CN109504632B - 一株枯草芽孢杆菌及其应用 - Google Patents
一株枯草芽孢杆菌及其应用 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12N1/205—Bacterial isolates
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Abstract
本发明提供了一株枯草芽孢杆菌是(Bacillus subtilis)XWS‑8,该菌株已于2011年12月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.5590。并提供了降解纤维素领域中的应用。试验表明,枯草芽孢杆菌(Bacillus subtilis)XWS‑8 CGMCC No.5590产纤维素酶的活力明显高于其他细菌,其对纤维素降解能力强。
Description
技术领域
本发明涉及一株芽孢杆菌及其应用,尤其涉及一株枯草芽孢杆菌及其在降解纤维素领域中的应用。
背景技术
秸秆是纤维组分含量很高的农作物残留物,利用纤维素分解菌将纤维素降解成短链糖,提供牲畜对秸秆饲料的消化吸收率,是当前饲料业的一个发展方向。目前研究较多的纤维素降解菌是霉菌,其中木霉、曲霉、根霉和青霉具有较强的酶活力,尤以绿色木霉、里氏木霉、康氏木霉为典型,是目前公认的较好的纤维素酶生产菌。但霉菌为好气性微生物,而饲料发酵是在少氧或缺氧环境中进行的,故产纤维素酶活高的厌氧及兼性厌氧细菌更具有应用上的现实意义。产芽孢的细菌因芽孢的形成,抗逆性强,能抵御不良环境,在耐酸、耐碱、耐高温等方面有明显的优势,更利于实际操作和工业生产,因此成为目前研究产纤维素酶微生物的一个重要方向。但是目前研究出的产纤维素酶且适用于秸秆发酵制备动物饲料的菌株并不多,且存在产酶成本高、酶活性差、适用的pH范围小等问题,因此急需寻找更多产酶活性高且稳定,作用范围更广泛微生物菌株。
发明内容
本发明的目的是提供一株能够降解纤维素的枯草芽孢杆菌。
本发明所提供的枯草芽孢杆菌是(Bacillus subtilis)XWS-8,该菌株已于2011年12月14日 保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.5590。
枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590 菌体菌体呈杆状,革兰氏染色呈阳性,菌体长约3.1μm,宽约0.9μm,芽孢呈椭圆形中生,不膨大,长约2.0μm,宽约0.9μm,生长温度30~37℃。生理生化特性如表1所示:
表1 枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590的生理生化性质
注:“+”表示阳性,“–”表示阴性
枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590的基因组DNA具有如下特异序列:
AGGTGACCGT CGGGGTGCTG AAGAATATCG GAGATACAGG GTCTGACTTTGCATCACTGA 60
CAGATGACAT AGAGGAAGTG ACGCTGGATA AGGGCAGCCC CGGCAGTAAAATCGCCGTGC 120
TTGAAGTGGA CGGCACGATT GAGGATAACG GCGGGTCCGC TGGCCTGCTCAGCTCAGGCG 180
GGTATGATCA CAGATCATTT TTAAAACAGG TTGAGCGTGC GAAAGAAGAC AAAAGCGTCA240。
本发明的另一个目的是提供枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCCNo.5590在降解纤维素领域中的应用,尤其是将该菌株用于秸秆发酵中降解纤维素以制取动物饲料。
枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590产纤维素酶的活力明显高于其他细菌,其对纤维素降解能力强。
附图说明
图1 菌体形态照片;
图2 纤维素酶活力标准曲线图。
具体实施方式
实施例一:枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590的筛选与保藏
1、培养基
CMC-Na培养基(g/L):CMC-Na 5.0,蛋白胨5.0,酵母膏0.5,KH2PO4 1.5,MgSO4 0.2,NaCl 5.0,琼脂15.0g,pH7.0,蒸馏水配制。
刚果红纤维素钠培养基(g/L):CMC-Na 5.0,蛋白胨5.0,酵母膏0.5,KH2PO4 1.5,MgSO4 0.2,NaCl 5.0,琼脂15.0g,刚果红0.2,pH7.0,蒸馏水配制。
NA培养基(g/L):牛肉膏5.0,蛋白胨10.0,NaCl 5.0,pH7.2~7.4,琼脂20,蒸馏水配制,用于菌株的固体培养。
NB培养基(g/L):牛肉膏5.0,蛋白胨10.0,NaCl 5.0,pH7.2-7.4,蒸馏水配制,用于菌株的固体培养。
2、微生物菌群来源
新鲜牛粪,取自河北农业大学牧场健康奶牛直肠或粪便。
3、初筛
1)取新鲜牛粪10.0g于试管中,在水浴锅内80℃水浴10分钟,杀死菌体。
2)称取5.0g处理过的牛粪于150mL NB培养基中37℃170r/min培养48小时。将培养后的发酵液进行梯度稀释,依次稀释为10-1、10-2、10-3、10-4、10-5、10-6。分别取稀释到10-4、10-5、10-6的发酵液20-30μL到刚果红纤维素钠平板中,三角刮涂布,37℃恒温箱中倒置培养48小时。
3)观察平板,标记出圈的菌种、测量透明圈直径并记录试验结果。
本试验筛选具有水解圈的菌株,经筛选得到既产纤维素酶又产芽孢的菌株17株。
4、复筛
对初筛菌株中水解圈直径较大的菌株进行液体发酵,测定发酵48小时后发酵上清液的酶活性,结果见表2(酶活力测定方法如实施例四所示)。
表2 复筛测定的酶活力结果
由表2可见,菌株XWS-8酶活力最强。该菌株已于2011年12月14日 保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.5590。
实施例二:枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590的种属鉴定
根据菌株的菌落形态特征、菌体形态特征、革兰氏染色性状、芽孢染色性状,有关生理生化鉴别试验,参照《常见细菌系统鉴定手册》、《伯杰氏细菌鉴定手册》进行属和种的鉴定。
1、菌落特征和菌体形态特征
根据NA培养基上生长的菌株XWS-8的菌落形态,初步判断其为细菌菌落,培养24h的菌落为不透明的暗黄色,呈近圆形,边缘不整齐,表面有皱褶,中间有突起。
菌株XWS-8经染色后置于光学显微镜下观察,菌体呈杆状,革兰氏染色呈阳性,菌体长约3.1μm,宽约0.9μm,芽孢呈椭圆形中生,不膨大,长约2.0μm,宽约0.9μm,如图1所示。
2、生理生化性质鉴定
糖、醇类发酵培养基:(NH4)2HPO4 1.0g,KCl 0.2g,MgSO4 0.2g,酵母0.2g,琼脂5~6g,糖或醇类10g,蒸馏水1000mL,溴甲酚紫(0.04%)15mL,pH7.0~7.2。
甲基红(M.R)试验培养基:蛋白胨5.0g,葡萄糖5.0g,NaCl 5.0g,蒸馏水1000mL,pH7.0~7.2。
V-P试验培养基:蛋白胨5.0g,葡萄糖5.0g,NaCl 5.0g,蒸馏水1000mL,pH7.0~7.2。
淀粉水解培养基:蛋白胨10.0g,牛肉膏5.0g,NaCl 5.0g,蒸馏水1000mL,可溶性淀粉2.0g,pH7.2~7.4。
硝酸盐还原试验培养基:KNO3 1.0g,蛋白胨10.0g,NaCl 5.0g,牛肉膏3.0g,蒸馏水1000mL,pH7.0~7.6。
脲酶培养基:NaCl 5.0g,KH2PO4 2.0g,蛋白胨1.0g,葡萄糖1.0g,酚红(0.2%酚红溶液)6.0mL,琼脂20.0g,蒸馏水1000mL,pH6.8~6.9。
吲哚试验培养基:1.0%胰胨水溶液,pH7.2~7.6。
苯丙氨基酸脱氨酶培养基:NaCl 5.0g,Na2HPO4 1.0g,DL-苯丙氨酸2.0g(或L-苯丙氨酸)1.0g,酵母膏3.0g,琼脂12.0g,蒸馏水1000mL,pH7.0。
明胶液化试验培养基:蛋白胨5.0g,明胶100~150g,蒸馏水1000mL,pH7.2~7.4。
参照东秀珠等的《常见细菌系统鉴定手册》对菌株进行生理生化实验。主要进行H2S产生试验、吲哚试验、明胶液化试验、过氧化氢酶(接触酶)试验、氨基酸脱羧酶试验、淀粉水解、V-P(乙酰甲基甲醇)试验、M.R(甲基红,Methyl Red)试验、柠檬酸盐利用、石蕊牛奶分解、糖、醇发酵、苯丙氨酸脱氨酶、产氨试验、脲酶(尿素水解)试验、亚硝酸盐还原试验、硝酸盐还原试验和荧光色素试验。
试验结果见表1。
3、DNA提取和16S rDNA基因扩增
参考Kim等和Rainey等的方法提取细菌总DNA。1%琼脂糖电泳检测。
引物为通用引物,正向引物为27F:5’-AGAGTTTGATCCTGG CTCAG-3’,反向引物为1495R:5’-CTACGGCTACCTTGTT ACGA-3’。
PCR反应体系:DNA(70ng/μL)模板2μL;dNTP Mixture(2.5 mmol/L)2.5μL;27F(20μmol/L)1.5μL;1495F(20 μmol/L)1.5μL;10×ExTaq Buffer(Mg2+ pluse)5μL;ExTaq DNA聚合酶 0.2μL;补足ddH2O到50μL。
PCR条件为:94℃预变性3min;然后94℃变性1min、55℃退火1min、72℃延伸3min,共30 个循环;最后72℃延伸5min。PCR 产物经试剂盒纯化后,送上海生工生物工程技术服务有限公司测序。
将所测得的16S rDNA序列用BLAST软件与GenBank数据库进行相似性分析,并与GenBank中的相近序列在Clustal X(1.8)程序包中进行多重序列匹配排列(Multiplealignments)分析,最后形成一个多重序列匹配排列阵,其中形成的缺口用横杠“-”填补,用Neighbor- Joining法构建系统发育树。
4、种属鉴定
通过对XWS-8菌株的形态特征和生理生化特性鉴定的结果,对照《伯杰氏细菌鉴定手册》和《常见细菌系统鉴定手册》,鉴定其为枯草芽孢杆菌(Bacillus amyloliquefacien),16S rDNA序列相似度达99.92%、生理生化特征相似度达98%。
表3 菌株XWS-8与参比菌株的16S rDNA序列相似性
实施例三:分子标记
1、菌株DNA提取及RAPD特异标记的筛选
用CTAB法提取各实验菌株和参试标准菌株的基因组DNA。利用琼脂糖凝胶电泳和紫外分光光度计检测样品的纯度及浓度,然后用TE缓冲液稀释成100 ng·μL-1备用。采用上海生工生物工程有限公司合成的30条随机引物对各菌株进行RAPD扩增。用筛选到的16个引物,以各实验菌株和参试菌株(表1)基因组DNA为模板进行PCR扩增。PCR反应体系:总体系为20μL,其中ddH2O 14.2μL,10×PCR Buffer(with Mg2+)2μL,10μmol/L引物1μL,5U/μlTap DNA聚合酶0.2μL,2.5mmol/L dNTP1.6μL,模板DNA1.0μL。扩增反应过程为:95℃预变性5min,94℃变性30s,36℃退火40s,72℃延伸1.5min,循环40次,最后72℃延伸5min。取扩增产物5μL电泳检测,筛选各菌株的RAPD特异条带。
2、特异片段的回收及克隆
使用DNA胶回收试剂盒切胶回收筛选到的RAPD特异条带,电泳检测后16℃水浴连接到pMD19-T载体上,然后热激法转化感受态细胞DH5α,均匀涂布于含100μg/mL氨苄西林钠(Amp)的LB琼脂平板上,挑取单个菌落进行菌落PCR。将菌落PCR阳性的克隆送宝锐通生物科技(北京)有限公司测序。
3、SCAR标记的转化和验证
通过对测序结果分析利用Primer Premier5.0软件,设计一对SCAR引物N1/N2。利用合成的引物N1/N2,以各菌株和参试菌株(表1)基因组DNA为模板进行PCR扩增,验证SCAR标记的准确性,同时以细菌16S rDNA片段作为对照。取扩增产物5μL进行电泳检测。
4、结果
枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590的基因组DNA具有如下特异序列:
AGGTGACCGT CGGGGTGCTG AAGAATATCG GAGATACAGG GTCTGACTTTGCATCACTGA 60
CAGATGACAT AGAGGAAGTG ACGCTGGATA AGGGCAGCCC CGGCAGTAAAATCGCCGTGC 120
TTGAAGTGGA CGGCACGATT GAGGATAACG GCGGGTCCGC TGGCCTGCTCAGCTCAGGCG 180
GGTATGATCA CAGATCATTT TTAAAACAGG TTGAGCGTGC GAAAGAAGAC AAAAGCGTCA240。
实施例四:对菌株发酵液进行CMC(羧甲基纤维素)酶活力测定,为1677.41U/mL
1、所用试剂:0.05M醋酸-醋酸钠缓冲液(g/L):醋酸钠3.35,冰醋酸1.3mL,pH 4.6,蒸馏水配制。
3,5-二硝基水杨酸显色液(DNS)(g/L):重结晶的3,5-二硝基水杨酸10,NaOH2O,酒石酸钾钠200,重蒸苯酚2,无水亚硫酸钠0.5,蒸馏水配制。
0.5%羧甲基纤维素钠溶液:CMC 5,醋酸-醋酸钠缓冲液配置。
2、所用方法:
葡萄糖标准曲线的绘制:
分别取1mg/mL葡萄糖标准液0,0.2,0.4,0.6,0.8,1.0,1.2mL依次加入7支25mL的比色试管中,以蒸馏水补加到2.0mL,再加入DNS试剂1.5mL,在沸水中煮沸5min,流水冷却,加蒸馏水21.5mL摇匀,在540nm处进行比色测定,用空白管溶液调零点,记录吸光度值,以葡萄糖浓度为横坐标,吸光度值为纵坐标绘制出标准曲线。标准曲线如图2所示。
发酵液CMC(羧甲基纤维素)酶活力测定
1)将初筛的菌种分别接种于不含葡萄糖的NB培养基中37℃ 180 r/min培养48h。
2)分别吸取各个菌种的发酵液1.5mL于Ependoff管中,10000 r/min离心10min后上清液即为粗酶液。
3)试管中加入20.00mg/mL CMC溶液1.5 mL,加入适当稀释的粗酶液0.5 mL,在50℃水浴中精确反应30min,加入1.5 mL DNS溶液,置沸水浴5min,取出,流水冷却,加入蒸馏水5 mL,540 nm处分光光度计测定其OD值(0.5 cm比色杯)。(空白是用未接菌的NB培养基代替粗酶液)。
酶活力定义:1 mL酶液在pH 4.6,50℃条件下每分钟水解CMC所产生的还原糖微克数为一个酶活单位,以u/mL表示。
计算公式:
酶活力(葡萄糖微克数/克,毫升)=OD×H×N×2×1000/30
式中:30-换算成每分钟;
H——标准曲线系数
N——酶液稀释倍数
2——换算成每毫升酶液
1000——葡萄糖毫克数换算成微克数
实施例五:玉米秸秆、小麦秸秆、水稻秸秆纤维素降解能力考察
中性洗涤液:在加热的条件下,在烧杯中加入18.61 g EDTA二钠盐和6.81 g四硼酸钠,加入约150 mL蒸馏水,使之溶解。将30g月桂基硫酸钠(化学纯)和乙二醇独乙醚(化学纯)溶于700 mL热水中,合并上述二溶液。再将4.56 g无水磷酸氢二钠溶于约150 mL热水中,再并入上述溶液,用磷酸调节pH值到6.9~7.1,加水至1000mL。
2 mol/L盐酸溶液:取167mL浓盐酸(密度1.19g/mL)置于盛有800 mL水的1 L容量瓶中,用水定容至刻度。
72 %硫酸溶液:取665 mL浓硫酸(密度1. 84 g/ mL)置于盛有300 mL水的1 L容量瓶(容量瓶外用冷水浴)中,待冷却至室温后,用水定容至刻度。
秸秆发酵:称取粉碎后的秸秆粉200Kg,用蒸汽灭菌15min后,放入3,000mL大三角瓶中,冷却至常温后,接入50mLOD600=2.0的种子液。搅拌均匀后放入培养箱中,以堆积状态(厚度30cm)进行培养。堆积放置过程中,为防止秸秆因发酵生热、温度升高,应控制温度27~32℃。
纤维素含量的测定方法:准确称取55℃烘干过夜至恒重的发酵玉米秸秆样品1.0000g(W1),加入2mol/L HCl 70mL,105℃保温50min后依次用95%乙醇、无水乙醇和丙酮抽滤洗涤2次,抽滤之前准确称量滤纸重量。将残渣连同滤纸置于烘箱中,60℃干燥至恒重减去滤纸重为W2。将残渣完全转移于烧杯中,加入10mL 72%硫酸,20℃降解4h后,加入蒸馏水90mL过夜。次日,将样品抽滤,蒸馏水将残渣洗至pH值为6.5,将残渣连同滤纸置于烘箱中,60℃干燥至恒重减去滤纸重为W3。
用以下公式计算木质素的含量与降解率:
纤维素含量(%)=(W3-W2)/W1×100%
纤维素降解率(%)=(对照玉米秸秆中纤维素含量-发酵处理后玉米秸秆纤维素含量)/对照玉米秸秆纤维素含量×100%。
结果:玉米秸秆纤维素降解能力达25.68%;小麦秸秆纤维素降解能力达23.51%;水稻秸秆纤维素降解能力达24.01%。
序列表
<110> 河北农业大学
<120> 一株枯草芽孢杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 240
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 1
aggtgaccgt cggggtgctg aagaatatcg gagatacagg gtctgacttt gcatcactga 60
cagatgacat agaggaagtg acgctggata agggcagccc cggcagtaaa atcgccgtgc 120
ttgaagtgga cggcacgatt gaggataacg gcgggtccgc tggcctgctc agctcaggcg 180
ggtatgatca cagatcattt ttaaaacagg ttgagcgtgc gaaagaagac aaaagcgtca 240
Claims (3)
1.枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590。
2.枯草芽孢杆菌(Bacillus subtilis)XWS-8 CGMCC No.5590在降解纤维素领域中的应用。
3.根据权利要求2所述的应用,其特征在于,所述菌株用于秸秆发酵中降解纤维素以制取动物饲料。
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