CN101619296B - 乳酸链霉菌及其应用 - Google Patents
乳酸链霉菌及其应用 Download PDFInfo
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- CN101619296B CN101619296B CN200910040622.6A CN200910040622A CN101619296B CN 101619296 B CN101619296 B CN 101619296B CN 200910040622 A CN200910040622 A CN 200910040622A CN 101619296 B CN101619296 B CN 101619296B
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- streptomyces lactis
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Abstract
本发明公开了一种乳酸链霉菌Streptomyces lactis GIMN4.001，该链霉菌已在中国典型培养物保藏中心保藏，保藏日期为2008年11月10日，保藏编号为CCTCC No.M208214。本发明的乳酸链霉菌的代谢产物上清液中有乳酸存在，能够产生乳酸，该菌可用于生产乳酸。
Description
技术领域
本发明是属于微生物技术领域，具体地说，涉及一种新的链霉菌及其应用。
背景技术
土壤中存在着大量的放线菌，放线菌对人类贡献极大。目前发现的几千种抗生素中，有一半以上是由放线菌产生的。另外，它的菌落颜色鲜艳，呈放射状，对人体无害，因此，人们常用它作食品染色剂，既美观，又安全。利用放线菌还可以生产维生素B12、蛋白酶和葡萄糖异构酶等医药用品。因此积极地从土壤中筛选具有不同功能的放线菌，具有重要的意义。
发明内容
本发明的目的是提供一种能够产生乳酸的新种链霉菌——乳酸链霉菌。
本发明所述的链霉菌命名为乳酸微杆菌Streptomyces lactis GIMN4.001，保藏编号为CCTCC No.M208214。
本发明所述的乳酸链霉菌是由广州白云区番茄根际土中采集的土壤，经分离培养获得。该菌培养条件为：
(1)培养基：采用高氏合成1号琼脂培养基。
具体配方为：可溶性淀粉20.0g，硝酸钾(KNO3)1.0g，磷酸氢二钾(K2HPO4)0.5g，硫酸镁(MgSO4.7H2O)0.5g，氯化钠(NaCl)0.5g，硫酸亚铁(FeSO4.7H2O)0.01g，琼脂20.0g，水1000ml，pH7.2～7.4；
(2)培养温度：28℃。
本发明乳酸链霉菌(Streptomyces lactis GIMN4.001)的分子分类地位的确定。
该菌株的16SrDNA序列如SEQ ID NO：1所示。
本发明的乳酸链霉菌与其序列同源性最高的师岗链霉菌ATCC 19166T的DNA-DNA杂交率为14.43％，与熏衣草灰链霉菌ATCC 13306T的DNA-DNA杂交率为20.00％。
综合16SrDNA序列分析和DNA-DNA杂交结果(1987年国际系统细菌学委员会ICSB规定，DNA同源性小于70％或杂交分子的热解链温度差小于或等于2℃为细菌种的界线，Wayne etal，1987)，本发明的乳酸链霉菌是一种新的放线菌，命名为乳酸链霉菌Streptomyces lactisGIMN4.001。
本发明乳酸链霉菌具有以下有益效果：
本发明的乳酸链霉菌的代谢产物上清液中有乳酸存在，能够产生乳酸，该菌可用于生产乳酸。
本发明菌株乳酸链霉菌Streptomyces lactis GIMN4.001已于2008年11月10日保藏于国家知识产权局指定的保藏单位中国典型培养物保藏中心(CCTCC，武汉大学校内)，保藏编号为No.M208214。
附图说明
图1是本发明乳酸链霉菌孢子的扫描电子显微镜照片(1μm)；
图2是本发明乳酸链霉菌产生晶体的照片(A：30μm B：10μm)；
图3是本发明乳酸链霉菌及部分相关菌株依据16SrDNA序列构建的系统发育树；
图4和图5是本发明乳酸链霉菌气质联用分析产乳酸的结果图。
具体实施方式
以下通过具体实施例来详细说明本发明。
实施例1：本发明乳酸链霉菌的分离培养方法
从广州白云山森林土壤中采集植物根际0-30cm土样，取10g土壤放入装有90mL无菌水的三角瓶中(带玻珠)，在180rpm转速下振荡30分钟，静置10分钟后取上层液稀释10倍，取0.1mL均匀涂布在高氏合成1号琼脂培养基板上，翻转平板置于28℃培养箱中培养3天，即可获得本发明所述的乳酸链霉菌Streptomyces lactis GIMN4.001，CCTCC No.M208214。
高氏合成1号琼脂培养基：
可溶性淀粉20.0g，硝酸钾(KNO3)1.0g，磷酸氢二钾(K2HPO4)0.5g，硫酸镁(MgSO4.7H2O)0.5g，氯化钠(NaCl)0.5g，硫酸亚铁(FeSO4.7H2O)0.01g，琼脂20.0g，水1000ml，pH7.2～7.4。
本发明的乳酸链霉菌Streptomyces lactis GIMN4.001具有下述微生物学特征：
A.形态特征
在扫描电镜下可见，该菌株孢子少，孢子链柔曲，孢子长柱形，表面光滑(见图1)。
B.培养学上的特征
该菌株在酵母精麦芽精琼脂(ISP2)、燕麦粉琼脂(ISP3)和高氏合成一号琼脂培养基等不同的培养基上的培养特征见表1。
表1 乳酸链霉菌在不同培养基上的培养特征
| 培养特征 | 酵母膏麦芽膏琼脂培养基(ISP2) | 燕麦琼脂培养基(ISP3) | 无机盐-淀粉琼脂培养基(ISP4) | 甘油天门冬素琼脂培养基(ISP5) | 淀粉琼脂培养基 | 高氏合成1号琼脂培养基 | 马铃薯琼脂培养基 | 葡萄糖天门冬素培养基 |
| 生长情况 | 好，菌落中间凸起，有沟纹 | 不长 | 好，菌落毛绒状 | 一般 | 不长 | 弱 | 好 | 一般 |
| 产孢情况 | 一般，孢子灰白色 | 一般，孢子堆灰绿色 | 一般 | 一般 | 孢子灰白色 | 一般，粉灰白色 | ||
| 气生菌丝颜色 | 黄白色 | 灰黄色 | 灰白色 | 黄白色 | 灰白色 | 白色 | ||
| 基内菌丝颜色 | 灰棕褐色 | 黄绿色 | 黄褐色 | 黄白色 | 暗灰黄色 | 黄色 | ||
| 可溶性色素颜色 | - | - | - | - | - | - |
本发明的乳酸链霉菌在培养基上所产生的晶体(见图2)具有以下特性：典型的双锥型，菌体越老晶体越多，晶体形成在菌丝外面，可能由代谢物形成；晶体不溶于水、丙酮、乙醇、醋酸、甲醇、甲醛、乙酸乙酯；溶于10％盐酸(有气泡产生)、20％硫酸(有气泡产生)、10％NaOH、10％EDTA、醋酸(有气泡产生)、草酸；含有晶体的培养液能使KMnO4退色。
C.生理生化特征
该菌株兼性厌氧，革兰氏阳性，接触酶阳性，不产生类黑色素和H2S，牛奶凝固，明胶不液化，不水解淀粉，硝酸盐还原阳性。能利用葡萄糖、D-果糖、木糖、肌醇为碳源，但不能利用D-半乳糖、蔗糖。
D.其它性质
该菌株最适生长温度28℃；细胞壁含有LL二氨庚二酸，属细胞壁I型；细胞水解物中含有甘露糖、半乳糖和核糖。甲基萘醌的主要类型是MK-7，含有Q-10。细胞壁脂肪酸种类和含量如下：
表2 细胞壁脂肪酸种类和含量
| 脂肪酸类型 | 含量(％) | 脂肪酸类型 | 含量(％) |
| 13:0ISO | 0.64 | 16:0ISO | 12.55 |
| 13:0ANTEISO | 0.52 | 16:1CIS 9 | 0.84 |
| 14:0ISO | 4.64 | 16:0 | 8.54 |
| 14:0 | 1.35 | 16:09METHYL | 0.34 |
| 15:1ISO G | 0.47 | 17:1ANTEISO C | 0.78 |
| 15:1ANTEISO A | 0.69 | 17:0ISO | 2.85 |
| 15:0ISO | 9.75 | 17:0ANTEISO | 11.63 |
| 15:0ANTEISO | 38.70 | 17:0CYCLO | 2.25 |
| 15:0 | 1.81 | 17:0 | 0.45 |
| 16:1ISO H | 0.60 | 18:0 | 0.40 |
实施例2：本发明乳酸链霉菌菌株16SrDNA的序列测定及系统发育树的构建具体按照下述操作进行：
1.所用试剂
(1)5M NaCl
(2)TE1(10mM Tris，25mM EDTA)pH8.0；TE2(10mM Tris，1mM EDTA)pH8.0
(3)10％SDS
(4)氯仿∶异戊醇(24∶1)
(5)异丙醇
(6)CTAB/NaCl
(7)蛋白酶K(上海宝生物公司)母液20mg/ml
2.DNA提取方法
(1)将菌丝转移至灭过菌的研钵中，充分研磨。
(2)加567μl TE悬浮，并加入30μl 10％SDS，3μl 20mg/ml蛋白酶K，混匀，37℃温育1小时；
(3)加100μl 5mol/L NaCl，混匀；
(4)加80μl CTAB/NaCl溶液，混匀，65℃温育20分钟；
(5)用等体积酚∶氯仿∶异戊醇(25∶24∶1)抽提，10,000rpm离心10分钟，将上清液移至干净离心管；
(6)用等体积氯仿∶异戊醇(24∶1)抽提，取上清液移至干净管中；
(7)加0.6倍体积异丙醇，颠倒混合，室温下静止1小时，沉淀DNA；
(8)14,000rpm离心20分钟，收集DNA沉淀；
(9)DNA沉淀加500μl70％乙醇漂洗，自然风干；
(10)加50μl TE溶解DNA，-20℃保存。
3.PCR扩增方法
利用通用引物：正向引物F27(5’-AGAGTTTGATCCTGGCTCAG-3’)和反向引物1522R(5’-AAGGAGGTGAT CCAGCCGCA-3’)，引物序列的设计参照Edwards & Rogall(1989)的文章(Edwards U，Rogall T，Blocker HE.1989.Isolation and direct complete determination of entiregenes.Nucleic Acids Res，17：7843～7853)，引物由上海英骏公司合成，扩增所用的试剂盒购买于大连宝生物公司。
PCR扩增体系为：
成分 终浓度
浓缩反应缓冲液 1×工作浓度(50mM/L KCL，10mM/L Tris-HCl，1.5mM/L MgCl2)
dNTP混合物 每种碱基0.2mmol/L
Taq DNA聚合酶 0.5～1.0U/50μl
正向引物 1μmol/L
反向引物 1μl mol/L
模板 102～105ng/50μl
无菌水 将反应体积补足至50μl
PCR反应程序：
16SrDNA的序列测定委托上海英俊生物科技有限公司进行。
将本发明乳酸链霉菌GIMN4.001的16SrDNA序列登录GenBank数据库(登录号是GQ184344)，与GenBank数据库中的已知序列进行BLAST比较，确定与菌株GIMN4.001亲缘关系最近的种属，并从数据库获得相关种属的16S rDNA序列，构建系统发育树。序列比对用CLUSTAL X1.83program，进化树的构建用Neighbour-joining方法，进化树分枝模式的稳定性用SEQBOOT和CONSENSE program进行bootstrap分析，重复次数为1000，计算各分支的置信度。本发明乳酸链霉菌及部分相关菌株依据16SrDNA序列构建的系统发育树见图3。
本发明所述的乳酸链霉菌其形态特征与其他的链霉菌有显著差异，与基因序列同源性达98％-99％的链霉菌相比，也完全不同。本发明的乳酸链霉菌与部分相关菌株依据16SrDNA序列构建了系统发育树，本发明的乳酸链霉菌与师岗链霉菌(Streptomyces morookaensis)和熏衣草灰链霉菌(Streptomyces lavenduligriseus)的亲缘关系最近，师岗链霉菌与本发明的乳酸链霉菌16SrDNA序列同源性最高有98.9％，但师岗链霉菌在4种ISP培养基和淀粉合成琼脂培养基上都能生长，不产生晶体。而本发明的乳酸链霉菌在ISP3和淀粉琼脂培养不能生长，在马铃薯琼脂培养基上生长茂盛；师岗链霉菌硝酸盐不还原，而本发明的乳酸链霉菌硝酸盐还原阳性；熏衣草灰链霉菌与本发明的乳酸链霉菌16SrDNA序列同源性最高有98.7％，但它们之间的生理生化特征有区别，熏衣草灰链霉菌硝酸盐还原弱，产生类黑色素和H2S，而本发明的乳酸链霉菌不产生类黑色素和H2S，而且本发明的乳酸链霉菌能产生大量双锥型晶体。用马铃薯葡萄糖液体培养基发酵能产生乳酸。乳酸链霉菌与其同源性最高的链霉菌菌株微生物学特性比较见表3。
表3 乳酸链霉菌与其同源性最高的链霉菌菌株微生物学特性比较
注：表中+表示阳性，-表示阴性，W表示反应弱，ND表示没有测定。
实施例3：本发明的乳酸链霉菌产乳酸能力的测定
将本发明乳酸链霉菌菌株接种于马铃薯葡萄糖液体培养基中发酵14天后，发酵液7000rpm离心5min，取上清。
取1ml样品，用N2吹干，加入BSTFA(99％)，TMLS(1％)70℃衍生30min，然后取1μl样品上气质联用分析仪(GC-MS)上分析。仪器为Thermo Finnigan Trace DSQ，所用色谱柱为DB-5ms，规格为30mx0.25mmx0.25μm。
升温程序：75℃保持1min，然后以每分钟10℃的速度升温，一直到300℃，保持10min。
质谱条件：40-500质量扫描范围，离子源温度230℃，传输线温度280℃，进样口温度250℃(不分流)。
测量结果表明，发酵上清液中存在乳酸，结果见图4和图5。其中，乳酸的保留时间为5.41min。图4中纵坐标为相对峰度，横坐标为保留时间。
序列表
<110>广东省微生物研究所
<120>乳酸链霉菌及其应用
<160>1
<170>Patent In version 3.1
<210>1
<211>1425bp
<212>rDNA
<213>Streptomyces lactis
<400>1
gctaccatgc agtcgacgat gatccggttt cggccgggga ttagtggcga 50
acgggtgagt aacacgtggg caatctgccc tgcactctgg gacaagccct 100
ggaaacgggg tctaataccg gatacgacct gccaaggcat gtcggggggt 150
ggaaagctcc ggcggtgcag gatgagcccg cggcctatca gcttgttggt 200
ggggtgatgg cctaccaagg cgacgacggg tagccggcct gagagggcga 250
ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300
gtggggaata ttgcacaatg ggcgaaagcc tgatgcagcg acgccgcgtg 350
agggatgacg gccttcgggt tgtaaacctc tttcagcagg gaagaagcgt 400
gagtgacggt acctgcagaa gaagcgccgg ctaactacgt gccagcagcc 450
gcggtaatac gtagggcgca agcgttgtcc ggaattattg ggcgtaaaga 500
gctcgtaggc ggcttgtcgc gtcggatgtg aaagcccggg gcttaacccc 550
gggtctgcat tcgatacggg caggctagag ttcggtaggg gagatcggaa 600
ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa caccggtggc 650
gaaggcggat ctctgggccg atactgacgc tgaggagcga aagcgtgggg 700
agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgttgggcac 750
taggtgtggg cgacattcca cgtcgtccgt gccgcagcta acgcattaag 800
tgccccgcct ggggagtacg gccgcaaggc taaaactcaa aggaattgac 850
gggggcccgc acaagcagcg gagcatgtgg cttaattcga cgcaacgcga 900
agaaccttac caaggcttga catacaccgg aaacggccag agatggtcgc 950
ccccttgtgg tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc 1000
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gttctgtgtt 1050
gccagcatgc ctttcggggt gatggggact cacaggagac tgccggggtc 1100
aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtct 1150
tgggctgcac acgtgctaca atggccggta caatgagctg cgatgccgtg 1200
aggtggagcg aatctcaaaa agccggtctc agttcggatt ggggtctgca 1250
actcgacccc atgaagttgg agttgctagt aatcgcagat cagcattgct 1300
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acgtcacgaa 1350
agtcggtaac acccgaagcc ggtggcccaa cccttgtgga gggagccgtc 1400
gaaggtggga ctggcgattg ggacg 1425
Claims (2)
1.一种乳酸链霉菌(Streptomyces lactis GIMN4.001)，其特征在于：其保藏编号为CCTCC No.M208214。
2.权利要求1所述的乳酸链霉菌(Streptomyces lactis GIMN4.001)在产乳酸中的应用。
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