地锦草内生放线菌生物活性及活性菌株L57的鉴定
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摘要
根据13株分离自药用植物地锦草(Euphorbia humifusa)的内生放线菌的酶活性以及抗菌、抗氧化等重要特征来首次探讨地锦草内生放线菌的生物活性,以期为开发新的活性物质提供参考依据。通过观察菌落周围是否有透明圈对分泌的胞外淀粉酶、脂肪酶、纤维素酶等常见酶进行活性测定;使用滤纸扩散法检测菌株抗菌活性;利用PCR特异性扩增检测菌株的PKS-Ⅰ,PKS-Ⅱ,NRPS基因;对菌株发酵液乙酸乙酯粗提物进行抑菌、抗氧化活性检测,并对高活性菌株进行形态观察及16S r DNA系统进化分析鉴定。结果表明:13株菌中至少12株菌具有一种酶活性,其中2株可同时产生以上3种酶。4株具有稳定且较强的抑菌能力,13株菌生物合成基因PCR筛查结果均为阴性。1株菌对DPPH自由基有较强清除作用(清除率≥50%),2株菌对·OH自由基有较强清除作用(清除率≥50%)。其中L57的活性物质粗提物具有广谱抗菌活性,对鲍曼不动杆菌的抑菌圈直径达22. 62 mm,并对DPPH自由基具有较强清除率,经形态观察及16S r DNA序列分析初步确定L57归属为噬油微杆菌(Microbacterium oleivorans)。研究证明地锦草内生放线菌可作为探寻新型活性物质的良好资源。
In 13 endophytic actinomycete strains isolated from Euphorbia humifusa,the feature of excreting enzyme,antimicrobial activity and antioxidant activity were explored in order to provide reference for the development of new active substances. The activities of enzymes such as amylase,lipase and cellulase were determined by observing the transparent circle. The antimicrobial activity was measured with the filter paper method,the secondary metabolite genes of PKSI/PKSⅡ and NRPS were detected by PCR amplification,the antimicrobial activity and antioxidant activity of the crude fermentation extract of highly active strains were also detected. The identification was also based on morphological characteristics and the 16 S r DNA gene sequence similarity analysis. Results indicated that:in total 13 endophytic actinomycete strains 12 strains could produce at least one enzyme,2 strains could simultaneously produce amylase,cellulase and lipase;4 strains showed stable and high activity against pathogenic bacteria;1 strain had a better DPPH radical scavenging activity(clearance rate≥50%);2 strains showed strong · OH free radical scavenging capacity(clearance rate≥50%);the results of PCR screening of the biosynthetic genes of 13 strains were all negative. Furthermore,the strain L57 which showed wide antimicrobial properties and strong DPPH radical scavenging activity was defined as Microbacterium oleivorans according to the characteristics of morphology and 16 s r DNA sequence analysis. The maximum suppression width of the L57 reached 22. 62 mm on Acinetobacter baumannii plates. Our results demonstrated that,the endophytic actinomycete from medical plant Euphorbia humifusa are good sources for discover the new active substances.
In 13 endophytic actinomycete strains isolated from Euphorbia humifusa,the feature of excreting enzyme,antimicrobial activity and antioxidant activity were explored in order to provide reference for the development of new active substances. The activities of enzymes such as amylase,lipase and cellulase were determined by observing the transparent circle. The antimicrobial activity was measured with the filter paper method,the secondary metabolite genes of PKSI/PKSⅡ and NRPS were detected by PCR amplification,the antimicrobial activity and antioxidant activity of the crude fermentation extract of highly active strains were also detected. The identification was also based on morphological characteristics and the 16 S r DNA gene sequence similarity analysis. Results indicated that:in total 13 endophytic actinomycete strains 12 strains could produce at least one enzyme,2 strains could simultaneously produce amylase,cellulase and lipase;4 strains showed stable and high activity against pathogenic bacteria;1 strain had a better DPPH radical scavenging activity(clearance rate≥50%);2 strains showed strong · OH free radical scavenging capacity(clearance rate≥50%);the results of PCR screening of the biosynthetic genes of 13 strains were all negative. Furthermore,the strain L57 which showed wide antimicrobial properties and strong DPPH radical scavenging activity was defined as Microbacterium oleivorans according to the characteristics of morphology and 16 s r DNA sequence analysis. The maximum suppression width of the L57 reached 22. 62 mm on Acinetobacter baumannii plates. Our results demonstrated that,the endophytic actinomycete from medical plant Euphorbia humifusa are good sources for discover the new active substances.
引文
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[16]Kobayashi K,Fukuda T,Usui T,et al.Bafilomycin L,a new inhibitor of cholesteryl ester synthesis in mammalian cells,produced by marine-derived Streptomyces sp.OPMA00072[J].J Antibiot,2015,68(2):126-132.
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[2]Ragazzi A,Moricca S,Capretti P,et al.Endophytic presence of Discula quercina on declining Quercus cerris[J].Journal of Phytopathology,1999,147(7-8):437-440.
[3]Hardoim P R,van Overbeek L S,Berg G,et al.The hidden world within plants:ecological and evolutionary considerations for defining functioning of microbial endophytes[J].Microbiol Mol Biol Rev,2015,79(3):293-320.
[4]Qin S,Xing K,Jiang J H,et al.Biodiversity,bioactive natural products and biotechnological potential of plantassociated endophytic actinobacteria[J].Appl Microbiol Biotechnol,2011,89(3):457-473.
[5]Chinese pharmacopoeia commission.Pharmacopoeia of The People.s Republic of China[M].Beijing:China Medical Science and Technology Press,2010:118.国家药典委员会编.中华人民共和国药典[M].北京:中国医药科技出版社,2010:118.
[6]Khas-Erdene,Wu Z Y M.Research progress on Mongolian medicine Euphorbia humifusa[J].World Latest Medicine Information,2017,17(68):38-40.哈斯额尔敦,吴张玉马.蒙药材地锦草的研究进展[J].世界最新医学信息文摘,2017,17(68):38-40.
[7]Dong X Z,Cai M Y.Manual for systematic identification of common bacteria[M].Beijing:Science Press,2001:364-398.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:364-398
[8]Cai L,Ou Y Q,Hou X Z,et al.Isolation and identification of endophytic bacteria from Fenghuangdancong tea[J].Science and Technology of Food Industry,2016,37(10):246-250.蔡丽,欧燕青,侯小桢,等.凤凰单丛茶内生拮抗细菌的筛选与鉴定[J].食品工业科技,2016,37(10):246-250.
[9]Gontang E A,Gaudêncio S P,Fenical W,et al.Sequence based analysis of secondary-metabolite biosynthesis in marine actinobacteria[J].Appl Environ Microbiol,2010,76(8):2487-2499.
[10]Banik J J,Brady S F.Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary[J].Proc Natl Acad Sci USA,2008,105(45):17273-17277.
[11]Fu Y H,Chen G,Yao X C,et al.Screening of antioxidant activity of four marine symbiotic actinomycetes and microencapsulation[J].Asia-Pacific Traditional Medicine,2014,10(4):64-66.傅玉鸿,陈钢,姚香草,等.四株海洋放线菌抗氧化活性初筛与微囊化包埋研究[J].亚太传统医药,2014,10(4):64-66.
[12]Zhang X G,Tang P,Liu Y J,et al.Antioxidant activity of endophyte extracts isolated from six medicinal plants[J].Modern Food Science and Technology,2016,32(4):66-74.张新国,唐鹏,刘英娟,等.6种药用植物内生菌提取物的抗氧化活性研究[J].现代食品科技,2016,32(4):66-74.
[13]Jiang X,Jia L M,Zhang L,et al.Comparison of several methods of extracting genomic DNA of Bifidobacterium[J].Chinese Journal of Health Laboratory Technology,2004,14(5):641-642.江晓,贾力敏,张磊,等.双歧杆菌基因组DNA提取几种方法的比较[J].中国卫生检验杂志,2004,14(5):641-642.
[14]Stierle A,Strobel G,Stierle D.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew[J].Science,1993,260(5105):214-216.
[15]Jia L,Chen S Y,Zhai Y G,et al.Recent advances in studies on endophytes and their associated bioactive products[J].Chinese Traditional and Herbal Drugs,2007,38(11):1750-1754.贾栗,陈疏影,翟永功,等.近年国内外植物内生菌产生物活性物质的研究进展[J].中草药,2007,38(11):1750-1754.
[16]Kobayashi K,Fukuda T,Usui T,et al.Bafilomycin L,a new inhibitor of cholesteryl ester synthesis in mammalian cells,produced by marine-derived Streptomyces sp.OPMA00072[J].J Antibiot,2015,68(2):126-132.
[17]Sasaki T,Igarashi Y,Saito N,et al.TPU-0031-A and B,new antibiotics of the novobiocin group produced by Streptomyces sp.TP-A0556[J].Antibiot,2001,54(5):441-447.
[18]Carr G,Williams D E,Diaz-marrero A R,et al.Bafilomycins produced in culture by Streptomyces spp.isolated from marine habitats are potent inhibitors of autophagy[J].J Nat Prod,2010,73(3):422-427.
[19]Wang Y,Dai C C.Advances in studies on metabolic transformation of bioactive substances by endophytic fungi[J].Chinese Traditional and Herbal Drugs,2009,40(9):1496-1499.王宇,戴传超.内生真菌对生物活性物质代谢转化作用的研究进展[J].中草药,2009,40(9):1496-1499.