CN116536230A - 一株可利用铵盐发酵产surfactin的枯草芽孢杆菌及其应用 - Google Patents
一株可利用铵盐发酵产surfactin的枯草芽孢杆菌及其应用 Download PDFInfo
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- CN116536230A CN116536230A CN202310154815.4A CN202310154815A CN116536230A CN 116536230 A CN116536230 A CN 116536230A CN 202310154815 A CN202310154815 A CN 202310154815A CN 116536230 A CN116536230 A CN 116536230A
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Abstract
本发明公开了一株可利用铵盐发酵产surfactin的枯草芽孢杆菌及其应用,该枯草芽孢杆菌分类命名为Bacillus subtilis LJ‑33,保藏编号为CCTCC NO:M 20222078。该菌株可以利用廉价无机氮NH4Cl作为唯一氮源发酵生产surfactin,具有发酵原料不受管控,成本低廉、高产和高生产速率等优势,具有较好的应用潜力。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株能够高效利用NH4Cl作为氮源的枯草芽孢杆菌菌株及其和应用。
背景技术
在绝大多数的工业微生物生产中,生产原料碳源和氮源往往占主要原料成本的50-90%。对于氮源而言,可分为有机氮源和无机氮源,其价格差异显著。通常,有机氮源价格较为昂贵,主要以蛋白胨和酵母粉为主,在10000-40000元/t;而无机氮源价格便宜,主要以铵盐和硝酸盐为主,其中氯化铵是无机氮源中最为廉价的,价格通常在600-1000元/t。硝酸盐如硝酸钠、硝酸铵等作为易制爆化学品,在采购、存储、使用等方面都受到严格监管,将其作为发酵氮源时必然产生较多的管理成本。因此,能高效利用氯化铵为氮源的生产菌株具有显著的低成本优势。目前从自然界分离的各种脂肽生产菌株多为芽孢杆菌,但优化后的发酵氮源通常为硝酸铵、硝酸钠或有机氮源。对模式菌株枯草芽孢杆菌168菌株进行基因工程改造后也可以获得脂肽surfactin的高产菌株;其发酵氮源同样为硝酸铵、硝酸钠或有机氮源。
微生物细胞对无机氮的同化作用涉及到谷氨酰胺合成酶和谷氨酸合成酶的联合反应,将NH4 +转化为谷氨酰胺和谷氨酸,成为生成其他氨基酸的氨基供体。然而,每种微生物都有自身一套特定的氮同化系统,该系统中涉及到许多的调控蛋白。例如,全局调节蛋白GlnR、TnrA和CodY都参与了相关酶的转录调控,介导氮同化。因此,氮同化是一个复杂的代谢调控系统,难以通过一个或几个基因的改造来提高氮的同化能力。
发明内容
本发明的第一目的在于提供一株可利用铵盐发酵产surfactin的枯草芽孢杆菌。
为实现上述技术目的,本发明采用如下技术方案:
一株可利用铵盐发酵产surfactin的枯草芽孢杆菌,其分类命名为Bacillussubtilis LJ-33,保藏编号为CCTCC NO:M 20222078。
本发明的另一目的在于提供上述枯草芽孢杆菌在发酵产surfactin中的应用。
作为一种优选的实施方式,发酵氮源为铵盐。
作为一种优选的实施方式,以氯化铵为唯一氮源发酵所述枯草芽孢杆菌产surfactin。
作为一种优选的实施方式,在发酵培养的培养基中添加金属离子;所述金属离子为Fe2+、Mn2+或Mg2+中的一种或多种;优选同时添加三种金属离子。
作为一种优选的实施方式,金属离子的添加浓度为:Fe2+:0.25-1.25mM;Mn2+:0.002-0.500mM;Mg2+:0.5-4mM。
作为一种优选的实施方式,在发酵培养的培养基中添加L-亮氨酸。
作为一种优选的实施方式,发酵培养的培养基由以下成分组成:蔗糖、NH4Cl、L-亮氨酸,磷酸氢二钾,Fe2+,Mn2+,Mg2+,消泡剂。
作为一种优选的实施方式,将所述枯草芽孢杆菌进行种子培养后,培养物再次接种到种子培养液中培养至细胞密度达到预定值,将两次培养后的种子液接种至发酵罐发酵。
作为一种优选的实施方式,将所述枯草芽孢杆菌的种子液接种至发酵罐中,在通气量0.02vvm,搅拌速度300rpm,37℃,pH 6.5条件下发酵。
本发明以枯草芽孢杆菌B.subtilis 168为出发菌,利用NH4Cl作为唯一氮源,基于适应性实验室进化技术筛选出一株可高效利用NH4Cl的优势菌株,记为LJ-3;通过优化发酵培养基的微量金属元素,获得了最适培养体系。在此基础上,将该菌株通过基因工程改造成脂肽surfactin合成菌株,具体包括:将由强启动子Pveg控制的来源于Bacillus velezensisBS-37(高产surfactin野生菌)的sfp基因进整合到LJ-3的同一位点中,恢复菌株合成surfactin的能力,得到重组菌LJ-31;紧接着,将由Pveg控制的来源于Bacillus velezensisBS-37的长链脂肪酸CoA连接酶(long-chain fatty acid-CoA ligase)编码基因lcfA整合到枯草芽孢杆菌LJ-31中的cydBC(编码细胞色素醌醇氧化酶)位点,得到重组菌,记作LJ-32;最后,将由Pveg控制的来源于B.subtilis 168的亮氨酸渗透酶编码基因yvbW整合到枯草芽孢杆菌LJ-32中的解淀粉酶编码基因amyE位点,得到重组菌LJ-33。本发明所获得的脂肽生产菌株LJ-33菌株的最大优势在于可以利用廉价氮源NH4Cl来生产脂肽surfactin,大幅降低了发酵原料成本,且具有高产量、高生产速率等优势,有很大的应用潜力。
附图说明
图1为底盘菌株Bacillus subtilis LJ-3与出发菌株Bacillus subtilis 168利用NH4Cl为唯一氮源时的生长情况。
图2为底盘菌株Bacillus subtilis LJ-3与出发菌株Bacillus subtilis 168在优化培养体系中的生长情况。
图3为用于本发明内容中基因工程操作的反向筛选标记方法,其中LF代表替换基因上游片段~1000bp,RF代表替换基因自身~800bp,DR代表基因下游~400bp,IG表示想插入基因组的目标片段,PC cassette由质粒pTPC扩增出来。
图4为基因工程菌LJ-31,LJ-32,LJ-33生产脂肽surfactin的发酵效果。
图5为基因工程LJ-33发酵产脂肽surfactin的HLPC图谱。
本发明所述的生物材料,其分类命名为Bacillus subtilis LJ-33,已于2022年12月23日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:M 20222078,保藏地址:中国武汉。
具体实施方式
所述实施例中所使用的实验方法如无特殊说明,均为常规方法,具体可参照《分克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行;下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
基因插入
基因的插入方法为反向筛选标记法,流程如图3所示。基本原理是利用含有突变型苯丙氨酰转移tRNA合成酶α亚基基因(pheS*)的枯草芽孢杆菌利用培养基中的对氯苯丙氨酸(p-Cl-Phe)会致死的特性,制备成反向筛选盒子(含有氯霉素cat基因和pheS*基因,命名为PC片段)。使用PCR仪扩增出替换基因上游前~1000bp(LF),替换基因下游后~400bp(DR),插入基因IG,替换基因~800bp(RF)和反向筛选盒子(PC)。随后,将片段按照LF-IG-DR-PC-RF的顺序进行融合PCR。随后将融合后的片段导入到感受态细胞进行基因插入。利用氯霉素和对氯苯丙氨酸筛选出阳性克隆子。
重组菌构建方式
扩增体系:
ddH2O 20μL,高保真酶混合物25μL,引物1-2μL,引物2-2μL,模板1μL。
PCR程序:
95℃预变性,5min,(95℃变性,15s,退火Tm温度,15s,72℃延伸,30s/kb)30个循环,72℃,5min。
枯草芽孢杆菌感受态制备:
采用GM I-GM II转接培养的方法制备成感受态细胞,具体步骤为:(1)从-80℃的超低温冰箱中取出冻存管进行三区划线,分离单菌落。(2)挑取单菌落接入5mL的GM I溶液中,在30℃,150rpm振荡培养过夜(12h)。(3)次日,取2mL菌液转接到18mL GM I培养基中,在37℃,200rpm振荡培养3.5h。(4)取10mL菌液转接到90mL GMⅡ培养基中,37℃,100rpm振荡培养2h。随后利用离心机,在6000rpm,4℃条件下离心10min收集菌体。(5)用5mL的上清液悬浮菌体,并加入2.5mL 30%的无菌甘油,使甘油浓度降到10%,随后以0.5mL的量分装到2mL的无菌离心管中,存放于-80℃超低温冰箱,已备转化时使用。
菌株转化过程:
(1)先向制备好的0.5mL的感受态中加入1μg的DNA片段,37℃,100rpm培养1h,进行第一次同源重组。(2)涂布氯霉素抗性平板,37℃,培养12h。然后菌落PCR验证转化子。挑取阳性克隆子接种于加了无抗性的LB,培养4h进行第二次同源重组。(3)培养物稀释适当浓度,取200μL涂布含对氯苯丙氨酸的MGY-Cl平板上,37℃培养12h,菌落PCR验证出整合了目标片段的阳性克隆子,测序正确阳性克隆子。
10×低盐溶液:磷酸氢二钾140g/L,磷酸二氢钾60g/L,硫酸铵20g/L,柠檬酸三钠10g/L,硫酸镁2g/L。
GM I溶液配方(100mL):10×低盐溶液,10mL;10%葡萄糖溶液,5mL;0.2%L-trp,2.5mL;5%酸水解酪蛋白,0.5mL;10%酵母汁,1mL;无菌水,81mL。
GM II溶液配方(100mL):10×低盐溶液,10mL;10%葡萄糖溶液,5mL;0.2%L-Trp,0.5mL;5M CaCl2,0.5mL;5M MgCl2,0.5mL;5%酸水解酪蛋白,0.8mL;10%酵母汁,0.4mL;无菌水,83mL。
MGY-Cl培养基配方:葡萄糖5g/L,酵母粉提取物4g/L,硝酸铵1g/L,氯化钠0.5g/L,磷酸氢二钾1.5g/L,磷酸二氢钾0.5g/L,硫酸镁0.2g/L,DL-4-氯-苯丙氨酸5mM。
Surfactin的检测
发酵结束后,取1mL发酵液在10,956×g条件下离心5min除去细胞。将所得到的上清液利用无水乙醇稀释适合浓度,在10,956×g条件下离心5min后,用0.22μm的有机膜过滤后制备为待测样品。使用岛津LC-20液相的紫外检测器surfactin进行定量,液相参数为:采用venusll XBP C18(4.6×150mm,5μm)色谱柱进行分离;流动相为90%的色谱级甲醇与10%的甲酸-水(含有0.05%的甲酸),进样体积20μL,流速为0.6mL/min,柱温35℃,检测器为紫外检测器,波长为214nm。
底物含量测定
蔗糖通过HPLC测定。使用岛津LC-20液相RID检测器的进行定量。液相参数为:色谱柱:Aminex HPX-87H,300mm×7.8mm柱(250mm×4.6mm,5μm);流动相:10mmol/L硫酸;流速:0.5mL/min;柱温:30℃,进样体积为10μL。
培养基中的NH4 +的测定采用靛酚蓝比色法测定。在强碱性的介质中,NH4 +与苯酚和次氯酸钠发生反应生成稳定的水溶性染料靛酚蓝。称取1g的苯酚,用纯水溶解,加入2.7mL的硝普钠(1.25%),定容至100mL的容量瓶中,配制成A液。精确称取0.5g NaOH和0.4g柠檬酸三钠溶液用纯水溶解,加入5mL次氯酸钠(0.1mol/L),定容至100mL的容量瓶中,配制成B液。分别吸取500uL的A液,100uL待测NH4 +溶液和500uL的B液,振荡混匀,在37℃水浴30min后取出,吸取200uL样液加到酶标板上,在625nm条件下,利用酶标仪测定吸光度。通过NH4 +浓度和吸光度成正相关性测定NH4 +的浓度。
实施例1通过实验室适应性进化获得高效利用NH4Cl的底盘菌株
适应性进化培养基的组分为:蔗糖20g/L,NH4Cl 50mM,磷酸二氢钾3g/L,磷酸氢二钾10g/L,硫酸镁0.5g/L,硫酸亚铁0.05g/L。固态培养基需要加入琼脂20g/L。
第一步:将适应性进化培养基分别配制成液态和固态。
第二步:将B.subtilis 168菌株从-80℃超低温冰箱中取出,利用接种环在固态适应性进化培养基平板上进行三区画线,37℃培养24h。
第三步:从平板上挑取若干单菌落,分别转接到含有50mL液态适应性进化培养基的250-mL三角烧瓶中。随后,在37℃200rpm摇床中培养24h。最后,测定细胞密度(OD600),挑选出细胞密度最高的菌液进行下一步实验。
第四步:将第三步中细胞密度最高菌液进行适当稀释,用涂布棒涂布于固态适应性进化培养基平板上,在37℃培养24h。
第五步:通过重复第三和第四步的步骤进行实验室适应性进化。其中固态到液态培养的过程记为一个周期。经过20个周期的适应性实验室进化后,将生长OD值最高的培养物稀释后涂布在固态适应性进化培养基中分离获得单菌落,在液体适应性培养基中验证单菌落的生长性能,对具有最好生长性能的菌株进行保藏,记为Bacillus subtilis LJ-3。如图1所示,在NH4Cl为唯一氮源的培养基中,Bacillus subtilis LJ-3的生长OD值相比出发菌株168菌株提高了191.6%。
实施例2优化培养基中的金属离子种类及浓度促进菌株LJ-3利用无机氮生长
以实施例1中获得的优势底盘菌株LJ-3进行筛选金属离子的单因素实验,从5种金属离子(Fe2+,Mn2+,Mg2+,Cu2+,Zn2+)中筛选出促进LJ-3生长和无机氮吸收的金属离子。
第一步:将LJ-3接种到种子培养基中37℃过夜培养24h。
第二步:将培养好的种子液按4%的接种量接种到金属离子筛选培养基中,在37℃培养30h。
第三步:通过测定细胞密度(OD600),残余NH4 +和蔗糖浓度,筛选出对LJ-3生长和底物吸收的有益金属离子。
种子培养基组分为:蛋白胨10g/L,酵母粉提取物5g/L,氯化钠10g/L。
金属离子筛选培养基组分为:以蔗糖20g/L,50mM NH4Cl,磷酸二氢钾3g/L,磷酸氢二钾10g/L为基础培养基组分。通过添加不同浓度的金属离子到基础培养基中成为最终金属离子筛选培养基。
结果如表1-表5所示:Fe2+,Mn2+和Mg2+可以促进菌株LJ-3的生长和底物吸收,其影响程度为Fe2+>Mn2+>Mg2+;Zn2+没有显著影响,Cu2+有抑制作用。因此,各种离子的优选浓度分别为:0.5mM Fe2+,0.01mM Mn2+,2mM Mg2+。
表1不同浓度亚铁离子对LJ-3生长和底物吸收的影响
表2不同浓度锰离子对LJ-3生长和底物的吸收的影响
表3不同浓度镁离子离子对LJ-3生长和底物的吸收的影响
表4不同浓度锌离子离子对LJ-3生长和底物的吸收的影响
表5不同浓度铜离子对LJ-3生长和底物的吸收的影响
将优选的微量金属元素及浓度添加到基础培养基组分中,将蔗糖浓度增加到50g/L,考察LJ-3在该发酵培养基的生长效果。
第一步:将LJ-3接种到种子培养基中37℃过夜培养24h。
第二步:将培养好的种子液按4%的接种量接种到发酵培养基中,在37℃培养。
第三步:测定细胞密度和底物含量。
种子培养基组分:蛋白胨10g/L,酵母粉提取物5g/L,氯化钠10g/L。
发酵培养基组分:蔗糖50g/L,50mM NH4Cl,磷酸二氢钾3g/L,磷酸氢二钾10g/L,FeSO4 0.5mM,MnSO4 0.01mM,MgSO4 2mM。
结果如图2所示:在加入复合金属离子(FeSO4 0.5mM,MnSO4 0.01mM,MgSO4 2mM)培养24h后,B.subtilis LJ-3对底物NH4 +和蔗糖分别摄取了50.1%和84.4%,细胞密度(OD600)到达9.36;相比出发菌株B.subtilis 168,底物摄取量分别提高87.6%和154.2%,细胞密度(OD600)提高了291.61%。
实施例3枯草芽孢杆菌LJ-31的构建方法
以实施例1筛选出的LJ-3为出发菌株,将来源于B.velezensis BS-37(高产surfactin野生菌)的sfp基因在将Pveg启动子的控制下,Pveg-sfp整合到LJ-3的相同位点中,恢复菌株合成surfactin的能力,得到重组菌LJ-31。采用反向筛选标记法,如图3所示,往下的实施例中涉及基因构建的均使用该方法。
通过引物对分别扩增出LF,Pveg,sfp,DR,PC和RF片段。随后,通过融合PCR将6个片段融合,构建成打靶片段LF-Pveg-sfp-DR-PC-RF。
LF-F:TTTGTGATTTTCAGCGTGATTGAAAACCT
LF-R:CTGTGTAAGATAGATCTCTAGATCCTCCGTCTGCAAAAGATTGT
Pveg-F:AGACGGAGGATCTAGAGATCTATCTTACACAGCATCACACTGG
Pveg-R:ACTCCGTAAATCTTCATGTTTGTCCTCCTTATTAGTTAATCTACATTTA
sfp-F:AATAAGGAGGACAAACATGAAGATTTACGGAGTATATATGGACCGC
sfp-R:GCGCACTGAAAAGGAATTATAACAGCTCTTCATACGTTTTCATCTCAATC
DR-F:GAGATGAAAACGTATGAAGAGCTGTTATAATTCCTTTTCAGTGCGCCTGC
DR-R:TCATTTGTATACATACTTTAAAAATAGATTATCCGAAAGAAAATCTATTA
PC-F:TAATAGATTTTCTTTCGGATAATCTATTTTTAAAGTATGTATACAAATGA
PC-R:ATAAATTCCGTAAATCTTCATTTATAAAAGCCAGTCATTAGGCCTATCTG
RF-F:CCTAATGACTGGCTTTTATAAATGAAGATTTACGGAATTTATATGGACCG
RF-R:TCTCCTTGAGGCGATAGACCG
实施例4枯草芽孢杆菌LJ-32的构建方法
以实施例3中的LJ-31为出发菌株,引入在启动子Pveg调控下的来自贝莱斯芽孢杆菌(B.velezensis BS-37)的长链脂肪酸CoA连接酶(long-chain fatty acid-CoA ligase)编码基因lcfA,Pveg-lcfA整合到枯草芽孢杆菌LJ-31中cydBC(编码细胞色素醌醇氧化酶)位点,得到重组菌,记作LJ-32。
通过引物对分别扩增出LF,Pveg,lcfA,DR,PC和RF片段。随后,通过融合PCR将6个片段融合,构建成打靶片段LF-Pveg-lcfA-DR-PC-RF。
LF-F:CTCGTATCATTCGGAACGATCATGTCA
LF-R:CTGTGTAAGATAGATCGGTATACCTCCTGACTAAATGGATCTGTTGA
Pveg-F:AGTCAGGAGGTATACCGATCTATCTTACACAGCATCACAC
Pveg-R:GCCATGGCTTTTCAGACTGCATGTTTGTCCTCCTTATTAGTTAA
lcfA-F:ATAAGGAGGACAAACATGCAGTCTGAAAAGCCATGGC
lcfA-R:AGGTCTTTTCCCATTAAGGCACCTTGTTTTCACGGGAAG
DR-F:CAAGGTGCCTTAATGGGAAAAGACCTGTTTCGATATAAA
DR-R:TGTATACATACTTTAAAAATATTTTCGGCAGAAACAG
PC-F:GAGCTGTTTCTGCCGAAAATATTTTTAAAGTATGTATACAAATGATGAA
PC-R:CATGAAGAGATGCCATTTATAAAAGCCAGTCATTAGGCCT
RF-F:TGACTGGCTTTTATAAATGGCATCTCTTCATGATCTTTGGTTTATACT
RF-R:TTAATAAGTCATAGGCTCCTTATGGCTGAC
实施例5枯草芽孢杆菌LJ-33的构建方法
以实施例4中的LJ-32为出发菌株,引入在启动子Pveg调控下的来自B.subtilis168的亮氨酸渗透酶编码基因yvbW,Pveg-yvbW整合到枯草芽孢杆菌LJ-32中解淀粉酶编码基因amyE位点,得到重组菌LJ-33。
LF-F:TATTCCGTATGTCAAGTGGCTG
LF-R:CTGTGTAAGATAGATCTCTTGACACTCCTTATTTGATTTTTTG
Pveg-F:TCAAATAAGGAGTGTCAAGAGATCTATCTTACACAGCATCACAC
Pveg-R:TGTCGTTTTTCATGTTTGTCCTCCTTATTAGTTAATCTACAT
yvbw-F:ACTAATAAGGAGGACAAACATGAAAAACGACAATCAAACGT
yvbw-R:AGCCTTGCCCTTACTGATGCTTGCGTCCT
DR-F:AAGCATCAGTAAGGGCAAGGCTAGACG
DR-R:CATCATTTGTATACATACTTTAAAAATTTTGTACGCATCGTTTTTCTC
PC-F:CGATGCGTACAAAATTTTTAAAGTATGTATACAAATGATGAATAAATTTTAA
PC-R:CGTTTTGCAAACATTTATAAAAGCCAGTCATTAGGCCT
RF-F:CTAATGACTGGCTTTTATAAATGTTTGCAAAACGATTCAAAAC
RF-R:TCAATGGGGAAGAGAACCG
实施例6重组菌株LJ-31,LJ-32,LJ-33生产surfactin的效果
考察实施例3,4,5构建的重组菌LJ-31,LJ-32和LJ-33在5L搅拌式发酵罐上生产surfactin的效果。
第一步:种子需要进行两次预培养,预培养的过程与实施例2中的种子培养过程相同。第二次预培养是将第一次培养物按4%的接种量再次接种到种子培养液中,培养到细胞密度(OD600)达到3.5左右,按4%接种量接种到发酵罐中。
第二步:发酵生产surfactin。使用5L搅拌式发酵罐,相应的工程参数为:工作体积3.5L,通气量(90-95%氧气)0.02vvm,搅拌速度300rpm,温度37℃,使用1M NaOH或1M HCl控制pH为6.5。
Surfactin发酵培养基配方:50g/L蔗糖,150mM NH4Cl,10mM L-Leu,磷酸氢二钾1g/L,0.5mM FeSO4,0.01mM MnSO4,2mM MgSO4,添加1‰(v/v)消泡剂。
结果如图4所示:工程菌LJ-31在发酵罐中利用NH4Cl合成surfactin产量达到了3.8g/L,细胞密度(OD600)为14.05。经过实施例4强化了长链脂肪酸CoA连接酶得到的工程菌LJ-32,surfactin产量达到了4.9g/L,与LJ-31相比提高了28.3%,在发酵培养基中添加10mM L-Leu后,LJ-32的surfactin产量达到了5.3g/L。经过实施例5,强化亮氨酸渗透酶获得工程菌LJ-33,在发酵培养基中添加10mM L-Leu后,其surfactin的产量达到了6.2g/L。本发明所获得的工程菌LJ-33生产性能较好,利用无机氮源发酵时生产速率达到了0.248g.L-1.h-1。表6所示,目前已报道的芽孢杆菌发酵产surfactin的氮源多为硝酸盐和有机氮,本发明提供了一株能够高效利用NH4Cl合成surfactin的菌株B.subtilis LJ-33及其发酵体系。
表6不同枯草芽孢杆菌利用不同培养基合成surfactin
实施例7重组菌株LJ-33发酵产surfactin的组分分布
使用HPLC分析实施例6中重组菌株LJ-33在5L搅拌式发酵罐上发酵生产的surfactin组分。结果如图5所示,重组菌株LJ-33发酵合成surfactin产物,其主要组分与surfactin标准品(CAS:24730-31-2;Sigma公司)相同,但各组分的丰度有显著差异。基于文献数据分析,surfactin的主要组分分别是(a)iso-C13-surfactin,(b)iso-C14-surfactin,(c)nC14-surfactin,(d)iso-C15-surfactin。重组菌株LJ-33发酵合成的脂肽surfactin的4个主要组分的占比分别为:(a)iso-C13-surfactin 13.3±1.5%,(b)iso-C14-surfactin20.09±1.4%,(c)nC14-surfactin 23.93±1.7%,(d)iso-C15-surfactin 32.79±1.6%。
Claims (10)
1.一株可利用铵盐发酵产surfactin的枯草芽孢杆菌,其特征在于,其分类命名为Bacillus subtilis LJ-33,保藏编号为CCTCC NO:M 20222078。
2.权利要求1所述枯草芽孢杆菌在发酵产surfactin中的应用。
3.根据权利要求2所述的应用,其特征在于,发酵氮源为铵盐。
4.根据权利要求3所述的应用,其特征在于,以氯化铵为唯一氮源发酵所述枯草芽孢杆菌产surfactin。
5.根据权利要求2所述的应用,其特征在于,在发酵培养的培养基中添加金属离子;所述金属离子为Fe2+、Mn2+或Mg2+中的一种或多种;优选同时添加三种金属离子。
6.根据权利要求2或5所述的应用,其特征在于,金属离子的添加浓度为:Fe2+:0.25-1.25mM;Mn2+:0.002-0.500mM;Mg2+:0.5-4mM。
7.根据权利要求2所述的应用,其特征在于,在发酵培养的培养基中添加L-亮氨酸。
8.根据权利要求2所述的应用,其特征在于,发酵培养的培养基由以下成分组成:蔗糖、NH4Cl、L-亮氨酸,磷酸氢二钾,Fe2+,Mn2+,Mg2+,消泡剂。
9.根据权利要求2所述的应用,其特征在于,将所述枯草芽孢杆菌进行种子培养后,培养物再次接种到种子培养液中培养至细胞密度达到预定值,将两次培养后的种子液接种至发酵罐发酵。
10. 根据权利要求2或9所述的应用,其特征在于,包括:将所述枯草芽孢杆菌的种子液接种至发酵罐中,在通气量 0.02vvm,搅拌速度 300rpm, 37℃,pH 6.5条件下发酵。
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