CN117821348A - 一种胞外分泌表达κ-卡拉胶酶的重组枯草芽孢杆菌 - Google Patents
一种胞外分泌表达κ-卡拉胶酶的重组枯草芽孢杆菌 Download PDFInfo
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Abstract
本发明公开了一种胞外分泌表达κ‑卡拉胶酶的重组枯草芽孢杆菌，属于基因工程和酶工程技术领域。本发明以食品级枯草芽孢杆菌为表达宿主，质粒pP43NMK为表达载体，实现κ‑卡拉胶酶的异源表达。本发明通过去除κ‑卡拉胶酶的信号肽，优化发酵工艺，成功实现κ‑卡拉胶酶胞外分泌表达，酶活最高可达458.94U/mL，高于目前所报道的大多数κ‑卡拉胶酶，较高的胞外分泌表达水平有利于后期的大规模生产和分离纯化。由本发明所述重组菌发酵获得的κ‑卡拉胶酶，可通过水解κ‑卡拉胶绿色高效生产功能性κ‑卡拉胶寡糖，为卡拉胶寡糖的高效制备提供了新的路径。
Description
技术领域
本发明涉及一种胞外分泌表达κ-卡拉胶酶的重组枯草芽孢杆菌，属于基因工程和酶工程技术领域。
背景技术
卡拉胶(Carrageenan)作为一种结构独特的天然海洋硫酸多糖，由D-半乳糖通过交替的α-1,3和β-1,4糖苷键连接而成，由于卡拉胶粘度较高，不易被人体吸收，这极大地限制了卡拉胶的应用。卡拉胶寡糖(Carrageenan oligosaccharide)是卡拉胶的降解产物，研究发现，其活性较卡拉胶有显著提高，而且其物理性能也得到明显改善，卡拉胶寡糖分子量相较于卡拉胶大大减小，这使得其溶解性以及抗氧化、抗病毒、免疫调节等多种生物活性得到了提升，在一定程度上拓宽了卡拉胶的应用范围。目前，制备卡拉胶寡糖的方法主要有物理法、化学法和酶法。
酶法制备卡拉胶寡糖具有条件温和、反应特异性强、环境污染少等优点，逐渐取代了传统的制备卡拉胶寡糖的方法。但是，从自然界分离的菌株直接发酵生产卡拉胶酶存在产量低、培养条件复杂、产酶不稳定、酶纯化成本高等问题。因此，采用基因工程方法对从自然界分离的卡拉胶酶基因进行异种表达，以提高卡拉胶酶的表达量，对于大规模制备卡拉胶酶和实现卡拉胶寡糖的产业化具有重要意义。根据底物专一性的不同，卡拉胶酶(Carrageenanase)可分为κ-卡拉胶酶、ι-卡拉胶酶、λ-卡拉胶酶。在实际应用中，κ-卡拉胶酶(κ-carrageenanse,简称CgkA酶)应用较多，但是目前κ-卡拉胶酶制备仍面临两个方面的问题：1)重组κ-卡拉胶酶大多以胞内酶的形式分泌表达，粗酶的制备必须进一步破碎处理后再离心获得，这在实际生产中不仅增加了生产成本还降低了效率；2)现有的研究大多采用大肠杆菌进行异源表达，大肠杆菌表达***在工业应用上存在低表达量、内毒素、形成包涵体以及目的蛋白质错误折叠等诸多问题，不利于酶在食品、药品等领域中的应用。现有技术中，专利CN101784660A公开了在枯草芽孢杆菌中表达κ-卡拉胶酶，但其活性和表达量仍较低。因此，亟待发掘能够高效胞外分泌表达κ-卡拉胶酶的表达***。
发明内容
为解决上述问题，本发明提供了一种胞外高效分泌表达κ-卡拉胶酶的重组枯草芽孢杆菌，建立了更安全的食品级表达体系，实现了κ-卡拉胶酶的异源胞外分泌表达。
本发明的第一个目的是提供了一种重组枯草芽孢杆菌，所述重组枯草芽孢杆菌表达了氨基酸序列如SEQ ID NO.1所示的κ-卡拉胶酶。
在一种实施方式中，所述重组枯草芽孢杆菌以枯草芽孢杆菌Bacillus subtilisWB600为宿主。
在一种实施方式中，氨基酸序列如SEQ ID NO.1所示的κ-卡拉胶酶是通过删除Zobellia galactanivorans来源的野生型κ-卡拉胶酶的信号肽序列得到的；所述Zobelliagalactanivorans来源的野生型κ-卡拉胶酶的氨基酸序列如SEQ ID NO.5所示，编码所述野生型κ-卡拉胶酶的信号肽的核苷酸序列如SEQ ID NO.4所示。
在一种实施方式中，所述κ-卡拉胶酶的基因的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中，所述表达所使用的载体为可以在所述重组枯草芽孢杆菌内自主复制的质粒，所述质粒包括但不限于质粒pP43NMK。
在一种实施方式中，所述质粒pP43NMK的核苷酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供一种生产κ-卡拉胶酶的方法，所述方法包括应用上述重组枯草芽孢杆菌进行发酵的步骤。
在一种实施方式中，所述的发酵为，将所述重组枯草芽孢杆菌在种子培养基中活化培养得到种子液，再按照1％-5％(v/v)的接种量将种子液转接至发酵培养基中进行发酵培养。
在一种实施方式中，所述的发酵为，将所述重组枯草芽孢杆菌在种子培养基中活化培养得到种子液，将种子液以1％-5％(v/v)的接种量转接至装有50mL发酵培养基的250mL锥形瓶中。
在一种实施方式中，所述种子液接种量优选为4％(v/v)。
在一种实施方式中，所述种子培养基的组成包括：酵母粉2～8g/L，胰蛋白胨5～15g/L，NaCl 5～15g/L。
在一种实施方式中，所述种子培养基的pH为6.5～7.5。
在一种实施方式中，所述发酵培养基的组成包括：胰蛋白胨5～10g/L，酵母粉25～35g/L，K2HPO4 15～20g/L，KH2PO4 1～3g/L，甘油5～10g/L。
在一种实施方式中，所述发酵培养基的pH为7～8。
在一种实施方式中，所述发酵培养的条件为20～37℃、180～280r/min。优选为25℃、180～280r/min。
在一种实施方式中，所述发酵培养的培养时间为12～96h。
本发明的第三个目的是提供所述重组枯草芽孢杆菌或所述方法在食品领域中的应用。
在一种实施方式中，所述应用包括所述重组枯草芽孢杆菌或所述方法在κ-卡拉胶寡糖制备中的应用。
本发明的有益效果：
本发明发现，野生型κ-卡拉胶酶(Zobellia galactanivorans来源)并不能在枯草芽孢杆菌中实现异源胞外分泌表达，野生型κ-卡拉胶酶胞外活力检测不到酶活力，本发明通过去除野生型κ-卡拉胶酶的信号肽序列，实现了其在枯草芽孢杆菌中的胞外分泌表达，最高酶活力为458.89U/mL。
附图说明
图1为κ-卡拉胶酶重组质粒的构建流程；其中，F2、R2为目的基因扩增引物，短线表示同源臂；
图2为发酵温度和时间对κ-卡拉胶酶摇瓶发酵的影响；
图3为κ-卡拉胶酶的SDS-PAGE蛋白电泳图；
图4为应用重组κ-卡拉胶酶制备酶解产物时的HPLC分析。
具体实施方式：
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中所涉及的酶活检测方法如下：
κ-卡拉胶酶的酶活性检测使用3,5-二硝基水杨酸法(简称DNS法)。按照以下条件和步骤：将0.1mL酶液添加到0.9mL底物(底物κ-卡拉胶用Tris-HCl(pH 7.5,50mM)配成终浓度为0.5％(w/v))，在40℃反应15min，加入1mL DNS停止反应，再放入沸水中煮沸5min后立即放入冰水中，加入4mL蒸馏水，在520nm条件下测定吸光值。根据D-半乳糖标准曲线，计算出酶活。以高温灭活的酶液作为空白对照。
一个酶活力单位(U)定义：在上述反应条件下，1min内生成1μg还原糖(以D-半乳糖计)所需的酶量。
下述实施例中所涉及κ-卡拉胶酶解产物的分析方法：
利用高效液相色谱(HPLC)分析酶解产物，具体分析条件：采用反相C18柱；检测器：紫外检测器；流动相：A相10mM乙酸铵(pH 4.5)；B相乙腈。0-60min，80-60％ A相、20-40％B相梯度洗脱；紫外波长254nm；柱温40℃；流速0.5mL/min；进样量10μL。
实施例1枯草芽孢杆菌分泌表达***的构建
表达载体CgkA/pP43NMK的构建过程如图1所示。用于扩增pP43NMK载体的引物为表1中F1/R1，用于扩增目的基因CgkA引物为表1中F2/R2。
表1枯草芽孢杆菌表达***构建所需引物
PCR体系为：Max Master Mix 25μL，正向引物(10M)2μL，反向引物(10M)2μL，模板DNA1μL，加入ddH2O至50μL。PCR扩增条件为：98℃预变性3min；再进行30个循环(98℃10s,60℃15s,68℃1min)；最后68℃保温10min。
利用表1中引物，以上述PCR体系扩增，得到的线性化载体pP43NMK和两端带有同源臂的目的基因，通过同源重组的方法进行连接。同源重组反应体系为：载体0.03pmol，目的基因0.06pmol，5×CE II Buffer 4μL，Exnase II 2μL，加入ddH2O至20μL，37℃反应30min。将连接产物转化至Escherichia coli JM109中，涂布具有氨苄青霉素的LB平板，挑取单菌落进行测序、菌落PCR验证，提取得到含有κ-卡拉胶酶基因的重组质粒CgkA/pP43NMK。将重组质粒转入表达宿主B.subtilis WB600中，得到重组基因工程菌B.subtilis WB600(CgkA/pP43NMK)。
实施例2重组κ-卡拉胶酶的发酵生产
LB培养基：酵母粉5g/L，胰蛋白胨10g/L，NaCl 10g/L，pH 7.0。
TB培养基：胰蛋白胨6g/L，酵母粉30g/L，K2HPO4 16.432g/L，KH2PO4 2.314g/L，甘油5g/L，pH 7.5。
(1)宿主菌活化培养：将依照实施例1所述方法得到的重组基因工程菌B.subtilisWB600(CgkA/pP43NMK)在LB固体培养基上进行划线活化，置于37℃恒温培养箱中过夜培养，挑取阳性单菌落接种于含有50mL LB液体培养基的250mL锥形瓶中。接种前添加终浓度为5μg/mL卡那霉素。锥形瓶置于200r/min的回转式摇床中，在37℃下培养14h。
(2)发酵培养条件优化：将种子液以4％(v/v)的接种量转接至装有50mL发酵培养基的250mL锥形瓶中。在分别于20℃、25℃、30℃、37℃摇床中振荡培养12～96h(转速为200r/min)，接种前添加终浓度为5μg/mL卡那霉素。结束发酵后，将发酵液于4℃、10000rpm条件下离心15min，收集上清液即得到粗酶液。测定粗酶液的酶活力，结果如图2所示，随着发酵时间的延长，胞外酶活逐渐提高。在25℃、200r/min条件下振荡培养24h后胞外酶活可达458.89(U/mL)。
实施例3重组κ-卡拉胶酶的分离纯化
用5个柱体积的Binding buffer(50mM Tris-HCl,500mM NaCl,pH 7.5)平衡镍柱；发酵粗酶液用0.45μm水系膜过膜处理后，开始上样，流速为1mL/min；用Binding buffer继续平衡镍柱后，先用Wash buffer(50mM Tris-HCl,500mM NaCl,20mM咪唑,pH 7.5)洗去部分杂蛋白，随后用Elution buffer(50mM Tris-HCl,500mM NaCl,500mM咪唑,pH 7.5)梯度洗脱目的蛋白。收集后的目的蛋白在透析液(50mM Tris-HCl,pH 7.5)中透析24h，得到纯酶。最后通过SDS-PAGE蛋白电泳及酶活测定的方式进行鉴定。SDS-PAGE蛋白电泳结果如图3所示。
实施例4重组κ-卡拉胶酶的应用
利用重组κ-卡拉胶酶酶解κ-卡拉胶，将其降解为一系列κ-卡拉胶寡糖。
将0.1mL纯酶与0.9mL底物浓度0.5％(w/v)，用灭活的酶液代替纯酶作为对照组，在40℃反应3h，水浴摇床转速200～300rpm。取0.5mL酶解产物，依次加入0.2mL 0.2M溶于甲醇的衍生化试剂AEC(3-氨基-9-乙基咔唑)、25μL 0.5M的NaBH3CN(氰基硼氢化钠)和50μL乙酸。将上述混合体系置于70℃水浴锅中保温1h，加入0.1mL 1M NaOH中止反应。最后再加入1mL氯仿萃取。在8000rpm离心5min取上清液，过0.22μm有机膜，进行HPLC分析。结果如图4显示，反应3h酶解产物主要是κ-卡拉胶二糖(DP2)、四糖(DP4)和八糖(DP8)。并且DP4占比最高，转化率62.16％、其次是DP2糖转化率15.49％，DP8转化率最低7.15％。整个反应体系的转化率达到84.80％。
对比例1
在SEQ ID NO.2所示的基因序列的第一个起始密码子之后加入核苷酸序列如SEQID NO.4所示的编码目的基因自身信号肽的核苷酸序列。加入信号肽编码序列后的基因编码的氨基酸序列如SEQ ID NO.5所示。通过基因合成，将其构建至质粒pP43NMK，并转化至宿主B.subtilis WB600中，经测序验证后获得重组菌株B.subtilis WB600(Signal-CgkA/pP43NMK)。
将重组菌株B.subtilis WB600(Signal-CgkA/pP43NMK)进行发酵，验证其胞外表达κ-卡拉胶酶的能力，发酵方法同实施例2，发酵结果显示，未检测到胞外酶活力，即胞外酶活力为0U/mL。
Claims (10)
1.一种重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌表达了氨基酸序列如SEQ ID NO.1所示的κ-卡拉胶酶。
2.根据权利要求1所述的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌以枯草芽孢杆菌Bacillus subtilis WB600为宿主。
3.根据权利要求1所述的重组枯草芽孢杆菌，其特征在于，所述的表达以质粒pP43NMK为表达载体，其核苷酸序列如SEQ ID NO.3所示。
4.一种生产κ-卡拉胶酶的方法，所述方法包括应用权利要求1-3任一所述的重组枯草芽孢杆菌进行发酵的步骤。
5.根据权利要求4所述的方法，其特征在于，所述发酵包括：将所述重组枯草芽孢杆菌在种子培养基中活化培养得到种子液，再按照1％-5％(v/v)的接种量将种子液转接至发酵培养基中进行发酵培养。
6.根据权利要求5所述的方法，其特征在于，所述种子培养基的组成包括：酵母粉2～8g/L，胰蛋白胨5～15g/L，NaCl 5～15g/L。
7.根据权利要求5所述的方法，其特征在于，所述发酵培养基的组成包括：胰蛋白胨5～10g/L，酵母粉25～35g/L，K2HPO4 15～20g/L，KH2PO4 1～3g/L，甘油5～10g/L。
8.根据权利要求5所述的方法，其特征在于，所述发酵培养的温度为20～37℃。
9.权利要求1-3任一所述重组枯草芽孢杆菌，或权利要求4-8任一所述的方法在食品领域中的应用。
10.根据权利要求9所述的应用，其特征在于，所述应用包括所述重组枯草芽孢杆菌或所述方法在κ-卡拉胶寡糖制备中的应用。
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