CN116731994A - 一种右旋糖酐蔗糖酶突变体及其制备方法与应用 - Google Patents
一种右旋糖酐蔗糖酶突变体及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种右旋糖酐蔗糖酶突变体及其利用麦芽糖为受体底物制备益生元的方法，所述右旋糖酐蔗糖酶突变体氨基酸序列如SEQ ID NO.1所示，编码所述酶的核苷酸序列如SEQ ID NO.2所示。突变后的葡聚糖蔗糖酶可将麦芽糖作为受体底物，催化蔗糖制备低聚糖，该方法具有操作简单，能耗低，效率高的特点，其制备得到的低聚糖可作为益生元，在食品医药等领域具有巨大的市场应用前景。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种右旋糖酐蔗糖酶突变体及其制备方法与应用。
背景技术
随着社会经济的快速发展，人们生活节奏不断加快，饮食不规律、熬夜等不良生活习惯使越来越多的人处在亚健康状态，健康问题日益成为国内外学者的研究重点。研究表明益生元在调节肠道菌群结构、改善肠道微生态、调节脂类代谢、降低胆固醇、增强机体免疫力等方面发挥重要作用；同时以功能性低聚糖为主的益生元作为甜味剂、膨松剂、稳定剂等方面显示出优越的物理性质而广泛应用在食品、饲料、化妆品和医药等领域。目前已研究开发出的益生元如菊糖、FOS、GOS等，即使年产量超万吨仍难以满足市场的需求。除了少数低聚糖如大豆低聚糖、水苏糖等从天然植物中提取，大部分低聚糖的生产采用酶法合成工艺，半乳糖苷酶、果糖苷酶等糖苷转移酶的应用实现了GOS、FOS的工业化生产，因此酶法合成新型益生元的研究具有广阔的市场前景和应用价值。
发明内容
本发明的目的在于提供一种催化生产低聚糖的右旋糖酐蔗糖酶突变体，所述右旋糖酐蔗糖酶突变体氨基酸序列(808AA)如SEQ ID NO.1所示：
PDGAQVKGAFQQVNGKNIYFDAQTGYARQNVGFLDGTAKGFDEQGNQIKSGIATDLSGNVYYFDASGKMLTGVQNIDGKKYYFDEQGHRRRNYAGVFNNEFIYFGLDGVGQSAIEYQFEKGLTSQNSVATSHNAAKSYDTKSFTNVDGFLTANSWYRPTDILRNGTKWEPSTETDFRPLLMTWWPDKEVQANYLNYMSALGLGDQKIYTGASSQLDLNNAALIVQEAIEKKISLEKSTKWLDDSIKSFIKSKRKDIQGNLVDTNPGWTIDSETGSTNHLQNGAFIFTNSPLVPEANAAEGNRLINRTPSQQTGNHISYASQPYSGDDWGYELLLGNDVDNSNPIVQAEQLNWIHYLMNFGTITAPQDPDAHLANFDSIRIDAVDNVDADLLQIAGDYFKAAYQVGENDKNANQHIHILEDWSPNDVWYNQQVNGNSQLTMDATMQNQLLASLTRPITSRDSMKSFTKDALLVHRTADNSYNQAVPNYSFIRAHDSEVQTIIAKIISDKHPDLYPTVDKALLAKDSALYDEAFTEYNADMQKISSQKQYTHNNMPSAYAILLTNKDTVPRVYYGDLFTDNGEYMANKTPYYDAITSLLTARTKFVSGGQSLSVDKNDVLTSVRYGKGALSATDNGSSDTRNQGIGVIVSNNPNLDLNNDKVTLSMGISHAHQAYRPLLLTNSQGIVAYATDSEVPQNLYKTTNDKGELTFDASEIKGYDTVQTSGYLAVWVPVGASDEQDARTIASTEKNNGNSVYHSNAALDSQLIYEGFSNFQTVPSKNASADEYANVIIAKHAADFNKWGVTSFQM
本发明的第二目的在于提供一种右旋糖酐蔗糖酶突变体的编码基因，其核苷酸序列(2424bp)如SEQ ID NO.2所示：
CCCGATGGTGCGCAAGTTAAAGGTGCTTTTCAACAAGTTAATGGAAAAAACATTTATTTTGATGCTCAAACTGGATACGCTAGACAAAATGTAGGATTTTTGGATGGTACAGCAAAAGGGTTTGATGAGCAAGGAAATCAGATTAAAAGTGGTATAGCCACTGATTTGTCAGGTAATGTTTACTATTTTGATGCTAGTGGAAAGATGTTAACAGGCGTTCAAAATATTGATGGCAAGAAATATTACTTTGATGAACAGGGACATCGTAGAAGAAATTATGCTGGTGTATTTAATAATGAATTTATTTACTTTGGATTAGATGGCGTTGGGCAAAGTGCCATTGAATACCAGTTTGAGAAAGGATTAACTTCACAAAACAGTGTTGCTACAAGTCATAATGCTGCAAAGTCTTATGATACCAAAAGTTTTACTAACGTGGATGGTTTTTTAACTGCTAATTCATGGTATCGACCTACCGACATTTTAAGAAATGGCACAAAGTGGGAGCCTTCGACAGAAACTGATTTTAGGCCACTGCTCATGACTTGGTGGCCTGATAAAGAAGTACAGGCGAATTATTTGAACTATATGTCTGCGCTAGGACTGGGTGATCAAAAAATATATACGGGGGCCTCGAGTCAATTAGACTTAAATAATGCTGCTTTGATTGTTCAAGAAGCCATTGAAAAAAAGATTAGTCTTGAAAAAAGCACAAAATGGTTAGACGATTCCATTAAAAGTTTTATTAAAAGCAAACGCAAAGATATTCAGGGAAACTTGGTAGACACCAACCCAGGGTGGACGATTGATAGTGAAACAGGCTCTACTAACCATTTGCAAAATGGGGCGTTTATCTTTACAAATAGTCCCTTAGTTCCTGAAGCAAATGCAGCAGAAGGTAACCGATTAATTAACAGGACACCTAGTCAACAGACGGGAAATCATATATCATATGCAAGCCAACCGTACAGCGGAGACGATTGGGGATATGAACTATTATTAGGCAATGATGTCGATAATTCTAATCCTATCGTACAAGCTGAACAACTAAACTGGATACATTATTTGATGAATTTTGGGACGATAACGGCGCCTCAGGATCCAGACGCACATTTAGCTAATTTTGATAGCATTCGAATTGACGCAGTAGATAATGTTGATGCTGACTTATTACAGATTGCCGGCGATTATTTTAAAGCTGCTTATCAGGTAGGAGAAAACGATAAAAATGCGAATCAACACATTCACATTTTAGAAGATTGGTCTCCTAATGACGTTTGGTATAACCAACAAGTTAATGGTAATAGCCAATTAACTATGGATGCCACGATGCAAAACCAATTGTTAGCATCATTAACGAGACCCATTACCAGTAGAGATTCTATGAAGAGTTTTACTAAAGACGCTCTGCTAGTTCATCGAACTGCTGATAATTCTTACAATCAGGCCGTACCCAATTACAGCTTTATTCGAGCTCATGATAGTGAGGTTCAGACAATAATTGCCAAAATTATTTCTGATAAGCATCCTGATTTATATCCCACTGTTGATAAGGCTTTACTGGCTAAGGATAGTGCCCTCTACGACGAAGCTTTTACAGAGTATAATGCTGACATGCAAAAGATTTCTTCACAAAAGCAGTATACGCATAATAATATGCCCAGTGCTTATGCAATTTTGTTAACTAATAAAGATACTGTGCCAAGAGTCTATTATGGTGATTTATTTACAGATAATGGTGAGTATATGGCTAATAAGACGCCTTATTACGATGCCATCACGAGTTTGCTTACCGCACGTACCAAATTTGTATCAGGTGGACAATCGCTTTCCGTAGATAAGAATGATGTGTTAACTAGTGTCAGATACGGAAAAGGTGCCTTGTCTGCAACGGATAACGGTAGTTCTGACACACGTAATCAAGGCATTGGTGTTATTGTCAGTAATAATCCTAATTTGGATTTAAATAACGATAAAGTGACTTTGAGCATGGGGATTAGTCATGCACATCAAGCATACCGGCCTTTATTATTAACTAACAGTCAGGGAATAGTGGCATATGCAACAGACAGCGAAGTACCACAGAATCTTTATAAAACAACTAATGATAAAGGTGAATTGACGTTTGATGCATCAGAGATAAAAGGTTATGATACTGTTCAGACATCTGGTTACTTAGCTGTATGGGTACCGGTAGGCGCTTCTGATGAACAAGATGCTAGAACCATAGCCAGTACTGAAAAAAATAATGGTAATTCTGTTTATCATTCTAATGCTGCATTGGATTCTCAACTTATCTATGAAGGATTTTCTAATTTTCAAACTGTCCCATCCAAAAATGCTTCGGCAGATGAATATGCCAACGTTATTATTGCAAAACATGCTGCAGACTTTAATAAATGGGGTGTTACAAGTTTCCAAATG
本发明的第三目的在于提供一种包含右旋糖酐蔗糖酶突变体编码基因的表达载体。
本发明的第四目的在于提供一种包含右旋糖酐蔗糖酶突变体编码基因的基因工程菌株。
本发明的第五目的在于提供一种催化生产低聚糖的右旋糖酐蔗糖酶突变体的制备方法，按以下步骤进行：
(1)将右旋糖酐蔗糖酶突变体编码基因和表达载体pET28a(+)相连接，并将连接产物转化至E.coli BL21(DE3)，获得包含pET28(+)-DsrM-TB12表达载体的基因工程菌株；
(2)于37℃的条件下培养基因工程菌株12h，传代转接继续培养至菌液的OD600达到0.6～0.8时，加入IPTG至终浓度为0.1～0.5mM，然后20～24℃低温诱导培养20～26h，离心收集菌体，用PBS缓冲溶液洗涤重悬；
(3)高压匀浆破碎含重组菌株的菌液，离心获得含右旋糖酐蔗糖酶突变体的粗酶液；
(4)对酶进行活性测定。
本发明还提供所述右旋糖酐蔗糖酶突变体利用麦芽糖作为受体底物制备低聚糖的应用。利用突变酶催化蔗糖制备低聚糖，反应条件：pH＝5.0～7.0、反应温度为30～35℃、酶添加量为1～10U、反应时间为8～12h。
本发明的有益技术效果是：与一般右旋糖酐蔗糖酶相比，右旋糖酐蔗糖酶突变体可以直接一步酶法合成分子量在3～5kDa之间的右旋糖酐，且催化较为稳定，分子量分布较为集中，免去了先催化生成高分子量右旋糖酐再水解生产微分子量右旋糖酐的步骤。在麦芽糖作为受体底物时，右旋糖酐蔗糖酶突变体可以催化蔗糖合成多种低聚糖，进而制备为益生元。本发明的右旋糖酐蔗糖酶突变体可应用于食品医药等领域。
附图说明
图1不同IPTG浓度对酶活力的影响
图2不同反应时间右旋糖酐蔗糖酶突变体催化受体反应合成产物的薄层层析结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行完整的描述，显然，所描述的实施例仅是本发明一部分实施例。基于本发明的实施例，本领域的普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
实施例1产右旋糖酐蔗糖酶突变体基因工程菌株的构建
步骤1：肠膜明串珠菌(Leuconostoc mesenteroides)NRRL B-1299基因组的获取
将肠膜明串珠菌(L.mesenteroides)NRRL B-1299进行试管培养活化，试管装液量为5ml，接种量为5％，培养温度为30℃，培养时间为10-12h，得到菌液后，12000rpm离心获取菌泥，利用革兰氏阳性菌的提基因组试剂盒提取肠膜明串珠菌(L.mesenteroides)NRRL B-1299的基因组作为PCR模板。
步骤2：产右旋糖酐蔗糖酶突变体DsrM-TB12基因的获取及线性化克隆载体的制备
上游引物：
5’-ATGGGTCGCGGATCCGAATTCCCCGATGGTGCGCAAGTTA-3’
下游引物：
5’-TGCGGCCGCAAGCTTGTCGACCATTTGGAAACTTGTAACACC-3’
以来源于肠膜明串珠菌(L.mesenteroides)NRRL B-1299的基因组DNA为模板，设计突变体引物，利用PCR扩增得到DsrM-TB12基因片段。PCR扩增产物经1％琼脂糖凝胶电泳检验后，设计合适的酶切体系，加入Dpn I去除带甲基化的原始模板。酶切4h后，将酶切后的PCR反应液进行PCR纯化回收，备用。
将pET28a(+)质粒用EcoR I和Sal I内切酶进行双酶切，酶切产物进行胶回收，作为线性化克隆载体备用。
步骤3：制备E.coli BL21(DE3)感受态细胞
取5ul E.coli BL21(DE3)菌液加入5ml的LB试管中，37℃，180rpm，摇床培养12h，取1ml试管中的菌液加入装有100ml LB培养基的摇瓶中，37℃，180rpm摇床培养，待菌液OD600为0.5～0.6时，取出摇瓶，冰上放置10min，4℃，4100rpm，离心10min，弃上清，用2ml含有15％甘油的0.05M CaCl2溶液重悬，分装后于-80℃冰箱中保存，备用。
步骤4：利用一步克隆技术构建目标菌株
将步骤2中回收得到的DsrM-TB12基因片段和线性化的pET28a(+)质粒载体，通过一步克隆转入E.coli BL21(DE3)中，具体方法如下：将装有100μl制备好的E.coli BL21(DE3)感受态细胞放在冰盒上解冻5-10min；向感受态细胞中加入10μl冷却的一步克隆反应体系，手指轻弹，混匀，冰上放置30min；后于42℃热激90s，迅速冰浴3min。向上述反应体系中加入900μl LB培养基，37℃，180rpm摇床培养1h进行细胞复苏。取100μl涂布在抗性平板上，37℃培养箱中培养12h，挑取单菌落，使用步骤2中的引物进行菌落PCR验证，能扩增出目的基因片段的菌落即为pET28a-DsrM-TB12质粒成功表达的菌株。
实施例2基因工程菌株发酵制备右旋糖酐蔗糖酶DsrM-TB12
种子培养基：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，121℃灭菌15min。
发酵培养基：蛋白胨12g/L，酵母粉24g/L，KH2PO4 2.31g/L，K2HPO416.43g/L，葡萄糖10g/L，121℃灭菌15min。
制备方式：取平板上单菌落接种于装液量为5ml的试管内，37℃，180rpm培养12h。后取其中1ml菌液接种于装有100ml培养基的500ml摇瓶中，180rpm，37℃培养0～4h后加入0.1～0.5mM IPTG诱导DsrM-TB12蛋白的表达，继续在20～24℃下培养20～26h，停止发酵后，将发酵液于8000rpm离心10min，弃上清，用pH为5.5的PBS缓冲溶液洗涤重悬，高压匀浆破碎细胞获得粗酶液。如图1所示，不同IPTG浓度诱导条件下，DsrM-TB12的最高酶活可达15.6U/ml。
实施例3右旋糖酐蔗糖酶突变体表达酶活性的测定
右旋糖酐蔗糖酶突变体的酶活力表示为在pH＝5.5，30℃的条件下，1min内反应生成1μmol果糖所需的酶量为一个酶活单位U。
超声破碎含右旋糖酐蔗糖酶突变体的菌液，破碎后离心收集上清液，取1ml酶液和测量酶活用的酶活反应体系2.5ml混合反应30min，反应后加入1.5ml DNS试剂，煮沸5min，使用分光光度计在540nm波长下测量，代入果糖标准曲线计算反应后果糖含量，判断酶活。
酶活反应体系为：乙酸钠1.46g/L，氯化钙0.05g/L，蔗糖120g/L，用20％盐酸调节pH至5.5。
具体方法为：
(1)取7支比色管编号0～6，分别加入浓度为1mg/ml的果糖标准液0ml、0.2ml、0.4ml、0.6ml、0.8ml、1.0ml、1.2ml，补足蒸馏水至2.0ml，加入1.5ml DNS试剂，配成不同果糖浓度的反应液，将比色管摇匀后在沸水浴中加热5min，取出，冷却至室温，蒸馏水定容至20ml，颠倒混匀，用0号管调零，540nm波长下测定吸光值，以吸光值为横坐标，果糖含量(μmol)为纵坐标制定标准曲线。
(2)取500μl待测酶液与2.5ml反应体系混合，混匀后放入30℃水浴中反应30min，采用DNS法测定反应体系中0min和30min的还原糖(即为右旋糖酐蔗糖酶突变体催化反应生成的果糖)含量。
(3)取右旋糖酐蔗糖酶突变体反应体系500μl，加蒸馏水定容至2ml，加入1.5mlDNS，沸水浴5min，取出冷却至室温，蒸馏水定容至20ml，颠倒混匀，测定540nm波长下的吸光值，对照标曲计算得到相应还原糖的含量(μmol)。
(4)
实施例4葡聚糖蔗糖酶受体反应合成低聚糖
在含1mM CaCl2的NaAc-HAc(50mM，pH＝5.4)缓冲液反应体系中，加入一定量的蔗糖、麦芽糖受体和酶液，使其终浓度分别为100mg/ml、100mg/ml和5U/ml，置于37℃恒温震荡水浴摇床(180rpm)中反应12h，煮沸10min终止反应。将反应液与等体积无水乙醇混合，静置离心(20min，8000rpm)除去多糖，然后用旋转蒸发仪对上清液进行浓缩并除去乙醇。
低聚糖(醇)产率：将受体反应中合成的低聚糖(醇)总产量占反应体系中总糖的百分比定义为低聚糖(醇)产率。
实施例5：酶催化反应体系各产物产量的变化趋势
在含1mM CaCl2的NaAc-HAc(50mM，pH＝5.4)缓冲液反应体系中，按照最适供受体比例加入最适浓度的蔗糖、麦芽糖和酶液，将整个反应置于37℃恒温震荡水浴摇床(120rpm)中反应8h，并在反应0、1、2、4、6、8、10、12h取样，样品煮沸10min终止反应。不同反应时间右旋糖酐蔗糖酶突变体催化受体反应合成产物的薄层层析结果如图2所示。
在反应过程中，作为底物的蔗糖的圆点逐渐变小至消失，麦芽糖的圆点不断变小，产物中依次出现三个圆点，并随反应进行逐渐变大，同时果糖的圆点不断变大。和标品对比，最先合成的较大的圆点是低聚三糖，随后出现的是低聚四糖和低聚五糖，每个圆点包含一种或以上同一聚合度的低聚糖。根据催化反应机理，推测葡聚糖蔗糖酶首先以麦芽糖为受体合成低聚三糖，然后低聚三糖作为受体同麦芽糖竞争葡糖基，合成低聚四糖，同理低聚四糖作为受体合成低聚五糖。随着低聚糖碳链的延长，其本身作为受体竞争葡糖基的能力逐渐下降，因此产物中的低聚三糖含量最高，低聚四糖次之，低聚五糖最少。
Claims (7)
1.一种右旋糖酐蔗糖酶突变体，其特征在于，所述右旋糖酐蔗糖酶突变体的氨基酸序列如SEQ ID NO. 1所示。
2.一种编码权利要求1所述右旋糖酐蔗糖酶突变体的编码基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO. 2所示。
3.根据权利要求2所述的包含右旋糖酐蔗糖酶突变体编码基因的表达载体。
4.根据权利要求2所述的包含右旋糖酐蔗糖酶突变体编码基因的基因工程菌株。
5.一种制备权利要求1所述右旋糖酐蔗糖酶突变体的方法，其特征在于，按以下步骤进行：
1）将右旋糖酐蔗糖酶突变体的编码基因和表达载体pET28a(+)相连接，并将连接产物转化至E.coli BL21(DE3)中，获得包含编码基因的基因工程菌株；
2）于37℃的条件下培养权利要求4所述的基因工程菌株12 h，传代转接继续培养至菌液的OD600达到0.6~0.8时，加入IPTG至终浓度为0.1~0.5 mM，然后20~24℃低温诱导培养20~26 h，离心收集菌体，用PBS缓冲溶液洗涤重悬；
3）高压匀浆破碎含基因工程菌株的菌液，离心获得含右旋糖酐蔗糖酶突变体的粗酶液；
4）对酶进行活性测定。
6.根据权利要求1~5任意一项所述右旋糖酐蔗糖酶突变体的应用，其特征在于，所述右旋糖酐蔗糖酶突变体催化蔗糖并利用麦芽糖作为受体底物制备低聚糖的应用。
7.根据权利要求6所述，其特征在于，利用突变酶催化蔗糖制备低聚糖，反应条件：pH为5.0~7.0、反应温度为30~35℃、酶添加量为1~10 U、反应时间为8~12 h。
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