CN117264861B - 一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用 - Google Patents
一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明涉及微生物基因工程技术领域,尤其涉及一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用。本发明涉及的枯草芽孢杆菌C6‑AEA3分类为Bacillus subtilis,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2023年8月31日,保藏编号为CCTCC NO:M 20231579。本发明得到的枯草芽孢杆菌C6‑AEA3具有高纤维素酶活性,能高效降解秸秆饲料中的纤维素,为农作物秸秆生物降解提供有效的菌种资源。
Description
技术领域
本发明涉及微生物基因工程技术领域,尤其涉及一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用。
背景技术
农作物秸秆,是大自然中最宝贵、最便宜的可再生资源之一,是潜在的动物饲料来源,同时也是规模最大的一类农业废弃物,全世界每年可产生超过五十亿吨的农作物残茬。农作物秸秆的主要成分木质素、纤维素、半纤维素等,均属于芳香族高分子化合物,具有难以在自然环境中被降解的复杂三维结构,阻碍纤维素和半纤维素被进一步利用,因而常对秸秆进行预处理以提高纤维素的利用率。预处理方法可分为物理法、化学法和生物法。相对于前两种方法,生物降解更能节省人力、物力和财力,是一种较为理想的降解纤维素的方法。纤维素酶可分为三类:内切葡聚糖酶(Endoglucanase,EG,EC:3.2.1.4)、外切葡聚糖酶(Exoglucanase,CBH,EC:3.2.1.91)和β-葡萄糖苷酶(β-glucosidase,BG,EC:3.2.1.21),它们协同作用于纤维素使其彻底降解为葡萄糖。
枯草芽孢杆菌作为一种被深入研究的模式菌,其代谢机理和遗传背景相对清晰,具有异源蛋白分泌能力强、低品质碳源上生长特性好、无明显密码子偏好性等特点,属于我国的饲用益生菌准用菌种,现已应用于构建酶、维生素、功能性糖等天然产物合成的微生物细胞工厂。虽然枯草芽孢杆菌本身具备良好的纤维素酶分泌能力,但因其纤维素酶系不完整,单菌直接作用于粗饲料的降解效果不佳,尤其体现在外切葡聚糖酶和木质素酶的缺乏方面,不能高效破坏植物细胞壁的纤维结晶结构。因此,通过对枯草芽孢杆菌的遗传改造或异源酶基因的高效表达,获得具有益生菌特性且能高效降解纤维素的枯草芽孢杆菌基因工程重组菌能够更加满足畜禽养殖业中饲用微生物添加的生产需求。
发明内容
本发明的目的在于提供一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一株共表达多元纤维素酶基因的枯草芽孢杆菌C6-AEA3,所述枯草芽孢杆菌C6-AEA3分类为Bacillussubtilis,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2023年8月31日,保藏编号为CCTCCNO:M 20231579。
本发明还提供了所述的枯草芽孢杆菌C6-AEA3的构建方法,包括如下步骤:
以野生型枯草芽孢杆菌为出发菌株,依次在所述出发菌株中敲入内切葡聚糖酶基因、外切葡聚糖酶基因和β-葡聚糖酶基因;
所述野生型枯草芽孢杆菌为Bacillus subtilis C6,野生型枯草芽孢杆菌Bacillus subtilis C6,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2022年9月26日,保藏编号为CCTCCNO:M 20221488。
作为优选,所述敲入的方法基于CRISPR/Cas9系统。
作为优选,所述内切葡聚糖酶基因为内切葡聚糖酶基因gene2006;所述内切葡聚糖酶基因gene2006的核苷酸序列如SEQ ID NO.1所示;
敲入所述内切葡聚糖酶基因的位点为野生型枯草芽孢杆菌的aprE位点。
作为优选,所述外切葡聚糖酶基因为外切葡聚糖酶基因Cel48S;所述外切葡聚糖酶基因Cel48S的核苷酸序列如SEQ ID NO.2所示;
敲入所述外切葡聚糖酶基因的位点为野生型枯草芽孢杆菌的epr位点。
作为优选,所述β-葡聚糖酶基因为β-葡聚糖酶基因gene4296;所述β-葡聚糖酶基因gene4296的核苷酸序列如SEQ ID NO.3所示;
敲入所述β-葡聚糖酶基因的位点为野生型枯草芽孢杆菌的amyE位点。
本发明还提供了所述的枯草芽孢杆菌C6-AEA3或者所述的构建方法构建得到的枯草芽孢杆菌C6-AEA3在制备降解纤维素的产品中的应用。
本发明还提供了所述的枯草芽孢杆菌C6-AEA3或者所述的构建方法构建得到的枯草芽孢杆菌C6-AEA3在制备降解秸秆中纤维素的产品中的应用;
所述秸秆为玉米秸秆、小麦秸秆和水稻秸秆中的一种或几种。
本发明还提供了一种降解秸秆中纤维素的方法,包括如下步骤:
将秸秆、浸润培养基与发酵菌液混合,发酵6~8d;
所述发酵菌液为所述的枯草芽孢杆菌C6-AEA3或者所述的构建方法构建得到的枯草芽孢杆菌C6-AEA3制备得到的发酵菌液;
所述发酵菌液的制备方法为:将枯草芽孢杆菌C6-AEA3接种于LB培养基中,培养15~17h,得到活化的菌液;将所述活化的菌液接种于LB培养基中,培养20~28h,得到发酵菌液。
作为优选,所述秸秆为玉米秸秆、小麦秸秆和水稻秸秆中的一种或几种;
所述秸秆的粒径≤1mm;
所述浸润培养基以水为溶剂,还包括如下浓度的组分:
硝酸铵4~6g/L、七水硫酸镁4~6g/L、氯化钠0.8~1.2g/L;
所述秸秆与浸润培养基的质量体积比为3~5g:15~25mL;
所述秸秆与发酵菌液的质量体积比为3~5g:1mL;
所述发酵的温度为36~38℃。
本发明提供的一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用,本发明的方案具有如下优点:
本发明获得的共表达多元纤维素酶基因枯草芽孢杆菌C6-AEA3具有高纤维素酶活,能高效降解秸秆饲料中的纤维素,为农作物秸秆生物降解提供有效的菌种资源。
本发明构建得到的枯草芽孢杆菌C6-AEA3的内切葡聚糖酶活、外切葡聚糖酶活等纤维素酶活相关指标较出发菌株显著升高。
本发明构建的纤维素酶基因枯草芽孢杆菌C6-AEA3能有效破坏多种秸秆饲料的纤维素表面形态结构,玉米秸秆、小麦秸秆和水稻秸秆的中性洗涤纤维(NDF)含量分别降低5.6%、2.8%、5.7%,酸性洗涤纤维(ADF)含量分别降低21.7%、17.3%、19.4%,半纤维素含量分别降低1.1%、4.3%、3.2%,木质素含量分别降低2.7%、8.7%、5.9%,纤维素结晶度分别降低3.1%,8.8%、12.3%。本发明构建的菌株C6-AEA3能够显著降解秸秆类纤维素生物质,为提高畜牧行业饲料的利用效率奠定基础。
附图说明
图1为基于CRISPR/Cas9系统依次敲入3种纤维素酶基因的基因编辑方法示意图。
图2为枯草芽孢杆菌C6-AEA3的酶学性质测定结果(A表示验证电泳图,1泳道表示aprE位点敲入内切葡聚糖酶基因gene2006后条带大小,2泳道表示aprE位点敲入内切葡聚糖酶基因gene2006前条带大小,3泳道表示epr位点敲入外切葡聚糖酶基因Cel48S后条带大小,4泳道表示epr位点敲入外切葡聚糖酶基因Cel48S前条带大小,5泳道表示amyE位点敲入β-葡聚糖酶基因gene4296后条带大小,6泳道表示amyE位点敲入β-葡聚糖酶基因gene4296前条带大小;B表示生长曲线图;C表示酶活性结果图)。
图3为枯草芽孢杆菌C6-AEA3对玉米秸秆、小麦秸秆或水稻秸秆中纤维素的降解能力。
图4为枯草芽孢杆菌C6-AEA3降解玉米秸秆、小麦秸秆或水稻秸秆中纤维素后的傅里叶变换红外光谱图。
图5为枯草芽孢杆菌C6-AEA3降解玉米秸秆、小麦秸秆或水稻秸秆中纤维素后的X-射线衍射图。
图6为枯草芽孢杆菌C6-AEA3降解玉米秸秆、小麦秸秆或水稻秸秆中纤维素后的扫描电镜图。
保藏说明
枯草芽孢杆菌C6-AEA3分类为Bacillus subtilis,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2023年8月31日,保藏编号为CCTCC NO:M20231579。
野生型枯草芽孢杆菌Bacillus subtilis C6,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2022年9月26日,保藏编号为CCTCCNO:M 20221488。
具体实施方式
在本发明中,所述内切葡聚糖酶基因为内切葡聚糖酶(EC:3.2.1.4)基因gene2006;所述内切葡聚糖酶(EC:3.2.1.4)基因gene2006的核苷酸序列如SEQ ID NO.1所示;敲入所述内切葡聚糖酶(EC:3.2.1.4)基因的位点为野生型枯草芽孢杆菌的aprE位点。
所述外切葡聚糖酶基因为外切葡聚糖酶(EC:3.2.1.91)基因Cel48S;所述外切葡聚糖酶(EC:3.2.1.91)基因Cel48S的核苷酸序列如SEQ ID NO.2所示;敲入所述外切葡聚糖酶(EC:3.2.1.91)基因的位点为野生型枯草芽孢杆菌的epr位点。
所述β-葡聚糖酶基因为β-葡聚糖酶(EC:3.2.1.21)基因gene4296;所述β-葡聚糖酶(EC:3.2.1.21)基因gene4296的核苷酸序列如SEQ ID NO.3所示;敲入所述β-葡聚糖酶(EC:3.2.1.21)基因的位点为野生型枯草芽孢杆菌的amyE位点。
在本发明中,所述野生型枯草芽孢杆菌筛选自圆唇散白蚁(ReticulitermesLabralis)肠道中。
所述敲入的方法基于CRISPR/Cas9系统进行设置的。使用在线设计工具CRISPROR设计靶向sgRNA,PCR产物与BsaI酶切后的穿梭质粒pJOE8999连接,上、下游同源臂序列及纤维素酶基因片段连接至SalI和XbaI酶切位点之间,转化至出发菌中,去除质粒,获得无抗性枯草芽孢杆菌。依次敲入内切葡聚糖酶(EC:3.2.1.4)基因gene2006、外切葡聚糖酶(EC:3.2.1.91)基因Cel48S和β-葡聚糖酶(EC:3.2.1.21)基因gene4296,获得多元纤维素酶基因枯草芽孢杆菌C6-AEA3。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
本发明实施例中所述的野生型枯草芽孢杆菌Bacillus subtilis C6菌株为CN116463238A《一株可降解纤维素的枯草芽孢杆菌及应用》中所述的Bacillussubtilis C6。
实施例1
共表达多元纤维素酶基因的枯草芽孢杆菌C6-AEA3的构建
根据野生型枯草芽孢杆菌Bacillus subtilis C6菌株(全基因组序列位于NCBI,GenBank:CP123621.1)上aprE位点、epr位点和amyE位点的基因序列,使用在线设计工具CRISPROR设计靶向sgRNA(表1),如SEQ ID NO.4~9所示。经BsaI酶切后的穿梭质粒pJOE8999与sgRNA退火产物连接,并转化至大肠杆菌感受态(E.coli JM109)中,用卡那霉素抗性的平板筛选,挑取单克隆用引物“JOE-sg-F/-R”(表1)SEQ ID NO.10~15所示,进行菌落PCR鉴定,将显示出的条带大小与预期相符的菌株摇菌,提质粒,得到sgRNA载体质粒,并测序鉴定。
表1CRISPR/Cas9基因敲入载体的sgRNA序列
根据Bacillus subtilis C6菌株的基因组信息,设计上、下游同源臂序列及纤维素酶基因序列(内切葡聚糖酶(EC:3.2.1.4)基因gene2006 SEQ ID NO.1、外切葡聚糖酶(EC:3.2.1.91)基因Cel48S SEQ ID NO.2、β-葡聚糖酶(EC:3.2.1.21)基因gene4296 SEQID NO.3的扩增引物如表2所示(SEQ ID NO.16~33)并回收。以sgRNA载体为骨架,在SalI和XbaI酶切位点进行双酶切,回收产物与上、下游同源臂序列及纤维素酶基因序列以无缝克隆的方式连接,转化至大肠杆菌感受态(E.coli JM109)中,用卡那霉素抗性的平板筛选,挑取单克隆用引物“JOE-TYB-F/-R”如表2所示(SEQ ID NO.34~35)进行菌落PCR鉴定,将显示出的条带大小与预期相符的菌株摇菌,提质粒,并测序鉴定。最终获得连接了sgRNA、对应同源臂片段和纤维素酶片段的基因敲入载体。
SEQ ID NO.1:
gctgcagggacaaaaacgccagtagccaagaatggccagcttagcataaaaggtacacagctcgttaaccgagacggtaaagcggtacagctgaaggggatcagttcacacggattgcaatggtatggagaatatgtcaataaagacagcttaaaatggctgagggacgattggggtatcaccgttttccgtgcagcgatgtatacggcagatggcggttatattgacaacccgtccgtgaaaaataaagtaaaagaagcggttgaagcggcaaaagagcttgggatatatgtcatcattgactggcatatcttaaatgacggtaatccaaaccaaaataaagagaaggcaaaagaattcttcaaggaaatgtcaagtctttacggaaacacgccaaacgtcatttatgaaattgcaaacgaaccaaacggtgatgtgaactggaagcgtgatattaaaccctatgcggaagaagtgatttccgttatccgcaaaaatgatccagacaacatcatcattgtcggaaccggtacatggagccaggatgtaaatgatgctgccgatgaccagctaaaagatgcaaacgttatgtacgcacttcatttttatgccggcacacacggccagtttttacgggataaagcaaactatgcactcagcaaaggagcgcctatttttgtgacagaatgggggacaagtgatgcttccggaaatggcggtgtattccttgatcaatcgcgggaatggctgaaatatctcgatagcaagaccattagctgggtgaactggaatctttctgataagcaggaatcatcctcagctttaaagccgggggcatctaaaacaggcggctggcagttgtcagatttatctgcttcaggaacattcgttagagaaagcattctcggcaccaaagattcgacgaaggacattcctgaaacgccagcaaaagataaacccacacaggaaaacggtatttctgtacaatacagagcaggggatgggagtatgaacagcaaccaaatccgtccgcagcttcaaataaaaaataacggcaataccacggttgatttaaaagatgtcactgcccgttactggtataaagcgaaaaacaaaggccaaaactttgactgtgactacgcgcagattggatgcggcaatgtgacacacaagtttgtgacgttgcataaaccaaagcaaggtgcagatacctatctggaacttgggtttaaaaacggaacgctggcaccgggagcaagcacagggaatattcagctccgtcttcacaatgatgactggagcaattatgcacaaagcggcgattattcctttttcaaatcaaatacgtttaaaacaacgaaaaaaatcacattatataatcatggaaaactgatttggggaacagaacccaaatagtttaacttttggcgggc。
SEQ ID NO.2:
ggcccgactaaagcgcccaccaaggacggcacatcttacaaggatttgttcttagagctttatggtaagataaaagatccgaagaacggatatttttcccccgatgaaggtatcccttatcattcaattgaaacattgatcgttgaagctccagattatggacatgtgacaacatcagaagcattctcttattacgtctggctggaggcaatgtacggcaatctaacgggaaactggtctggggtagaaacggcctggaaggtcatggaggactggattataccggactccactgagcagccaggaatgtcttcatataacccgaatagccctgcgacatacgcagacgaatacgaagatccctcgtattatccgagcgagctgaaatttgatacagtgcgtgttgggtcagatcctgttcataatgatctggtctcggcgtacggcccgaatatgtatctgatgcattggcttatggacgtagacaattggtacggtttcggcacgggaacaagagccacatttatcaacacgtttcagcgaggcgaacaggaatcaacatgggagacgattcctcacccatcaattgaagaatttaaatatggcggtccaaatggctttcttgatttatttactaaagacaggtcgtatgcaaaacaatggagatacaccaatgctcctgacgctgaggggagggccattcaagccgtgtattgggccaacaaatgggcaaaagagcaaggaaaaggttctgctgttgcgtcggtggtcagtaaggccgctaaaatgggagacttccttagaaatgacatgtttgacaaatactttatgaagatcggagcgcaagataaaactccggccacaggatatgattccgcacattatttaatggcgtggtatacggcgtggggagggggcataggcgccagctgggcttggaaaatcggatgcagccatgcccacttcggctatcaaaatccgtttcaaggctgggtctcagcaacgcagtctgattttgccccgaaatcttcaaacgggaaacgtgactggacaacatcttataaacgccagctggagttttaccagtggcttcaaagcgcagaaggcgggattgcgggcggggcaacaaacagttggaatggacggtatgaaaaatatccagcgggtacctcgacattttatggcatggcatatgtacctcacccggtttatgctgatccgggcagcaatcaatggtttggattccaagcatggagcatgcagcgggtcatggaatactatttggaaaccggcgacagctcagtgaaaaacttgattaaaaaatgggttgattgggtgatgtccgaaatcaaactgtatgatgacggaacctttgcaatcccaagcgatctggaatggtctggccagcctgatacctggacaggcacgtacactggaaatcctaatttgcatgttcgcgttacctcctacgggaccgatttgggggttgcaggttccctcgcgaatgccctcgccacatacgcagcagctacggaacgctgggaaggtaaattagacactaaggcgagagatatggcagcggaactcgtaaaccgcgcttggtataatttctattgtagtgaaggcaaaggagtagtcacagaggaggcgagggcagattacaaacgattttttgagcaagaagtatacgtgccggctggatggtctggtacgatgccgaacggcgataaaattcagccgggaataaagtttattgacattagaacaaagtatcgccaagacccgtattacgatattgtctaccaggcatacttacggggtgaagctcccgtattaaactatcaccgtttctggcatgaagtcgatcttgctgtggcgatgggagttctggctacctatttccctgatatgacatataaagtaccggggacgccgagtacaaagttatacggagatgtgaacgatgatgggaaagtcaacagcacggatgcggtcgctctcaagcgttatgtgctgcgttccggtattagcatcaatacagataatgcggacctaaatgaagatggcagagttaactcaactgaccttggcatcctgaagcggtatatcttaaaagaaattgatacgcttccttataaaaattaatgatgactcgagtaaggatctccaggca。
SEQ ID NO.3:
atgccttatctgaaacgagtgttgctgcttcttgtcactggattgtttatgagtttgtttgcagtcacttctactgcctcagctcaaacaggtggatcgttttttgacccttttaacggctataactccggtttttggcaaaaagcagatggttattcgaatggaaatatgttcaactgcacgtggcgggctaataacgtatcagtgacgtcattgggtgaaatgcgtttagcgctaacaagcccatcttataacaagtttgactgcggggaaaaccgttctgttcaaacatatggctatggactttatgaagtcagaatgaaaccagctaaaaacacagggatcgtttcatcgttcttcacttacacaggtccaacagatggaactccttgggatgagattgatatcgaatttttaggaaaagatacaacaaaggttcaatttaactattatacaaatggtgcaggaaaccatgagaagattgttgatctcgggtttgatgcagccaatgcctatcatacgtatgcattcgattggcagccaaactctattaaatggtatgtcgatgggcaattaaaacatactgcaacaaaccaaattccgacaacacctggaaagatcatgatgaacttgtggaatggcacgggtgtcgatgaatggcttggctcctacaatggtgtaaatccgctatacgctcattatgactgggtgcgctatacaaaaaaataa。
表2同源臂及纤维素酶基因片段扩增引物
将上述基因敲入载体转化至枯草芽孢杆菌Bacillussubtilis C6中,涂布至含有0.2%甘露糖和卡那霉素抗性的平板上。挑取形态大小正常的单克隆进行菌落PCR鉴定,PCR鉴定时的验证引物如表3所示。将测序鉴定为预期片段的阳性单克隆菌体在50℃、220rpm条件下摇菌16h后,在无抗LB平板划线,于37℃静置培养12h后,挑取形态大小正常的单克隆分别在无抗LB平板和卡那霉素抗性LB平板点接,验证菌株是否成功去除质粒。枯草芽孢杆菌中纤维素基因的敲入为依次敲入,敲入的顺序为:在aprE位点敲入内切葡聚糖酶基因gene2006,在epr位点敲入外切葡聚糖酶基因Cel48S,在amyE位点敲入β-葡聚糖酶基因gene4296。最终获得敲入上述3种纤维素酶基因的枯草芽孢杆菌C6-AEA3,具体敲入的示意图如图1所示。验证结果如图2A所示。
表3菌落PCR验证基因敲除的引物
实施例2
枯草芽孢杆菌C6-AEA3的酶学性质
将野生型枯草芽孢杆菌Bacillus subtilis C6作为对照,对枯草芽孢杆菌C6-AEA3的生长曲线和纤维素降解相关酶活性进行测定。
过夜活化的Bacillus subtilis C6和C6-AEA3菌液以1%接种量至200μL LB液体培养基中,37℃,震荡培养,测定24h内600nm下的吸光度值,结果如图2B(C6-WT代表野生型枯草芽孢杆菌Bacillussubtilis C6,C6-AEA3代表枯草芽孢杆菌C6-AEA3)。
过夜活化的Bacillus subtilis C6和C6-AEA3菌液以1%接种量接种至LC液体培养基,37℃,220rpm摇床中培养24h,于9000rpm,4℃离心10min获得上清粗酶液,作为后续酶活性测定的样品,每株菌设定3个生物学重复。将粗酶液分别与1%的CMC-Na溶液、1mg/mL的pNPC溶液、5mmol/L的pNPG溶液、1%的玉米芯木聚糖溶液、1*6cm规格的无菌滤纸条在水浴锅中分别孵育10min、30min、180min、30min、30min后,终止反应,读取对应的吸光度值,计算其内切葡聚糖酶活、外切葡聚糖酶活、β-葡萄糖苷酶活、木聚糖酶活和滤纸酶活,结果如图2C所示。活化为在LB培养基中进行活化。
图2显示,三种纤维素酶基因的敲入对枯草芽孢杆菌C6-AEA3的生长无显著影响,此外,内切葡聚糖酶活、外切葡聚糖酶活、β-葡萄糖苷酶活、木聚糖酶活、滤纸酶活分别达到26.3U/mL,9.8U/mL,3.9U/mL,19.6U/mL和2.4U/mL,分别是出发菌株Bacillus subtilis C6的3.1倍、6.6倍、3.0倍、1.2倍、1.8倍。因此,菌株C6-AEA3是一株纤维素酶高产枯草芽孢杆菌。
实施例3
枯草芽孢杆菌C6-AEA3对秸秆中纤维素的降解效果
将野生型枯草芽孢杆菌Bacillus subtilis C6和枯草芽孢杆菌C6-AEA3在2mL LB培养液中37℃、220rpm摇菌16h,得到活化的菌液。以1%的接种量将活化的菌液接种至50mLLB培养液中,37℃、220rpm摇菌24h获得发酵菌液。以过1mm筛的无菌玉米秸秆、小麦秸秆、水稻秸秆样品为材料,将15g秸秆样品与75mL浸润培养基(所述浸润培养基以水为溶剂,还包括如下浓度的组分:硝酸铵5g/L、七水硫酸镁5g/L、氯化钠1.0g/L)、3.75mL的发酵菌液均匀混合,以无菌浸润培养基等量替代发酵菌液作为空白对照组,装于带有气阀的发酵袋中,于37℃发酵7d。培养结束后各组分别取1g秸秆样品加入4mL去离子水,在220rpm条件下震荡混匀30min后得到浸提液,测定浸提液中还原糖含量和内切葡聚糖酶活,将剩余秸秆样品于65℃烘干16h至恒重,以用于后续指标测定。中性洗涤纤维和酸性洗涤纤维含量的测定方法参照试验方法参照ANKOM 200i型(美国)半自动纤维分析仪操作说明书。半纤维素含量的测定方法参照Solarbio半纤维素含量检测试剂盒。木质素含量的测定方法参照范式(VanSoest)纤维素洗涤法。通过场发射扫描电子显微镜,观察玉米秸秆、小麦秸秆、水稻秸秆发酵前后表面形态结构的变化。通过X-射线衍射仪和傅里叶变换红外光谱仪,测定玉米秸秆、小麦秸秆、水稻秸秆发酵前后纤维素的降解程度。结果如图3~6所示(C6-WT代表野生型枯草芽孢杆菌Bacillussubtilis C6,C6-AEA3代表枯草芽孢杆菌C6-AEA3)。
将菌株Bacillus subtilis C6和C6-AEA3与秸秆混合发酵7d后,C6-AEA3相比Bacillus subtilis C6的纤维素降解能力显著提升。结果如图3所示,在C6-AEA3组中玉米秸秆、小麦秸秆、水稻秸秆的中性洗涤纤维含量分别由72.7%、66.0%、71.6%下降至67.1%、63.2%、65.9%,酸性洗涤纤维含量分别由61.5%、58.6%、66.1%下降至39.8%、41.3%、46.7%,木质素含量分别由26.2%、22.6%、23.0%下降至23.5%、13.9%、17.1%,半纤维素含量分别由11.6%、14.2%、13.4%下降至10.5%、9.9%、10.2%,纤维素结晶度分别由31.17%、29.65%、35.29下降至28.07%、20.81%、23.00%,还原糖含量分别达到8.8mg/mL、16.5mg/mL、13.1mg/mL,新鲜发酵样品中的内切葡聚糖酶活分别为6.5U/mL、7.0U/mL、6.9U/mL。而在C6-WT组中,玉米秸秆、小麦秸秆、水稻秸秆的中性洗涤纤维含量分别降低3.4%、2.6%、4.4%,酸性洗涤纤维含量分别降低18.9%、16.4%、17.7%,木质素含量分别降低1.8%、7.9%、4.4%,半纤维素含量分别降低0.1%、2.5%、2.7%,纤维素结晶度分别降低0.9%,3.6%、10.7%,还原糖含量分别为5.3mg/mL、14.2mg/mL、10.1mg/mL,内切葡聚糖酶活分别为3.7U/mL、3.4U/mL、2.4U/mL。C6-AEA3组相较于Bacillus subtilis C6组的中性洗涤纤维降解率分别提升64.7%、7.7%、29.5%;酸性洗涤纤维降解率分别提升14.8%、5.5%、9.6%;小麦秸秆和水稻秸秆中的半纤维素降解率分别提升72.0%、18.5%;木质素降解率分别提升50.0%、10.1%、34.1%;还原糖含量是Bacillus subtilis C6的1.7倍、1.2倍和1.3倍;内切葡聚糖酶活性是Bacillus subtilis C6的1.8倍、2.1倍和2.9倍。上述各物质降解率提升的计算说明如下:C6-AEA3组中各物质含量降低的百分比减去C6-WT组中各物质含量降低的百分比得到差值,然后用该差值除以C6-WT组中各物质含量降低的百分比,结果再乘以百分之百即得到降解率提升值或变化倍数。傅里叶变换红外光谱结果如图4和X-射线衍射结果如图5。图4和图5均表明,相较于对照组,3种秸秆发酵前后的纤维素均得到了显著降解。扫描电镜结果如图6,图6显示3种秸秆表面结构变得疏松多孔,说明菌株C6-AEA3可以有效破坏纤维素结构。本发明说明获得的共表达多元纤维素酶基因枯草芽孢杆菌C6-AEA3具有高纤维素酶活,能高效降解秸秆饲料中的纤维素,为植物秸秆生物降解提供有效的菌种资源。
由以上实施例可知,本发明提供了一株共表达多元纤维素酶基因的枯草芽孢杆菌及其构建方法与应用。本发明构建的纤维素酶基因枯草芽孢杆菌C6-AEA3能有效破坏多种秸秆饲料的纤维素表面形态结构,玉米秸秆、小麦秸秆和水稻秸秆的中性洗涤纤维(NDF)含量降低降低5.6%、2.8%、5.7%,酸性洗涤纤维(ADF)含量降低21.7%、17.3%、19.4%,半纤维素分别降低1.1%、4.3%、3.2%,木质素含量降低2.7%、8.7%、5.9%,纤维素结晶度分别降低3.1%,8.8%、12.3%。本发明构建的菌株C6-AEA3能够显著降解秸秆类纤维素生物质,为提高畜牧行业饲料的利用效率奠定基础。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一株共表达多元纤维素酶基因的枯草芽孢杆菌C6-AEA3,其特征在于,所述枯草芽孢杆菌C6-AEA3分类为Bacillus subtilis,保藏于中国典型培养物保藏中心,地址为中国武汉武汉大学,保藏日期为2023年8月31日,保藏编号为CCTCC NO:M 20231579。
2.权利要求1所述的枯草芽孢杆菌C6-AEA3在制备降解纤维素的产品中的应用。
3.权利要求1所述的枯草芽孢杆菌C6-AEA3在制备降解秸秆中纤维素产品中的应用;
所述秸秆为玉米秸秆、小麦秸秆和水稻秸秆中的一种或几种。
4.一种降解秸秆中纤维素的方法,其特征在于,包括如下步骤:
将秸秆、浸润培养基与发酵菌液混合,发酵6~8d;
所述发酵菌液为权利要求1所述的枯草芽孢杆菌C6-AEA3制备得到的发酵菌液;
所述发酵菌液的制备方法为:将枯草芽孢杆菌C6-AEA3接种于LB培养基中,培养15~17h,得到活化的菌液;将所述活化的菌液接种于LB培养基中,培养20~28h,得到发酵菌液。
5.根据权利要求4所述的方法,其特征在于,所述秸秆为玉米秸秆、小麦秸秆和水稻秸秆中的一种或几种;
所述秸秆的粒径≤1mm;
所述浸润培养基以水为溶剂,还包括如下浓度的组分:
硝酸铵4~6g/L、七水硫酸镁4~6g/L、氯化钠0.8~1.2g/L;
所述秸秆与浸润培养基的质量体积比为3~5g:15~25mL;
所述秸秆与发酵菌液的质量体积比为3~5g:1mL;
所述发酵的温度为36~38℃。
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