CN117363484A - D-乳酸发酵菌株的筛选和d-乳酸脱氢酶基因的检测方法 - Google Patents
D-乳酸发酵菌株的筛选和d-乳酸脱氢酶基因的检测方法 Download PDFInfo
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Abstract
本申请提供了D‑乳酸发酵菌株的驯化筛选方法和D‑乳酸脱氢酶基因的检测方法。D‑乳酸发酵菌株的驯化筛选方法包括以下步骤:(1)制备驯化培养基和鉴别分离培养基;(2)分别称取青、黄贮饲料,在无菌环境下放入装有驯化培养基的离心管中,35.2℃条件下连续培养24小时;(3)取出培养箱中的离心管,吸取培养液放入另一新的装有驯化培养基中,35.2℃条件下连续培养24小时;(4)取出连续培养7天的样品,使用接种环蘸取培养液,并划线至鉴别分离培养基表面,35.2℃条件下倒置培养,及时挑取周围产透明圈的单个菌落,接种至培养基中连续培养至底部出现较多沉淀。通过采用本发明的方法,有利于驯化分离携带D‑LDHs的菌株,为D‑乳酸发酵提供菌株资源。
Description
技术领域
本发明涉及微生物领域,更具体地,涉及D-乳酸发酵菌株的筛选和D-乳酸脱氢酶基因的检测方法。
背景技术
D-乳酸(D-lactic acid)是L-乳酸(L-lactic acid)的立体异构体,也是一种基本有机化工原料,作为合成多种手性物质的重要前体,光学纯度大于98%的高光学纯度D-乳酸在医药、农药、化工等方面具有广泛应用,以高光学纯D-乳酸为原料聚合而成的聚乳酸材料能够替代普通化工产品聚合而成的可降解环保塑料、纤维制品,应用于医用骨内固定物、香烟过滤头、纺织用高级纤维等高端消费领域,同时也是3D打印技术的主要原材料。D-乳酸可通过化学合成和微生物发酵获得,化学合成存在环境污染、成本昂贵、光学纯度较低等问题,难以满足应用要求。
在微生物的乳酸发酵代谢通路中,乳酸脱氢酶催化丙酮酸直接生成乳酸,此步反应是可逆的,当利用乳酸作为碳源时,乳酸脱氢酶也可催化乳酸底物生成丙酮酸,根据发酵产生的乳酸构象不同,乳酸脱氢酶分为L型乳酸脱氢酶(L-lactate dehydrogenase,L-LDHs)和D型乳酸脱氢酶(D-lactate dehydrogenase,D-LDHs),D-LDHs主要在变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)中存在,乳酸杆菌(Lactobacillus)、嗜热链球菌(Streptococcusthermophilus)、芽孢杆菌(Bacillus)、肠膜明串珠菌(Leuconostoc mesenteroides)等菌株均具有发酵生产D-乳酸的潜质。
由于D-乳酸通常并非微生物代谢的必需产物,因此如何筛选并分离驯化携带D-LDHs的微生物成为D-乳酸发酵工业和合成生物学领域的难点问题。
发明内容
本发明通过利用乳酸脱氢酶催化反应可逆的原理,从青、黄贮饲料中获取多种D-乳酸发酵菌株种质资源,为其应用于生物发酵和合成生物学底盘细胞方面打下基础。
本发明提供了一种D-乳酸发酵菌株的驯化筛选方法,包括以下步骤:
(1)制备驯化培养基和鉴别分离培养基:
驯化培养基:胰蛋白胨10g、D-乳酸5g、氯化钠5g、超纯水1000mL,使用氢氧化钠和盐酸调节pH至5.7,121℃灭菌20分钟,分装至10mL无菌离心管,每管8.5mL;
鉴别分离培养基:蛋白胨10g、葡萄糖20g、酵母浸出物5g、碳酸钙5g,如需固体培养基则加入琼脂14g,超纯水1000mL,pH值6.8,121℃灭菌20分钟;
(2)分别称取1g青、黄贮饲料,使用剪刀剪成2-3mm细小碎片,在无菌环境下放入装有驯化培养基的10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时;
(3)24小时后取出培养箱中的离心管,多次上下颠倒混匀后,吸取200μL培养液放入另一新的装有驯化培养基10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时,该操作在7天内,每日重复进行;
(4)取出连续培养7天的样品,使用接种环蘸取培养液,并划线至鉴别分离培养基表面,封口膜封好皿盖,35.2℃条件下倒置培养,每间隔12小时观察一次,并及时挑取周围产透明圈的单个菌落,接种至置于10mL离心管的8.5mL液体鉴别分离培养基中连续培养至底部出现较多沉淀。
本发明还提供了一种D-乳酸脱氢酶基因的检测方法,包括以下步骤:
(1)使用添加碳酸钙的固体鉴别分离培养基,筛选产透明圈的单个菌落,通过革兰氏染色观察细胞形态;
(2)PCR模板的制备
使用处于生长期的细菌悬浊液,配制为108cfu/mL浓度,直接进行菌液PCR;
(3)16S rDNA PCR扩增
D-乳酸发酵菌株16S rDNA PCR扩增所用引物为细菌16S rDNA通用引物,上游引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’;下游引物1492R:5’-TACGGCTACCTTGTTACGACTT-3’;
PCR扩增体系:Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL;PCR扩增程序为:95℃预变性5min,94℃变性1min,55℃退火1min,72℃延伸1min,30个循环,72℃延伸10min;
PCR产物使用1.5%琼脂糖凝胶电泳检测并胶回收1400bp处条带;
16S rDNA PCR胶回收产物进行测序,测序所得基因序列提交至NCBI进行在线BLAST比对以确认细菌种属;
(4)D-乳酸脱氢酶基因PCR扩增
将经16s rDNA测序确认种属的乳酸发酵菌株在鉴别分离培养基中连续培养至对数生长期,对培养基进行二代测序,以获得覆盖度≥98%,测序深度≥50X的全基因组序列,并对其中存在的D-乳酸脱氢酶基因进行注释分析;
根据测序结果,设计4种D-乳酸脱氢酶基因引物,其分别为D-dld型,上游引物D-dld_F:5’-TTGAAGCCGATGCCAATGAGACC-3’,下游引物D-dld_R:5’-GTGCTGCCAAATATAGATGCCCAAC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,55.5℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小354bp;
D-ldhA-1型,上游引物D-ldhA-1_F:5’-ATTGGCGGATACGGTCAGTTATGTG-3’,下游引物D-ldhA-1_R:5’-GCGTTTGACTGCGGTCTTCTCC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,56.6℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小281bp;
D-ldhA-2型,上游引物D-ldhA-2_F:5’-ATTGTGAACCTTGAAGCCGCTACC-3’,下游引物D-ldhA-2_R:5’-TAACCGTATCCGCCAGCTCAGG-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,57.8℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小316bp;
D-ldh型,上游引物D-ldh_F:5’-TCATTGATTTGGACCAGGCTAA-3’,下游引物D-ldh_R:5’-ACAATCGTATTCTCGCCTTCAA-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,51.0℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小993bp;
PCR扩增体系为:Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL;PCR产物使用2.5%琼脂糖凝胶电泳检测并分别胶回收354bp、281bp、316bp、993bp处条带;
PCR胶回收产物进行测序,测序所得基因序列提交至NCBI进行在线BLAST比对以确认相应片段所属的乳酸脱氢酶类型。
通过采用本发明的方法,有利于驯化分离携带D-LDHs的菌株,为D-乳酸发酵提供菌株资源。另外,通过采用本发明提供的D-乳酸脱氢酶基因的检测方法,能够有效且快速鉴别4种不同类型的D-乳酸脱氢酶同工酶基因序列,避免了传统检测方法费时费力且结果准确性差的缺点。
附图说明
图1示出了根据一些实施例的经过48h培养筛选得到的副干酪乳酪杆菌(Lacticaseibacillusparacasei)的图。
图2示出了根据一些实施例的副干酪乳酪杆菌的革兰氏染色图。
图3示出了根据一些实施例的奶酪乳酪杆菌(Lacticaseibacillus casei)的PCR结果图,其中:①D-dld型;②D-ldhA-1型;③D-ldhA-2型;④D-ldh型。
图4示出了根据一些实施例的PCR胶回收产物所属的乳酸脱氢酶类型,其中:①D-dld型;②D-ldhA-1型;③D-ldhA-2型;④D-ldh型。
具体实施方式
下面的实施例可以使本领域技术人员更全面地理解本发明,但不以任何方式限制本发明。
本申请提供一种D-乳酸发酵菌株的驯化筛选方法。本发明的D-乳酸发酵菌株筛选方法解决了D-乳酸发酵菌株筛选困难的问题。本发明首次利用乳酸脱氢酶催化反应可逆的原理,以LB培养基为基础,将LB培养基中作为碳源的酵母提取物替换为D-乳酸,将饲料样本放入后连续传代培养7天,培养结束后将培养液转接至添加碳酸钙的YPD培养基表面,根据是否出现透明圈以确认是否有乳酸产生。
本发明提供的D-乳酸发酵菌株的驯化筛选方法包括以下步骤:
(1)制备培养基
改良LB液体培养基(驯化培养基):胰蛋白胨10g、D-乳酸5g、氯化钠5g、超纯水1000mL,使用氢氧化钠和盐酸调节pH至5.7,121℃灭菌20分钟,分装至10mL无菌离心管,每管8.5mL;
YPD培养基(鉴别分离培养基):蛋白胨10g、葡萄糖20g、酵母浸出物5g、碳酸钙5g,如需固体培养基则加入琼脂14g,超纯水1000mL,pH值6.8,121℃灭菌20分钟;
(2)分别称取1g青、黄贮饲料样品(购自优宜邦生物科技(上海)有限公司),使用剪刀剪成2-3mm细小碎片,在无菌环境下放入装有改良LB液体培养基的10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时;
(3)24小时后取出培养箱中的离心管,多次上下颠倒混匀后,吸取200μL培养液放入另一新的装有改良LB液体培养基10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时,该操作在7天内,每日重复进行;
(4)取出连续培养7天的样品,使用接种环蘸取培养液,并划线至YPD固体培养基表面,封口膜封好皿盖,35.2℃条件下倒置培养,每间隔12小时观察一次,并及时挑取周围产透明圈的单个菌落,接种至置于10mL离心管的8.5mL YPD液体培养基中连续培养至底部出现较多沉淀即可。
本发明还提供了一种D-乳酸脱氢酶基因的检测方法。具体地,本发明提供了一种基于PCR技术检测D-乳酸发酵菌株携带的D-乳酸脱氢酶基因方法,结合一、二代测序技术,对分离菌株进行鉴定,能够有效且快速鉴别4种不同类型的D-乳酸脱氢酶同工酶基因序列,避免了传统检测方法费时费力且结果准确性差的缺点。
本发明提供的D-乳酸脱氢酶基因的检测方法包括以下步骤:
(1)使用添加碳酸钙(0.5wt%)的YPD固体培养基,在48小时内筛选出产透明圈较大的单一菌落,透明圈直径为3mm(图1);副干酪乳酪杆菌是革兰氏阳性菌,运用革兰氏染色法在电子显微镜下观察呈蓝紫色的杆状菌(图2)。
(2)PCR模板的制备
使用处于生长期的细菌悬浊液,配制为108cfu/mL(OD600=0.1)浓度,直接进行菌液PCR;
(3)16S rDNA PCR扩增
D-乳酸发酵菌株16S rDNA PCR扩增所用引物为细菌16S rDNA通用引物,上游引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’;下游引物1492R:5’-TACGGCTACCTTGTTACGACTT-3’;
PCR扩增体系(50μL):Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL。PCR扩增程序为:95℃预变性5min,94℃变性1min,55℃退火1min,72℃延伸1min,30个循环,72℃延伸10min;
PCR产物使用1.5%琼脂糖凝胶电泳检测并胶回收1400bp处条带;
16S rDNA PCR胶回收产物送北京睿博兴科生物技术有限公司广州分公司测序。测序所得基因序列提交至NCBI(http://blast.ncbi.nlm.nih.gov)进行在线BLAST比对以确认细菌种属;
(4)D-乳酸脱氢酶基因PCR扩增
将经16s rDNA测序确认种属的乳酸发酵菌株在YPD培养基中连续培养至对数生长期,培养基送至中国科学院深圳先进技术研究院进行二代测序,以获得覆盖度≥98%,测序深度≥50X的全基因组序列,并对其中存在的D-乳酸脱氢酶基因进行注释分析;
根据测序结果,设计4种D-乳酸脱氢酶基因引物,其分别为D-dld型,上游引物D-dld_F:5’-TTGAAGCCGATGCCAATGAGACC-3’,下游引物D-dld_R:5’-GTGCTGCCAAATATAGATGCCCAAC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,55.5℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小354bp;
D-ldhA-1型,上游引物D-ldhA-1_F:5’-ATTGGCGGATACGGTCAGTTATGTG-3’,下游引物D-ldhA-1_R:5’-GCGTTTGACTGCGGTCTTCTCC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,56.6℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小281bp;
D-ldhA-2型,上游引物D-ldhA-2_F:5’-ATTGTGAACCTTGAAGCCGCTACC-3’,下游引物D-ldhA-2_R:5’-TAACCGTATCCGCCAGCTCAGG-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,57.8℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小316bp;
D-ldh型,上游引物D-ldh_F:5’-TCATTGATTTGGACCAGGCTAA-3’,下游引物D-ldh_R:5’-ACAATCGTATTCTCGCCTTCAA-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,51.0℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小993bp。
PCR扩增体系(50μL)为:Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL;PCR产物使用2.5%琼脂糖凝胶电泳检测并分别胶回收354bp、281bp、316bp、993bp处条带。菌液PCR结果如图3所示。
PCR胶回收产物送北京睿博兴科生物技术有限公司广州分公司测序。测序所得基因序列提交至NCBI(http://blast.ncbi.nlm.nih.gov)进行在线BLAST比对以确认该片段所属的乳酸脱氢酶类型(图4)。表1示出了相应菌株的乳酸产量。
表1
本领域技术人员应理解,以上实施例仅是示例性实施例,在不背离本发明的精神和范围的情况下,可以进行多种变化、替换以及改变。
Claims (2)
1.一种D-乳酸发酵菌株的驯化筛选方法,其特征在于,包括以下步骤:
(1)制备驯化培养基和鉴别分离培养基:
驯化培养基:胰蛋白胨10g、D-乳酸5g、氯化钠5g、超纯水1000mL,使用氢氧化钠和盐酸调节pH至5.7,121℃灭菌20分钟,分装至10mL无菌离心管,每管8.5mL;
鉴别分离培养基:蛋白胨10g、葡萄糖20g、酵母浸出物5g、碳酸钙5g,如需固体培养基则加入琼脂14g,超纯水1000mL,pH值6.8,121℃灭菌20分钟;
(2)分别称取1g青、黄贮饲料,使用剪刀剪成2-3mm细小碎片,在无菌环境下放入装有驯化培养基的10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时;
(3)24小时后取出培养箱中的离心管,多次上下颠倒混匀后,吸取200μL培养液放入另一新的装有驯化培养基10mL离心管中,使用封口膜包裹离心管盖,35.2℃条件下连续培养24小时,该操作在7天内,每日重复进行;
(4)取出连续培养7天的样品,使用接种环蘸取培养液,并划线至鉴别分离培养基表面,封口膜封好皿盖,35.2℃条件下倒置培养,每间隔12小时观察一次,并及时挑取周围产透明圈的单个菌落,接种至置于10mL离心管的8.5mL液体鉴别分离培养基中连续培养至底部出现较多沉淀。
2.一种D-乳酸脱氢酶基因的检测方法,其特征在于,包括以下步骤:
(1)使用添加碳酸钙的固体鉴别分离培养基,筛选产透明圈的单个菌落,通过革兰氏染色观察细胞形态;
(2)PCR模板的制备
使用处于生长期的细菌悬浊液,配制为108cfu/mL浓度,直接进行菌液PCR;
(3)16S rDNA PCR扩增
D-乳酸发酵菌株16S rDNA PCR扩增所用引物为细菌16S rDNA通用引物,上游引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’;下游引物1492R:5’-TACGGCTACCTTGTTACGACTT-3’;
PCR扩增体系:Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL;PCR扩增程序为:95℃预变性5min,94℃变性1min,55℃退火1min,72℃延伸1min,30个循环,72℃延伸10min;
PCR产物使用1.5%琼脂糖凝胶电泳检测并胶回收1400bp处条带;
16S rDNA PCR胶回收产物进行测序,测序所得基因序列提交至NCBI进行在线BLAST比对以确认细菌种属;
(4)D-乳酸脱氢酶基因PCR扩增
将经16s rDNA测序确认种属的乳酸发酵菌株在鉴别分离培养基中连续培养至对数生长期,对培养基进行二代测序,以获得覆盖度≥98%,测序深度≥50X的全基因组序列,并对其中存在的D-乳酸脱氢酶基因进行注释分析;
根据测序结果,设计4种D-乳酸脱氢酶基因引物,其分别为D-dld型,上游引物D-dld_F:5’-TTGAAGCCGATGCCAATGAGACC-3’,下游引物D-dld_R:5’-GTGCTGCCAAATATAGATGCCCAAC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,55.5℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小354bp;
D-ldhA-1型,上游引物D-ldhA-1_F:5’-ATTGGCGGATACGGTCAGTTATGTG-3’,下游引物D-ldhA-1_R:5’-GCGTTTGACTGCGGTCTTCTCC-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,56.6℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小281bp;
D-ldhA-2型,上游引物D-ldhA-2_F:5’-ATTGTGAACCTTGAAGCCGCTACC-3’,下游引物D-ldhA-2_R:5’-TAACCGTATCCGCCAGCTCAGG-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,57.8℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小316bp;
D-ldh型,上游引物D-ldh_F:5’-TCATTGATTTGGACCAGGCTAA-3’,下游引物D-ldh_R:5’-ACAATCGTATTCTCGCCTTCAA-3’,PCR扩增程序为:94℃预变性5min,94℃变性30s,51.0℃退火30s,72℃延伸1min,30个循环,72℃延伸5min,目标片段大小993bp;
PCR扩增体系为:Premix TaqTM 25μL,上游引物27F 1μL,下游引物1492R 1μL,模板菌液5μL,ddH2O,18μL;PCR产物使用2.5%琼脂糖凝胶电泳检测并分别胶回收354bp、281bp、316bp、993bp处条带;
PCR胶回收产物进行测序,测序所得基因序列提交至NCBI进行在线BLAST比对以确认相应片段所属的乳酸脱氢酶类型。
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