进行分类学鉴定,确定所产表面活性剂物质并对各影响因素进行评价。【方法】利用液体石蜡为底物筛选降解性能良好的产生物表面活性剂菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等实验确定菌株的分类地位。通过排油圈活性、表面张力值、薄层层析等方法确定生物表面活性剂的性质,分析碳、氮源和温度、pH、盐浓度各因素对菌株产生物表面活性剂的影响。【结果】从大连新港采集的样品中分离得到一株产表面活性剂的嗜碱菌株3372,经分类鉴定表明其是Dietzia cercidiphylli的新菌株。嗜碱菌3372发酵液粗提物的排油直径为6.1 cm,表面张力可从67.62 mN/m降到32.95 mN/m,经薄层层析分析,初步鉴定为脂肽类表面活性剂。综合各因素对发酵液表面活性的影响,菌株3372在pH为9.0、适盐浓度为3%的培养基中,经30°C培养可将发酵液表面张力值降到最低。【结论】嗜碱菌3372是脂肽类生物表面活性剂产生菌的新成员,其在高盐碱条件下产生表面活性剂的特性在工业应用上有一定的潜力。     

新型抗菌肽——表面活性素、伊枯草菌素和丰原素

表面活性素（surfactin）、伊枯草菌素（iturin）和丰原素（fengycin）是一类主要由革兰阳性芽胞杆菌通过非核糖体合成途径产生的抗菌肽,一般是由1个β-羟基脂肪酸与7～10个氨基酸肽链以酰胺键连接而成的环肽,具有抗细菌、抗真菌、抗病毒、抗肿瘤等生物活性,在医疗方面具有良好的应用前景。目前,人们对这3种新型抗菌肽在医药领域中的研究进展所知甚少,故本文对其发现历史、结构特点、作用机制、生物合成和应用价值进行阐述,为后续研究提供借鉴。     

Bacillus natto TK-1产脂肽的纯化、抑菌活性及其表面活性剂特性

利用酸沉、醇提和薄层层析等方法从Bacillus natto TK-1 发酵液中分离得到脂肽。TLC结果表明,在迁移值Rf 0.58-0.65处出现单一紫红色条带其为脂肽粗提物。脂肽的临界胶束浓度约115mg/L。在浓度为512mg/L时,脂肽能将水的表面张力显著地降低到30.1mN/m。同时,通过体外抗粘连实验表明,脂肽能显著抑制沙门氏菌、大肠杆菌和金黄色葡萄球菌对96孔板固体表面的粘附,其中,对沙门氏菌的抗粘连效果较为明显。通过平板扩散法考察脂肽抑菌活性,结果表明脂肽具有较广泛的抑菌谱,对灰霉和镰胞霉的抑菌能力较强。     

Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase

A gene cluster for the non-ribosomal synthesis of a peptide of unknown structure has been identified in the partial genome sequence of Streptomyces coelicolor. Using molecular and computational analyses, the total structure of a tripeptide siderophore synthesized by the non-ribosomal peptide synthetase within the cluster has been deduced from the translated sequence of its encoding gene. This represents a novel method for the structural assignment of natural products from genome sequence data.     

Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.     

Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.     

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3′-bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor was monitored by pigment development in vitro and in living bacteria. The results were verified using genetic mutants to inactivate each domain. Finally, the results complemented the currently proposed biosynthetic pathway of BpsA.     

Solid-Phase Synthesis of Surfactin,a Powerful Biosurfactant Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>, and of Four Analogues

Using the Fmoc methodology, we report the chemical synthesis of surfactin and of four of its analogues, by stepwise solid-phase peptide synthesis (SPPS) on Sasrin resin. Formation of depsipeptide bond was performed with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In developing our strategy, surfactin was used as a model and we synthesized both its racemic mixture and its R isoform. (R) 3-hydroxy fatty acid was obtained using Candida antarctica lipase from the racemic fatty acid, allowing a further identification of both R and S isoforms in the racemic mixture. Analogues were synthesized as racemic linear lipopeptides. Then, both enantiomers were separated and purified by adsorption chromatography on silicic acid, following cleavage from the resin. Linear R lipoheptapeptides were identified by TLC. They exhibit, in all cases, higher R_f values than those of the corresponding S isoforms. Cyclization was then performed independently for each enantiomer, using a HATU/DIEA coupling in solution. The yields were highly dependent on the position and on the nature of the modified amino acids.     

The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides

Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems.     

Synthesis and antimicrobial activity of cysteine-free coprisin nonapeptides

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. CopA3 (LLCIALRKK-NH₂), a 9-mer peptide containing a single free cysteine residue at position 3 of its sequence, was derived from the α-helical region of coprisin and exhibits potent antibacterial and anti-inflammatory activities. The single cysteine implies a tendency for dimerization; however, it remains unknown whether this cysteine residue is indispensible for CopA3’s antimicrobial activity. To address this issue, in the present study we synthesized eight cysteine-substituted monomeric CopA3 analogs and two dimeric analogs, CopA3 (Dimer) and CopIK (Dimer), and evaluated their antimicrobial effects against bacteria and fungi, as well as their hemolytic activity toward human erythrocytes. Under physiological conditions, CopA3 (Mono) exhibits a 6/4 (monomer/dimer) molar ratio in HPLC area percent, indicating that its effects on bacterial strains likely reflect a CopA3 (Mono)/CopA3 (Dimer) mixture. We also report the identification of CopW, a new cysteine-free nonapeptide derived from CopA3 that has potent antimicrobial activity with virtually no hemolytic activity. Apparently, the cysteine residue in CopA3 is not essential for its antimicrobial function. Notably, CopW also exhibited significant synergistic activity with ampicillin and showed more potent antifungal activity than either wild-type coprisin or melittin.