CN116694497A - 一株转化甘油和甘氨酸的放线菌 - Google Patents
一株转化甘油和甘氨酸的放线菌 Download PDFInfo
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Abstract
本发明公开了转化甘油和甘氨酸的小单孢菌菌株—深邃小单孢菌Micromonospora profundi TRM 95458,2022年08月02日保藏于广东省微生物菌种保藏中心(GDMCC),保藏号为GDMCC NO:62600。同时还公布了利用该菌株转化甘油和甘氨酸高效生产ABAGG的种子培养基和发酵培养基及其制备工艺,利用甘油为唯一碳源发酵生产ABAGG等含有丙三醇基团的化合物,变废为宝,具有较好的工业应用前景。
Description
技术领域
本发明涉及工业微生物领域,具体涉及一株利用甘油为唯一碳源发酵生产含有丙三醇基团的代谢产物的放线菌,更具体为深邃小单孢菌及其在工业、农业和医药中应用。
背景技术
微生物能够利用甘油转化生产精细化学品,其中肺炎克雷伯氏菌、弗氏柠檬酸杆菌和丁酸梭菌的研究比较多(Sun Y Q, Shen J T, Yan L, et al. Advances inbioconversion of glycerol to 1, 3-propanediol: prospects and challenges[J].Process Biochemistry, 2018, 71: 134-146.)。近年来利用各种微生物转化粗甘油生产一些精细化学品,受到了国内外学者的关注。齐向辉等提供了一种微生物共培养体系,利用甘油发酵生产1,3-丙二醇的方法,提高了甘油的转化率和1,3-丙二醇的产量,降低了生产成本(CN111394396B,)。赵宗保等用于胞外无细胞体系或在微生物体内催化生物柴油副产物甘油产1,3-丙二醇,显著降低1,3-丙二醇生产成本,提高产物得率(CN110964753B)。
甘油是生物柴油酯交换生产过程中不可避免的一种副产物,随着生物柴油产业的发展而产量巨大。微生物转化法有条件温和、简单、易操作等特点,使甘油在发酵领域的应用受到广泛关注(Chilakamarry C R, Sakinah A M, Zularisam A W, et al.Bioconversion of glycerol into biofuels—opportunities and challenges[J].BioEnergy Research, 2022, 15(1): 46-61.)。利用微生物细胞工厂,转化可再生生物质资源生产精细化学品,具有重要的工业、农业及医药应用价值(Jathanna H M, Rao C V.Using Aspergillus ochraceus, a Native Fungus, to Convert Biodiesel-DerivedCrude Glycerol to Single Cell Oil for Commercial Applications[J]. Waste andBiomass Valorization, 2022, 13(6): 2831-2845.)。
发明内容
本发明从木垒县鹰嘴豆根际土中分离得到一株深邃小单孢菌Micromonospora profundi TRM95458,并对其次生代谢产物进行挖掘,发现了该菌株能以甘油为唯一碳源,生产新结构化合物ABAGG(即化合物B),以及化合物A,D,E。结构式如下:
因此,本发明所要解决的技术问题是:提供一种生产ABAGG的小单孢菌,为深邃小单孢菌Micromonospora profundi TRM95458,于2022年08月02日保藏于广东省微生物菌种保藏中心(GDMCC)(地址:广州市先烈中路100号59号楼5楼,广东省微生物研究所),保藏号为GDMCC NO: 62600,分类命名:Micromonospora profundi,经检测,存活。
所述的深邃小单孢菌在转化甘油和甘氨酸中的应用,其特征在于其利用甘油为唯一碳源发酵生产含有丙三醇基团的代谢产物,用于转化可再生生物质资源生产精细化学品,在工业、农业和医药中应用。
本发明提供所述的深邃小单孢菌在生产ABAGG中的应用。所述的应用,其特征在于包括下述步骤:在合适条件下培养所述小单孢菌,获得发酵液;从发酵液中分离制备得到ABAGG,培养所述放线菌的发酵培养基是以甘油为唯一碳源、甘氨酸等为氮源。
所述的深邃小单孢菌发酵的培养基配方和培养条件为:种子液培养基:淀粉10g,TSB 3g,葡萄糖4g,酵母膏4g,麦芽浸粉10g,微量元素1mL,琼脂17g,调节pH 7.0。将固体平板菌打菌饼(直径1cm),接种于500mL、装液量为150mL的种子培养基中(每瓶接3-5个菌饼),30℃,180rpm培养5天。发酵液培养基:甘油20mL,蛋白胨10g,甘氨酸10g,酵母浸粉10g,微量元素液1mL,调节pH 7.0。按4%的接种量接种于发酵培养基中,30℃、180rpm条件下在摇床上培养7天。
所述的从发酵液中分离制备ABAGG的方法具体为:发酵液离心后,湿法上样至D101大孔树脂色谱柱,然后分别用4倍柱体积的蒸馏水除去糖类、盐类及水溶性蛋白等杂质,之后用30%~80%甲醇进行洗脱;合并30%~80%甲醇洗脱液经旋转蒸发仪减压浓缩得到发酵液粗提物浸膏。
从发酵液中分离纯化ABAGG的方法进一步包括如下步骤:将发酵液粗提物浸膏烘干后用甲醇溶解,过ODS柱,分别用40%,60%,80%的甲醇洗脱,收集80%的甲醇洗脱液浓缩成浸膏,继续进行pHPLC制备获得ABAGG。
所述pHPLC制备条件为:A:30%MeOH-H2O(0.075% HCOOH);B:100%MeOH;流速 :10mL/min;梯度洗脱条件:0-40min 0-65%B;40-45min 65-100%B;45-50min 100%B;DAD检测器波长: 350nm,收集保留时间RT=21.85min峰得到ABAGG。
本发明还提供一种化合物ABAGG,具有调节细胞渗透压作用,能够提高生物耐盐性,其结构式如下:
。
进而本发明也提供所述化合物ABAGG在调节细胞渗透压或提高生物耐盐性中的应用。
本发明具有以下有益效果:深邃小单孢菌TRM95458能够以甘油为唯一碳源,生产含有丙三醇基团的代谢产物,用于转化可再生生物质资源生产精细化学品,在工业、农业和医药中应用。
附图说明
图1、深邃小单孢菌TRM95458的扫描电镜照片(示菌丝体和圆形孢子)。
图2、深邃小单孢菌TRM95458的分类学地位与系统发育树。
图3、菌株95458发酵产物制备高效液相色谱图。
图4、ABAGG的二维核磁共振HMBC远程相关关系图。
图5、ABAGG的核磁共振氢谱谱图。
图6、ABAGG的核磁共振碳谱谱图。
图7、ABAGG的核磁共振HMBC谱图。
图8、ABAGG的HPLC定量分析标准曲线。
图9、不同甘油添加量深邃小单孢菌TRM95458转化生成ABAGG的产量。
图10、不同甘氨酸添加量深邃小单孢菌TRM95458转化生成ABAGG的产量。
图11、盐胁迫与深邃小单孢菌TRM95458累积渗调物质ABAGG的关系。
具体实施方式
下面通过具体实施方式的详细描述来进一步阐明本发明,但并不是对本发明的限制,仅仅作示例说明。
实施例一、深邃小单孢菌TRM95458的分离与分类鉴定
1深邃小单孢菌TRM95458分离
从新疆木垒县鹰嘴豆根际0-30cm土壤样品中,采用稀释涂布平板法,用1/10 ISP2培养基(葡萄糖4g,酵母膏4g,麦芽浸粉10g,微量元素1mL,琼脂17g,调节pH 7.0),28℃恒温培养,分离放线菌。进一步以甘油—复合氨基酸培养基筛选,得到菌株TRM 95458。
2深邃小单孢菌TRM95458的分类鉴定
2.1形态学观察
电子显微镜观察:固体平板培养,用刀片切下菌块(大小约0.5cm×0.5cm),将多余培养基切掉,带有菌丝培养基薄且厚度均匀。用电子显微镜观察记录菌丝形态、气生菌丝和基内菌丝生长情况,菌丝体是否产生孢子丝及孢子丝的排列方式、形状;孢子形状和大小等,扫描电镜照片见图1。
深邃小单孢菌TRM95458在ISP2培养基上生长良好,菌落圆形扁平,表面干燥,菌落边缘整齐,气生菌丝丰富,基内菌丝黄色,有黄褐色的可溶性色素产生。
2.2菌株TRM95458的分子生物学鉴定
2.2.1菌株TRM95458基因组DNA的提取方法
收集培养平板上的TRM95458菌体放入1.5mL的无菌离心管中,加入480μL的1×TE缓冲液。加入20μL溶菌酶(50mg·m L-1),放入37℃水浴过夜。每管加入50μL 20%的SDS,加入5μL 20mg·mL-1的蛋白酶K,60℃水浴2h。加入550μL的酚:氯仿:异戊醇(25:24:1),12000rpm离心5min,取上清移入另一离心管,反复抽提2次。取上清,加入300μL的95%异丙醇,70μL的乙酸钠(3mol·L-1),12000rpm离心10min,弃上清。用500μL的70%乙醇清洗离心产物1次,12000rpm离心5min,弃上清,将乙醇挥发完全。用30μL无菌超纯水充分溶解底部的DNA,1%的琼脂糖凝胶电泳检测DNA提取质量,将提取的DNA放入-20℃冰箱中保存备用。
2.2.2菌株TRM95458 16S rRNA基因的扩增方法
用放线菌16S r RNA基因通用引物27F(5’-AGAGTTTGATCCTGGCTC-3’)和1492R(5’-CGGCTACCTTGTTACGACTT-3’)扩增放线菌基因组DNA中的16S r RNA基因片段。50μL的PCR反应体系为:dd H2O 34μL,10×Buffer (缓冲液含Mg2+)5μL,dNTPs 2.5μL,引物27F(10μmol·L-1)2μL,引物1492R(10μmol·L-1)2μL,50% DMSO 2μL,Taq DNA聚合酶0.5μL,模板DNA2μL。
PCR反应条件为:预变性94℃ 4min;变性94℃ 1min,退火56℃ 1min,延伸72℃2min,30次循环;总延伸72℃ 8min。反应完成后用1%琼脂糖凝胶电泳检测。符合条件的PCR产物进行序列测定。
2.2.3测序结果的比对分析方法
测序结果用DNAMAN5. 2软件拼接,序列通过BLAST与GenBank数据库中的己有效发表的菌株序列进行比对,下载相似度较高的已有效发表菌株的16S rDNA基因序列,用MEGA5.0软件对序列进建系统发育树的行构,确定菌株TRM95458的分类学地位。
2.2.4菌株TRM95458的16S rDNA序列测定结果
测序结果用DNAMAN5.2软件拼接,确定该片段由1381个碱基组成,得到TRM95458的16S rDNA 序列为:
GGCTCCCTCCACAAGGGTTGGGCCACCGGCTTCGGGTGTTGCCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTTTGGGATTCGCTCCACCTCACGGTATCGCAGCCCATTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTTCGATGAGTCCCCGCCATAACGCGCTGGCAACATCGAACGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTGACCGCCCCCGAAGGACCTCACATCTCTGCGAGTTTTGCGGCCATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTGCGGCACAGAGAACCGGAGAGGCTCCCCACACCTAGCGCCCAACGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTACCGAACTCTAGCCTGCCCGTATCGACCGCAGGCTTGGAGTTGAGCCCCAAGTTTTCACGGTCGACGCGACAAGCCGCCTACGAGCTCTTTACGCCCAATAAATCCGGACAACGCTCGCACCCTACGTCTTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTGCAGGTACCGTCACTCTCGCTTCGTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTCCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATCACCCCACCAACAAGCTGATAGGCCGCGAGCCCATCCCAAGCCGAAAAACTTTCCACCACCAGCCATGCGACCAGCAGTAATATTCGGTATTAGCCCCCGTTTCCGAGGGTTATCCCAAAGCTTGGGGCAGGTTGCTCACGTGTTACTCACCCGTTCGCCGCTCGAGTACCCCGAAGGGCCTTTCCGCTCGACTTGCA
2.2.5同源进化树构建
通过BLAST比对,在GenBank数据库中下载与待测放线菌16S rDNA基因序列相似度较近的序列,用MEGA5.0软件对放线菌16S rDNA基因序列进行多重序列比对,构建系统发育树(见图2)。菌株TRM95458与深邃小单孢菌(Micromonospora profundi)的16S rDNA基因序列(一致性为100.00%)聚在同一个系统进化分支上,故将该小单孢菌鉴定为深邃小单孢菌(Micromonospora profundi)TRM95458。
实施例三、利用深邃小单孢菌TRM95458发酵并制备ABAGG
接种少量菌丝体于ISP2培养基中,28℃培养5d,获得深邃小单孢菌TRM95458的种子液,取种子液(4%的接种量)接种于发酵培养基内发酵培养,发酵条件为:转速180rpm,初始pH 7.0-7.2,装液量150mL,发酵温度30℃,发酵7d,收集发酵液,离心取上清液湿法上样到大孔树脂色谱柱(发酵滤液每100L装填D101 2.0kg),然后分别用4倍柱体积的清水、30%甲醇溶液、80%甲醇溶液、95%甲醇溶液洗脱;减压浓缩去掉溶剂后得到富含深邃小单孢菌素的发酵液甲醇浸膏,将甲醇超声溶解的浸膏过ODS柱,分别用20%、40%、60%、80%的甲醇洗脱,收集洗脱液,进行HPLC分析。收集80%的甲醇洗脱浸膏,进行制备型液相色谱分离与制备(见图3),将收集后的样本使用1D&2D NMR技术手段对化合物的结构进行解析,得到新化合物ABAGG。新化合物ABAGG核磁检测的HMBC关系图、核磁共振氢谱、碳谱、HMBC谱图分别见图4,图5,图6和图7。归属ABAGG核磁共振氢谱和碳谱数据见表1。
表1、ABAGG核磁共振氢谱和碳谱数据
Position | δ C | δ H (J in Hz) |
1 | 112.47 | |
2 | 151.70 | |
3 | 112.34 | 6.64 d (7.5) |
4 | 135.64 | 7.38 t (7.5) |
5 | 151.70 | 6.65 t(7.5) |
6 | 133.23 | 7.94 d (7.5) |
7 | 171.78 | |
8 | 45.47 | 4.11 2H, s |
9 | 172.34 | |
10 | 66.10 | 4.23 2H, m |
11 | 73.40 | 4.23 m |
12 | 71.94 | 4.16 2H, m |
实施例四、ABAGG的定量分析与结构表征
采用高效液相色谱法对ABAGG进行定量分析。色谱条件为:4.6mmX 150mm C18色谱柱(0.5μm),80%甲醇-水作为流动相,流速为1.0Ml/min检测波长为254nm,280nm,330nm 和445nm。得到的四个化合物的结构如下:
。
其中,化合物B(即ABAGG)为新结构化合物。
实施例五、深邃小单孢菌TRM95458发酵生产ABAGG的转化甘油功能测定
1、将分离纯化所得到的ABAGG纯品配制成1.0 mg/mL母液,倍性稀释为1.0、0.5、0.25、0.125、0.0625、0.03125、0.0156 mg/mL等系列浓度,用HPLC通过面积归一法测定标准曲线(见图8)。ABAGG标准曲线方程为:y=4.2905x+0.3875,方程中x是相对峰面积,y是ABAGG的含量,根据R2=0.9929表明线性关系较好,可以用来测定ABAGG的含量。
2、在发酵液培养基(小米10g,豆芽100g,葡萄糖10g,蛋白胨5g,NaCl 2.5g,(NH4)2SO4 1g,调节pH 7.0)中添加不同含量甘油,甘氨酸得到不同含量的ABAGG。结果发现,当甘油添加量为0.1mL,甘氨酸添加量为1g时,ABAGG产量最高。其中,图9显示不同甘油添加量深邃小单孢菌TRM95458转化生成ABAGG的产量;图10显示不同甘氨酸添加量深邃小单孢菌TRM95458转化生成ABAGG的产量。
实施例六、盐胁迫与深邃小单孢菌TRM95458累积渗调物质ABAGG的关系
向深邃小单孢菌TRM95458发酵液中添加0g/L,2.5g/L,5g/L,10g/L,15g/L,20g/LNaCl,随着NaCl浓度的增大,发酵液中ABAGG的产量先增加后降低,当NaCl含量为10g/L时,ABAGG的产量最高(如图11所示)。说明在较高盐浓度下,深邃小单孢菌TRM95458通过转化甘油和甘氨酸并在细胞内累积渗透压调节物质ABAGG从而提高深邃小单孢菌TRM95458的耐盐性,ABAGG是一个新结构的具有渗透压调节功能的相容性物质。
Claims (10)
1.一种放线菌,其特征在于,其为深邃小单孢菌Micromonospora profundi,于2022年08月02日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC NO: 62600。
2.按照权利要求1所述的放线菌在甘油转化作用中的用途。
3.如权利要求2所述的用途,其特征在于,其利用甘油为唯一碳源发酵生产含有丙三醇基团的代谢产物。
4.按照权利要求1所述的放线菌在生产化合物含丙三醇和甘氨酸基团的化合物ABAGG中的应用,化合物ABAGG的结构式如下:
。
5.一种化合物ABAGG的制备方法,其特征在于,包括下述步骤:在合适条件下培养如权利要求1所述的放线菌,从发酵液中分离制备得到ABAGG;
优选地,培养所述放线菌的发酵培养基是以甘油唯一碳源、甘氨酸为氮源;
化合物ABAGG的结构式如下:
。
6.如权利要求5所述的制备方法,其特征在于:培养所述放线菌的培养基配方为:种子液培养基:淀粉10g,TSB 3g,葡萄糖4g,酵母膏4g,麦芽浸粉10g,微量元素1mL,琼脂17g,调节pH 7.0;发酵液培养基:甘油20mL,蛋白胨10g,甘氨酸10g,酵母浸粉10g,微量元素液1mL,调节pH 7.0。
7.如权利要求5所述的制备方法,其特征在于:培养所述放线菌的培养条件为:25-35℃,150-200rpm,优选为30℃、180rpm条件下在摇床上培养5-10天。
8.如权利要求5所述的制备方法,其特征在于:从发酵液中分离制备ABAGG的方法具体为:发酵液离心后,湿法上样至D101大孔树脂色谱柱;然后分别用4倍柱体积的蒸馏水除去糖类、盐类及水溶性蛋白杂质;之后用30%~80%甲醇进行洗脱;合并30%~80%甲醇洗脱液经旋转蒸发仪减压浓缩得到发酵液粗提物浸膏。
9.如权利要求8所述的制备方法,其特征在于:从发酵液中分离制备ABAGG的方法进一步包括如下步骤:将发酵液初提物浸膏烘干后用甲醇溶解,过ODS柱,分别用40%,60%,80%的甲醇洗脱,收集80%的甲醇洗脱液浓缩成浸膏,进行pHPLC制备,条件为:A:30%MeOH-H2O(0.075% HCOOH);B:100%MeOH;流速:10 mL/min;梯度洗脱条件:0-40min 0-65%B;40-45min65-100%B;45-50min 100%B;DAD检测器波长:350nm,收集保留时间RT=21.85min峰得到ABAGG。
10.一种化合物ABAGG及其在调节细胞渗透压和提高生物如植物耐盐性中的应用,其结构式如下:
。
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