前体物和诱导子对内生真菌CH1产乙基长春胺的影响
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摘要
为了提高内生真菌CH1 Geomyces sp.次级代谢产物乙基长春胺的产量,首先采用单因素试验法考查环氧合酶抑制剂萘普生、羟化酶诱导子过氧化氢、前体物色氨酸、色胺、马钱子苷及裂环马钱子苷对内生真菌CH1产乙基长春胺的影响;在单因素试验的基础上,采用SPSS(统计产品与服务解决方案)19.0设计L_(18)(3~6)正交试验,进一步优化各前体物和诱导子的质量浓度,并在最优条件下进行试验验证。研究结果表明:在内生真菌CH1发酵液中添加前体物和诱导子均对内生真产乙基长春胺有显著影响(显著性概率P小于0.05);当萘普生、色氨酸、色胺、马钱子苷和裂环马钱子苷质量浓度分别为8,100,95,105和25 mg/L,过氧化氢质量浓度为20μg/L时,能有效提高乙基长春胺的产量,在此条件下进行验证试验,乙基长春胺产量可达5.09mg/L,与不加前体物质相比提高2.63倍。
In order to increase the yield of ethyl vincamine produced by endophytic fungus CH1 Geomyces sp, the effect of cyclooxygenase inhibitor and hydroxylase elicitor along with various precursors for the production of ethyl vincamine were researched. Firstly, the effects of naproxen, tryptophan, tryptamine, loganin, secologanin and hydrogen peroxide on ethyl vincamine production of endophytic fungus CH1 were investigated by single factor experiment. In addition,orthogonal experiment design was used to optimize the concentration by statistical software SPSS(statistical package for the social sciences) 19.0.The results show that the optimum mass concentrations for ethyl vincamine produced by endophytic fungus CH1 are as follows: naproxen 8 mg/L, tryptophan 100 mg/L,tryptamine 95 mg/L,loganin 105 mg/L,secologanin 25 mg/L, hydrogen peroxide 20 μg/L. Under the conditions, the highest ethyl vincamine production is up to5.09 mg/L, which is 2.63 times higher than that before optimization. The addition of elicitors and precursors in the fermentation medium of endophytic fungus CH1 can significantly increase the production of ethyl vincamine(significance probability is less than 0.05), which lays foundation for the biosynthesis research of ethyl vincamine produced by endophytic fungus CH1.
In order to increase the yield of ethyl vincamine produced by endophytic fungus CH1 Geomyces sp, the effect of cyclooxygenase inhibitor and hydroxylase elicitor along with various precursors for the production of ethyl vincamine were researched. Firstly, the effects of naproxen, tryptophan, tryptamine, loganin, secologanin and hydrogen peroxide on ethyl vincamine production of endophytic fungus CH1 were investigated by single factor experiment. In addition,orthogonal experiment design was used to optimize the concentration by statistical software SPSS(statistical package for the social sciences) 19.0.The results show that the optimum mass concentrations for ethyl vincamine produced by endophytic fungus CH1 are as follows: naproxen 8 mg/L, tryptophan 100 mg/L,tryptamine 95 mg/L,loganin 105 mg/L,secologanin 25 mg/L, hydrogen peroxide 20 μg/L. Under the conditions, the highest ethyl vincamine production is up to5.09 mg/L, which is 2.63 times higher than that before optimization. The addition of elicitors and precursors in the fermentation medium of endophytic fungus CH1 can significantly increase the production of ethyl vincamine(significance probability is less than 0.05), which lays foundation for the biosynthesis research of ethyl vincamine produced by endophytic fungus CH1.
引文
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[18]MCCOY E,O’CONNOR S E.Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus[J].J Am Chem Soc,2006,128(44):14276-14277.
[19]GUO Zhigang,LIU Yun,GONG Meizhen,et al.Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus[J].Plant Cell Tiss Organ Cult,2013,112(1):43-54.
[20]邢世海,王荃,潘琪芳,等.长春花萜类吲哚生物碱的生物合成途径[J].西北植物学报,2012,32(9):1917-1927.XING Shihai,WANG Quan,PAN Qifang,et al.Research progress of terpenoid indole alkaloids(TIAs)biosynthetic pathway of Catharanthus roseus[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(9):1917-1927.
[21]MOLCHAN O,ROMASHKO S,YURIN V.L-tryptophan decarboxylase activity and tryptamine accumulation in callus cultures of Vinca minor,L[J].Plant Cell Tissue and Organ Culture,2012,108(3):535-539.
[22]NIVISON-SMITH L,ACOSTA M L,MISRA S,et al.Vinpocetine regulates cation channel permeability of inner retinal neurons in the ischaemic retina[J].Neurochemistry International,2014,66:1-44.
[23]KUMAR K,KUMAR S R,DWIVEDI V,et al.Precursor feeding studies and molecular characterization of geraniolsynthase establish the limiting role of geraniol in monoterpene indolealkaloid biosynthesis in Catharanthus roseus leaves[J].Plant Science,2015,239:55-66.
[2]SABRY S M,BELAL T S,BARARY M H,et al.A validated HPLC method for the simultaneous determination of vincamine and its potential degradant(metabolite),vincaminic acid:applications to pharmaceutical formulations and pharmacokinetic studies[J].J Food Drug Ana,2010,18(6):447-457.
[3]SAURABH C V,KISHOR N G.Vinpocetine:hype,hope and hurdles towards neuroprotection[J].Asian J Pharm Res Dev,2013,1(4):17-23.
[4]ALVES J C F,SIMAS A B C,COSTA P R R.Formal enantioselective synthesis of(+)-vincamine.The first enantioselective route to(+)-3,14-epivincamine and its enantiomer[J].Tetrahedron:Asymmetry,1999,10(2):297-306.
[5]NEMES A,SZANTAY C J,CZIBULA L,et al.Synthesis of18-hydroxyvincamines and epoxy-1,14-secovincamine:A new proof for the aspidospermane-eburnane rearrangement[J].Heterocycles,2007,71(11):2347-2362.
[6]ELES J,KALAUS G,LEVAI A,et al.Synthesis of vinca alkaloids and related compounds 98.oxidation with dimethyldioxirane of compounds containing the aspidospermane and quebrachamine ring system:a simple synthesis of(7S,20S)-(+)-rhazidigenine and(2R,7S,20S)-(+)-rhazidine[J].Journal of Heterocyclic Chemistry,2002,39(4):767-771.
[7]STIERLE A,STROBLE G,STIERLE D.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew[J].Science,1993,260(5105):214-216.
[8]陈建华,刘佳佳,臧巩固,等.紫杉醇产生菌的筛选与发酵条件的调控[J].中南大学学报(自然科学版),2004,35(1):65-69.CHEN Jianhua,LIU Jiajia,ZANG Gonggu,et al.Screening of taxol-producing endophytic fungi and regulation of fermentation conditions[J].Journal of Central South University(Science and Technology),2004,35(1):65-69.
[9]REN Na,LIU Jiajia,YANG Dongliang,et al.Sequence-related amplified polymorphism(SRAP)maker as a new method for identification of endophytic fungi from Taxus[J].World Journal of Microbiology and Biotechnology,2012,28(1):215-221.
[10]GARYALI S,KUMAR A,REDDY M S.Taxol production by an endophytic fungus,Fusarium redolens,isolated from Himalayan Yew[J].J Microbiol Biotechnol,2013,23(10):1372-1380.
[11]KUMAR V,RAI S,GAUR P,et al.Endophytic Fungi:Novel Sources of anticancer molecules[C]//VERMA V,GANGE A,eds.Advances in Endophytic Research.New Delhi:Springer,2014:389-422.
[12]YIN Hong,SUN Yuhong.Vincamine-producing endophytic fungus isolated from Vinca minor[J].Phytomedicine,2011,18(8):802-805.
[13]杨显志,张玲琪,郭波,等.一株产长春新碱内生真菌的初步研究[J].中草药,2004,35(1):79-81.YANG Xianzhi,ZHANG Lingqi,GUO Bo,et al.Preliminary study of a vincristine-producing endophytic fungus isolated from leaves of Catharanthus roseus[J].Chinese Traditional and Herbal Drugs,2004,35(1):79-81.
[14]REN Na,LIU Jiajia,YANG Dongliang,et al.Indentification of vincamine indole alkaloids producing endophyticfungi isolated from Nerium indicum,Apocynaceae[J].Microbiological Research,2016,192:114-121.
[15]苟莉,唐春强,杨淑慎.前体及诱导子和发酵条件对烟曲霉TMS-26产紫杉醇发酵体系优化[J].菌物学报,2015,34(6):1165-1175.GOU Li,TANG Chunqiang,YANG Shushen.Optimization of Aspergillus fumigatus TMS-26 taxol production fermentation system by precursors,elicitors and fermentation conditions[J].Mycosystema,2015,34(6):1165-1175.
[16]代文亮,程龙,陶文沂.响应面法在紫杉醇产生菌发酵前体优化中的应用[J].中国生物工程杂志,2007,27(11):66-72.DAI Wenliang,CHENG Long,TAO Wenyi.Application of response surface methodology in optimization of precursors for taxol production by Fusarium mairei K178[J].China Biotechnology,2007,27(11):66-72.
[17]VERMA P,KHAN S A,MATHUR A K,et al.Regulation of vincamine biosynthesis and associated growth promoting effects through abiotic elicitation,cyclooxygenase inhibition,and precursor feeding of bioreactor grown Vinca minor hairy roots[J].Applied Biochemistry and Biotechnology,2014,173(3):663-672.
[18]MCCOY E,O’CONNOR S E.Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus[J].J Am Chem Soc,2006,128(44):14276-14277.
[19]GUO Zhigang,LIU Yun,GONG Meizhen,et al.Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus[J].Plant Cell Tiss Organ Cult,2013,112(1):43-54.
[20]邢世海,王荃,潘琪芳,等.长春花萜类吲哚生物碱的生物合成途径[J].西北植物学报,2012,32(9):1917-1927.XING Shihai,WANG Quan,PAN Qifang,et al.Research progress of terpenoid indole alkaloids(TIAs)biosynthetic pathway of Catharanthus roseus[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(9):1917-1927.
[21]MOLCHAN O,ROMASHKO S,YURIN V.L-tryptophan decarboxylase activity and tryptamine accumulation in callus cultures of Vinca minor,L[J].Plant Cell Tissue and Organ Culture,2012,108(3):535-539.
[22]NIVISON-SMITH L,ACOSTA M L,MISRA S,et al.Vinpocetine regulates cation channel permeability of inner retinal neurons in the ischaemic retina[J].Neurochemistry International,2014,66:1-44.
[23]KUMAR K,KUMAR S R,DWIVEDI V,et al.Precursor feeding studies and molecular characterization of geraniolsynthase establish the limiting role of geraniol in monoterpene indolealkaloid biosynthesis in Catharanthus roseus leaves[J].Plant Science,2015,239:55-66.