CN101037660A - 含有嗜盐菌素的钠线嗜盐古菌及其富集培养方法和用途 - Google Patents
含有嗜盐菌素的钠线嗜盐古菌及其富集培养方法和用途 Download PDFInfo
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Abstract
本发明公开了一种含有嗜盐菌素的钠线嗜盐古菌及其富集培养方法和用途。钠线嗜盐菌(Natrinema sp.)CGMCC No.1873属于革兰氏阴性菌,好氧生长,最适生长NaCl浓度为20%,最适生长pH值为7.5,能利用酵母膏、葡萄糖、果糖、乳糖等多种糖类作为能源与碳源生长。经鉴定该菌属于Natrinema pallidum。Natrinema pallidum sp.CGMCC No.1873分泌的一种蛋白类抗生素——嗜盐菌素,可对包括五个属十株不同的嗜盐菌具有生长抑制作用,并表现出较高的热稳定性和盐耐受性。通过简单的分离方法,可从培养液中收集嗜盐菌素。本发明提供的嗜盐菌素性质稳定,制备工艺简单,成本低廉,可运用于工业生产或医药领域,以去除生产中有害的微生物。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种含有嗜盐菌素的钠线嗜盐古菌及其富集培养方法和用途。
背景技术
某些微生物可分泌蛋白类抗生素。此类抗生素可通过与细胞壁、细胞膜或细胞器上的靶位点相互作用,起到抑菌或杀菌功效。真核生物中的蛋白类抗生素可称为真核素(eucaryocin),细菌中称为细菌素(bacteriocin),古菌中称为古菌素(archaeocin)。嗜盐菌素(halocin)就是一类产自嗜盐古菌的蛋白类抗生素,它可以通过抑制其它菌株的生长或直接导致其他菌株细胞破裂死亡,实现自身生长环境的净化,营养物质的富集。
栖息于高盐环境中的嗜盐古菌属于极端环境微生物,它表达分泌的嗜盐菌素,属于新型的天然药物资源。嗜盐菌素分子量小,仅由30-70个氨基酸残基组成;耐受极端环境,例如高温(100℃)、高盐(>1.5M)、有机溶剂(甲醇、乙醇)和蛋白酶等,可长期低温保藏;此外,还能抑制多种类型微生物生长。尽管具有良好的应用前景和广阔的市场潜力,但与短菌肽等其它细菌蛋白类抗生素相比,嗜盐菌素的研究起步晚、发现种类少、应用报道更少。因此,对嗜盐菌素这种新型天然药物资源的利用,可实现创新性药物开发,也可实现嗜盐菌工业生产去处杂菌的目的。
发明内容
本发明的目的是提供一种含有嗜盐菌素的钠线嗜盐古菌及其富集培养方法和用途。
含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum)保藏于中国微生物菌种保藏管理委员会普通微生物中心,种保藏号为CGMCC No.1873。
含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum)的富集培养方法:将含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum)接种于pH值为7.0~7.5的发酵培养基中,在37~42℃温度下培养96~120小时,得到钠线嗜盐古菌(Natrinema pallidum)的富集培养液,富集培养液中含有嗜盐菌素。所述的发酵培养基的质量体积百分比组分为:氯化钠20%、水合硫酸镁2%、氯化钾0.2%、柠檬酸三钠0.3%、酵母膏1%、酪蛋白氨基酸0.75%、其余为水。钠线嗜盐古菌(Natrinema sp.)的接种量为10%。嗜盐菌素提取方法:将上述培养至对数生长末期的富集培养液4000rpm离心20min,上清70℃处理1h后4000rpm离心去沉淀,随后置于50g/l的NaCl-Tris,pH 8.0的溶液中透析24h,得到嗜盐菌素,4℃长期放置备用。嗜盐菌素能在100度处理30min后仍具有活力、具有20g/l以上的盐耐受性。含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum)能对不同的嗜盐菌产生抑制作用。
本发明具有的有益效果:含有嗜盐菌素的钠线嗜盐古菌可对包括五个属十株不同的嗜盐菌具有生长抑制作用。本发明提供的嗜盐菌素性质稳定,具有较高的热稳定性和盐耐受性,制备工艺简单,成本低廉,可运用于工业生产或医药领域。
具体实施方式
实施例1:嗜盐菌Natrinema pallidum CGMCC No.1873的培养及生物学特性嗜盐菌(Natrinema pallidum)CGMCC No.1873接种于复合培养基,培养基组成如下:氯化钠20,水合硫酸镁2,氯化钾0.2,柠檬酸三钠0.3,酵母膏1,酪蛋白氨基酸0.75,pH 7.0。固体培养基中加入1.5%的琼脂。121℃灭菌30分钟。分别于42℃、150rpm的摇床上振荡培养和42℃的培养箱恒温静止培养。该菌具有以下特征:(1)菌落形态:菌落呈圆形,表面光滑,边缘平整,凸起,由于含有菌红素而显红色。(2)细胞形态:革兰氏阴性菌,细胞显微镜下(Olympus,BX40)呈现运动性,形态为多形态,杆状居多(3-5μm×0.8-1.2μm),部分细胞呈现球形(1-1.5μm)或不规则形态,老龄细胞呈球形。(3)限制因子:生长NaCl浓度范围为10%-30%,最适20%,NaCl浓度低于5%细胞裂解;生长pH值范围是6.0-9.0,最适7.5;生长温度范围是25-55℃,最适37-42℃。(4)生理生化特征:好氧生长,氧化酶、接触酶阳性,吲哚反应阴性,不水解淀粉、酪素、纤维素,能利用酵母膏、葡萄糖、果糖、乳糖,不能利用核糖,对短菌肽、新生霉素敏感,抗青霉素、红霉素、氯霉素、四环素等多种抗生素。
实施例2:嗜盐菌Natrinema pallidum CGMCC No.1873的16S rRNA基因的PCR扩增与序列测定
Natrinema pallidum CGMCC No.1873接种于复合培养基,从斜面中直接挑取一环菌体,加入400μL无菌水混匀,70℃水浴5分钟,12000rpm离心5分钟,上清直接用于PCR。扩增16S rRNA基因的一对通用引物如下:正向引物:
5’-ATTCCGGTTGATCCTGC-3’,反向引物:
5’-AGGAGGTGATCCAGCCGCAG-3’。分别对应大肠杆菌的16S rRNA基因的6-22和1540-1521位碱基。PCR反应体系(50μL)为:10×buffer 5μL,10mMdNTPs 1μL,10mM引物各1μL,ddH2O 42μL,Taq酶0.5μL,模板DNA 0.5μL。PCR反应条件为:94℃变性45s;55℃退火45s;72℃延伸90s,30个循环后72℃延伸7min。PCR产物纯化与序列测定由上海南方基因中心完成。完成测定的16S rRNA基因部分片断长度为526bp,与菌株Natrinema pallidumNCIMB777T的相关序列(AJ002949)相似性为100%。菌株Natrinema pallidumCGMCC No.1873的16S rRNA序列具体可见序列表。
因此,参照《Bergey’s Manual of Systematic Bacteriology》第二版第一卷的内容,根据菌株的限制因子特点、形态特征和生理生化指标,结合16S rRNA基因序列相似性,鉴定菌株CGMCC No.1873为嗜盐菌Natrinema pallidum。
实施例3:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素提取及敏感菌株筛选
培养至对数生长末期的Natrinema pallidum CGMCC No.18734000rpm离心20min,弃沉淀,上清滴于涂布指示菌的双层平板上,37℃培养7-14d,观察是否产生抑菌圈。采用的指示菌株包括:Natrinema altunense AJ2T,Nnm.sp.AB53,Nnm.pellirubrum JCM 10476T,Nnm.pallidum JCM 8980T,Haloarcula sp.AJ4,Halobiforma lacisalsi AJ5T,Hbf.haloterrestris JCM 11672T,Hbf.sp.AB1,Halorubrum sp.AJ201,Hrr.saccharovorum NCIMB 2081T,Hrr.coriense ch2T,Hrr.distributum JCM 9100T,Haloterrigena thermotolerans JCM 11050T,Htg.saccharevitans AB14T,Htg.sp.AJ233,Haloferax sp.YT226,Hfx.sp.YT228,Hfx.gibbonsii.CGMCC 1.2148T,Hfx.volacnii CGMCC AS1.2150T,Natronobacteriumsp.AB26,Nbt sp.AB29。研究表明,菌株Natrinema pallidum CGMCC No.1873对包括五个属10株不同的嗜盐菌具有生长抑制作用,分别为:Hbf.haloterrestrisJCM 11672T,Hrr.sp.AJ201,Hrr.saccharovorum NCIMB 2081T,Hrr.coriensech2T,Hrr.distributum JCM 9100T,Htg.saccharevitans AB14T,Htg.sp.AJ233,Hfx.sp.YT226,Hfx.sp.YT228,Nbt sp.AB26,Nbt sp.AB29,其中对Hrr.sp.AJ201的生长抑制作用最显著。
实施例4:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素温度敏感性
培养至对数生长末期的Natrinema pallidum CGMCC No.1873 4000rpm离心20min,上清分别于40、70和100℃分别处理30min、3h和6h,分别滴于已涂布指示菌Hrr.sp.AJ201、Htg.saccharevitans AB14T或Hfx.sp.YT228的双层平板上,37℃培养7-14d,观察是否产生抑菌圈(表1)。Natrinema pallidumCGMCC No.1873分泌的嗜盐菌素具有较稳定的物理化学性质,可以耐受70℃6h或100℃ 30min仍有活力。
表1 嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素温度、有机溶剂敏感性及盐依赖性表
指示菌 | 温度敏感性 | 盐依赖性 | 有机溶剂敏感性 | |||||||||||||||||||
40℃ | 70℃ | 100℃ | 100g/l | 50g/l | 20g/l | 10g/l | 0g/l | 乙醇 | 异丙醇 | 乙酸 | ||||||||||||
0.5h | 3h | 6h | 0.5h | 3h | 6h | 0.5h | 3h | 6h | ○ | I | ○ | I | ○ | I | ○ | I | ○ | I | ||||
AB14 | + | + | + | + | + | + | + | - | - | - | + | - | + | - | + | - | - | - | - | - | - | - |
AJ201 | + | + | + | + | + | + | + | - | - | - | + | - | + | - | + | - | - | - | - | - | - | - |
YT228 | + | + | + | + | + | + | + | - | - | - | + | - | + | - | + | - | - | - | - | - | - | - |
注:○(outside),表示透析袋外溶液;I(inside),表示透析袋内的溶液;-,代表无抑菌圈;+,代表有抑菌圈。
实施例5:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素盐依赖性
对数生长末期的培养液4000rpm离心20min,上清分别置于100g/l,50g/l,20g/l,10g/l和0g/l的NaCl溶液中透析24h,检测是否产生抑菌圈(表1)。Natrinema pallidum CGMCC No.1873分泌的嗜盐菌素对盐有一定的依赖性,在盐浓度大于20g/l的环境中才能维持其功能。
实施例6:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素对有机溶剂敏感性
对数生长末期的培养液4000rpm离心20min,上清与同体积有机溶剂(乙酸,乙醇,异丙醇)混合室温放置1h,检测是否产生抑菌圈(表1)。Natrinemapallidum CGMCC No.1873分泌的嗜盐菌素对有机溶剂敏感。
实施例7:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素等电点
采用硫酸调pH为3.0-6.0沉淀蛋白质,分别测定上清中嗜盐菌素的活性,活性最低的一管的pH值为最接近嗜盐菌素的等电点,其等电点在pH 4.0-3.0之间。
实施例8:嗜盐菌Natrinema pallidum CGMCC No.1873嗜盐菌素的分离
培养至对数生长末期的Natrinema pallidum CGMCC No.1873 4000rpm离心20min,上清70℃处理1h后4000rpm离心去沉淀,随后置于50g/l的NaCl-Tris(pH 8.0)溶液中透析24h,4℃长期放置备用。
序列表
单位:浙江大学
题目:一种产嗜盐菌素的嗜盐菌
序列类型:DNA
序列长度:526
菌株名称:Natrinema pallidum sp.J1
GCTAGTTGCA CGAGTTCAGA CTCGTAGCAG ATAGCTCAGT AACACGTGGC CAAACTACCC 60
TATGGATCCA AATAACCTCG GGAAACTGAG GCTAATGTGG AATAGCGTTC ATCGCCTGGA 120
GTGGCACGAA CGCGAAACGT TACGGCGCCA TAGGATGTGG CTGCGGCCGA TTAGGTAGAC 180
GGTGGGGTAA CGGCCCACCG TGCCTATAAT CGGTACGGGT TGTGAGAGCA AGAGCCCGGA 240
GACGGTATCT GAGACAAGAT ACCGGGCCCT ACGGGGCGCA GCAGGCGCGA AACCTTTACA 300
CTGCACGCGA GTGCGATAAG GGGACTCCGA GTGCGAGGGC ATATAGTCCT CGCTTTTCAC 360
GACCGTAAGG TGGTCGTAGA ATAAGTGCTG GGCAAGACCG GTGCCAGCCG CCGCGGTAAT 420
ACCGGCAGCA CGAGTGATGA CCGCTATTAT TGGGCCTAAA GCGTCCGTAG CTGGCCACGC 480
AAGTCTATCG GGAAATCCGC GCGCTTAACG CGCGGGCGTC CGGTGG 526
Claims (7)
1.一种含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum),其特征在于,该菌保藏于中国微生物菌种保藏管理委员会普通微生物中心,种保藏号为CGMCC No.1873。
2.一种如权利要求1所述的含有嗜盐菌素的钠线嗜盐古菌(Natrinemapallidum)的富集培养方法,其特征在于:将含有嗜盐菌素的钠线嗜盐古菌(Natrinema pallidum)接种于pH值为7.0~7.5的发酵培养基中,在温度37~42℃下培养96~120小时,获得钠线嗜盐古菌(Natrinema pallidum)的富集培养液,该富集培养液中含有嗜盐菌素。
3.根据权利要求2所述的一种含有嗜盐菌素的钠线嗜盐古菌(Natrinemapallidum)的富集培养方法,其特征在于:所述发酵培养基的质量体积百分比组分为:氯化钠20%、水合硫酸镁2%、氯化钾0.2%、柠檬酸三钠0.3%、酵母膏1%、酪蛋白氨基酸0.75%、其余为水。
4.根据权利要求2所述的一种含有嗜盐菌素的嗜盐古菌(Natrinemapallidum)的富集培养方法,其特征在于,所述的钠线嗜盐古菌(Natrinema sp.)的接种量为10%。
5.根据权利要求2所述的一种含有嗜盐菌素的钠线嗜盐古菌(Natrinemapallidum)的富集培养方法,其特征在于:所述的嗜盐菌素提取方法,将上述培养至对数生长末期的富集培养液4000rpm离心20min,上清70℃处理1h后4000rpm离心去沉淀,随后置于50g/l的NaCl-Tris,pH 8.0的溶液中透析24h,获得嗜盐菌素,可4℃长期放置备用。
6.根据权利要求5所述的一种含有嗜盐菌素的钠线嗜盐古菌(Natrinemapallidum)的富集培养方法,其特征在于:所述的嗜盐菌素能在100度处理30min后仍具有活力、具有20g/l以上的盐耐受性。
7.一种如权利要求1所述的含有嗜盐菌素的钠线嗜盐古菌(Natrinemapallidum)的用途,其特征在于:它对不同的嗜盐菌产生抑制作用。
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