【作者】 郑金铠；

【导师】 朱伟明；

【作者基本信息】 中国海洋大学 ， 药物化学， 2010， 博士

【摘要】 耐盐微生物是一类在含盐介质中仍能生长的极端微生物,广泛存在于海洋、盐湖、盐场等环境样品中,由于其拥有独特的生态学分布、特异的生理机制以及广泛的应用,近年来逐渐成为研究的热点资源之一。研究表明,高盐所造成的高渗透压和营养剥夺可激活微生物体内的沉默基因,进而诱导出独特的生物代谢途径。“天才菌株”是指代谢产量较大、且能够产生系列新化合物的生物活性菌株。因此,从盐胁迫诱导的耐盐天才菌株次生代谢产物中,必将发现新的活性物质。本论文从采自福建莆田盐场和红树林等高盐环境的样品中获得4株耐盐天才菌株,对其在盐胁迫条件下产生的次生代谢产物进行了系统研究,并就盐胁迫对耐盐天才菌株代谢产物化学多样性的影响做了初步探讨。研究内容主要包括以下3个方面：1.耐盐天才菌株的筛选。从来源于莆田盐场等高盐环境的样品中分离纯化获得120株耐盐菌株。以海虾生物致死、P388肿瘤细胞增殖抑制、抑菌试验等作为生物活性筛选模型,TLC薄层色谱和HPLC指纹图谱等作为化学筛选模型,采用生物活性和化学评价相结合的集成筛选模式,最终获得4株耐盐天才菌株。2.盐胁迫诱导下的4株耐盐天才菌株次生代谢产物的研究。对耐盐天才菌株进行发酵条件摸索,如培养基、发酵天数、盐度等,选择最佳培养条件进行大发酵,获得活性浸膏。通过薄层色谱、硅胶柱色谱、Sephadex LH-20柱色谱、反相高效液相色谱等分离方法,从盐胁迫诱导的4株耐盐天才菌株次生代谢产物中,共分离得到86个单体化合物；应用波谱解析(UV, IR, NMR, MS, X-Ray等)并结合化学方法鉴定了80个单体化合物的化学结构(Fig.0-1)。其中新化合物32个：从耐盐天才菌株Aspergillus sclerotiorum PT06-1寡营养高盐条件代谢产物中获得了两个结构新颖的活性环六肽(1和2),该菌在富营养高盐条件下得到了12个棕曲菌素类环三肽(10-21)和1个线性二肽衍生物(23)；从Aspergillus terreus PT06-2高盐条件代谢产物中得到2个邻氨基苯甲酸聚合物(29-30)和1个二苯基-γ-丁内酯类化合物(33)；从Aspergillus flocculosus PT05-1高盐条件代谢产物中得到3个麦角甾醇类似物(47-49)和1个吡咯色素(51)；从Aspergilluscandidus 085241低盐条件代谢产物中得到10个补身烷型倍半萜(58,59,66-73)。其它已知化合物的结构类型还涉及多种生物碱、青霉酸内酯类似物、神经鞘胺醇糖苷、苯衍生物等。运用体外生物活性筛选模型,初步评价了单体化合物的肿瘤细胞增殖抑制活性、抗病毒活性和抑菌活性。肿瘤细胞增殖抑制活性测试表明：化合物2、3、32、47、48对HL-60细胞均有弱的抑制活性(IC50,分别为56,36,58,14,12μM)；此外48对BEL-7402肿瘤细胞株也具有弱的抑制活性(IC50,18μM)。抑菌活性结果表明：麦角甾醇48、49对白色念珠菌、铜绿假单胞菌、产气杆菌均具有较强的抑制作用(MIC,1.6-7.5μM)；环肽类化合物1、2、10、13、16、18、20对白色念珠菌具有抑制活性(MIC,分别为7.0,3.5,30,3.8,6.7,30,7.5μM)；此外,化合物2还对铜绿假单孢菌具有弱的选择性的抑制活性(MIC,38μM),29和31对金黄色葡萄球菌具有具有弱的抑制活性(MIC,分别为64μM和54μM),30和47对产气杆菌具有弱的抑制活性(MIC,分别为34μM和15μM)。抗病毒测试表明：青霉酸内酯3和土曲霉素43对H1N1流感病毒具有强的抑制活性,体外测试IC50值分别为4.6和6.6μM,与阳性对照利巴韦林相当,43的动物体内实验评价有效。3.初步评价了盐胁迫对耐盐菌株代谢产物化学多样性的影响。通过比较代谢产量、TLC、HPLC指纹图谱及生物活性等方法,就高盐胁迫对9株耐盐菌株的次生代谢产物的影响做了分析,利用该方法得到了一株在高盐条件下大量产生生物活性penicillenol A (81)的工程菌。首次尝试在培养基中加入不同浓度、不同种类的卤盐,并初步探讨了其对PT06-1代谢产物化学多样性的积极影响,证实了微生物的次生代谢存在卤盐效应。综上所述,通过对耐盐天才菌株在盐胁迫条件下次生代谢产物的系统研究,证明盐胁迫能增加耐盐微生物的化学多样性,产生新的活性代谢产物,高盐胁迫是诱导耐盐微生物产生含氮化合物的有效策略。我们推测其产生可能与耐盐微生物缓解并对抗环境压力有关。上述研究为充分利用耐盐微生物资源获得活性新化合物提供了有益的参考。更多还原

【Abstract】 Halotolerant microorganism has been considered as an important and promising type of extremophiles, possessing special ecology, specific physiological mechanism and extensive utilization. The special hypersaline environment where they lived could active silent gene and induces other specific metabolic pathway. Therefore, we planned to obtain more novel bioactive compounds from the secondary metabolites of halotolerant talented strains under salt stress. Studies include the screening for halotolerant talented strains, isolation and structural elucidation of secondary metabolites, preliminary bioactivity evaluation, and the salt effects on the chemodiversity of secondary metabolites.1. Screening for the halotolerant talented strains.120 strains of halotolerant fungi have been isolated from salt sediments in Putian sea salt field, Fujian and mangrove environment, Hainan. The EtOAc extracts of these fungi were evaluated with the combinatory method of chemical and bioactive screening.4 of them were determined as halotolerant talented strains of cytotoxicity or antibiotic activities.2. Study on bioactive constituents of four halotolerant talented strains under salt stressAfter optimization of fermentation conditions under salt stress, these strains were fermented, respectively. The whole broths were extracted with ethyl acetate to give active extracts, which were subjected to extensive silica gel column chromatography, Sephadex LH-20 and HPLC.86 compounds were isolated from four halotolerant talented strains under salt stress,80 structures of which (see Fig.0-1) were determined by means of spectral analysis (UV, IR, NMR, MS, X-Ray) and chemical methods. Among them,32 compounds were new ones, including two novel cyclic hexapeptides (1 and 2) obtained from Aspergillus sclerotiorum PT06-1 cultured in a nutrient-limited medium containing 10% salts, twelve cyclic tripeptides (10-21) and a dipeptide derivative (23) from PT06-1 cultured in a nutrient-suffient medium containing 10% salts, two anthranilate condensation compound (29 and 30) and butyrolactone I analog (33) from Aspergillus terreus PT06-2 cultured in a hypersaline medium, three steroids (47-49) and a red pyrrole-containing pigment (51) from Aspergillus flocculosus PT05-1 cultured with 10% sea salt, ten drimane-type sesquiterpenes (58,59,66-73) from Aspergillus candidus 085241 cultured with 1% sea salt. Compounds 2,3,32,47 and 48 exhibited weak cytotoxicity against HL-60 cell line with IC50 values of 56,36,58,14, and 12μM, respectively; 48 also had weak cytotoxicity against BEL-7402 cell line with an IC50 value of 18μM. Compounds 1,2, 10,13,16,18,20,29,30,31,47,48 and 49 showed antibiotic activities against Candida albicans, Pseudomonas aeruginosa, Enterobacter aerogenes or Staphylococcus aureus with MIC values ranged from 1.6μM to 64μM. Additionally, two known compounds 3 and 43 were evaluated with significant antiviral activity against H1N1 virus in vitro with IC50 values of 4.6 and 6.6μM, respectively.3. Preliminary exploration of halide salt effects on chemodiversity of secondary metabolites from halotolerant strainsTo further explore the halide salt effects on chemodiversity of secondary metabolites of halotolerant talented strains, secondary metabolites from 9 halotolerant talented strains under different culture conditions containing different halide salts at different concentration were analyzed by comparing their weight, TLC, HPLC and bioactivities. The strain HK01 was found to mainly produce bioactive penicillenol (81) cultured in a hypersaline medium. In addition, we established that the different halide salts were also able to influence the secondary metabolites of halotolerant strains.In a word, novel bioactive compounds can be obtained from secondary metabolites of halotolerant strains under salt stress, and more nitrogen-containing compounds produced when stains cultured in the hypersaline media. It was supposed that the specific products probably be metabolized by halotolerant microorganisms to relieve or resist the salt stress from enviroment. Above studies provided a good example for obtaining bioactive novel compounds from halotolerant strains.更多还原