CN115491366A - 一种特异性产生木寡糖的木聚糖酶BgXyn8A及其基因与应用 - Google Patents
一种特异性产生木寡糖的木聚糖酶BgXyn8A及其基因与应用 Download PDFInfo
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- CN115491366A CN115491366A CN202110681603.2A CN202110681603A CN115491366A CN 115491366 A CN115491366 A CN 115491366A CN 202110681603 A CN202110681603 A CN 202110681603A CN 115491366 A CN115491366 A CN 115491366A
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- Prior art keywords
- bgxyn8a
- xylanase
- ala
- enzyme
- oligosaccharide
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Abstract
本发明涉及一种能特异性产生木寡糖的耐热木聚糖酶BgXyn8A及其基因和应用。所述耐热木聚糖酶BgXyn8A氨基酸序列如SEQ ID NO.2所示，其编码基因BgXyn8A核苷酸序列如SEQ ID NO.5所示。该木聚糖酶BgXyn8A具有分解木聚糖产生低聚糖的作用，且能特异性产生低聚糖，不产生木糖，产物的组分为木二糖至木六糖及长链寡糖，还产生了大量阿拉伯糖基木聚糖和聚合度大于6的长链木寡糖，在低聚糖生产上和农林废弃物的资源化利用上拥有独特的优势。而且该酶最适温度60℃，最适pH值为6.0，常温下，在酸性、中性和碱性范围内均具有较高活性；在55℃和50℃下的半衰期分别约为20min和3.5h，在pH3.0–10.0范围内于25℃放置12h后仍能保持80％以上的酶活性，因此该酶具有很好的稳定性、适用条件温和，应用前景好。
Description
技术领域
本发明涉及生物技术领域，具体地，涉及一种能特异性产生木寡糖的内切 -β-1,4木聚糖酶BgXyn8A的蛋白、编码该蛋白的基因、含有该基因的表达质粒和重组菌株、该蛋白的应用及改造。
背景技术
低聚木糖或称木寡糖(xylo-oligosaccharides，XOS)，是由2–10个木糖通过糖苷键连接形成主链的低聚合度糖的总称。低聚木糖是一种益生功能超强的功能性低聚糖，在食品、医药、饲料、农业和化妆品等领域具有重要的应用价值，市场需求量大，发展前景广阔。除了XOS以外，阿拉伯糖基木寡糖 (arabinoxylo-oligosaccharides，AXOS)也是人体均衡饮食中的主要膳食纤维之一，可以作为人体肠道微生物所需的益生元。
木聚糖(xylan)是制备低聚木糖的底物，目前工业上广泛采用木聚糖酶水解法来生产低聚木糖，即对富含木聚糖的农业废弃物(如玉米芯等)进行预处理，然后通过木聚糖酶水解、产物的分离提取、干燥等工艺便可获得低聚木糖。
植物细胞壁主要由纤维素、半纤维素和木质素组成。半纤维素是自然界中含量仅次于纤维素的一大类可再生生物质资源，其来源广泛，包括各种农业残渣 (秸秆、壳、茎)、落叶和针叶林、城市固体废物、制浆造纸工业废物和草本能源作物。木聚糖是半纤维素中含量最丰富的一种，是植物细胞壁的重要组成成分，大量存在于硬木(15–30％)、软木(7–10％)及一年生植物(<30％)中。木聚糖酶能通过随机切割木聚糖分子内部的β-1,4-糖苷键，通常会产生低聚木糖及少量单糖。
木聚糖酶是低聚木糖制备的关键。目前所鉴定的木聚糖酶水解作用所产生的多为聚合度(DP)2–6的木寡糖，并且绝大多数酶都会或多或少地产生一些单糖，迄今鲜见能特异性产生低聚木糖且组分中大量含有DP>6的长链木寡糖的木聚糖酶。能特异性产生低聚木糖，意味着酶能将底物最大限度地转化成低聚木糖，而且有利于生产高纯度的低聚木糖产品。另外，低聚木糖的平均聚合度越高，其清除羟自由基的作用越显著，抗氧化活性越强。
我国每年产生了大量的玉米芯、甘蔗渣及农作物秸秆等农业废弃物，长期以来一直未能得到充分的重视和开发利用。利用木聚糖酶解技术将其中的木聚糖制备成高附加值的低聚木糖等产品，不仅将促进我国低聚糖工业的发展，而且还可以变废为宝，保护环境，具有十分重大的经济效益和社会效益。然而，当前低聚木糖的成本过高，阻碍了其被更广泛的使用。因此，挖掘能高效制备低聚木糖的新型木聚糖酶具有重要的意义。
发明内容
本发明的目的是提供一种能高效特异性产生木寡糖的新型木聚糖酶。
本发明的再一目的是提供编码上述新型木聚糖酶的基因。
本发明的另一目的是提供包含上述基因的重组载体和重组菌株。
本发明的另一目的是提供一种制备上述新型木聚糖酶的基因工程方法。
本发明的另一目的是提供上述新型木聚糖酶的应用。
本发明的另一目的是获得上述新型木聚糖酶的改良基因。
本发明上述目的通过以下技术方案实现：
本发明从芽孢杆菌Bacillus sp.KW1中分离克隆得到一种新的木聚糖酶基因BgXyn8A，DNA全序列分析结果表明，其核苷酸序列如SEQ ID NO.4，木聚糖酶 BgXyn8A的编码基因BgXyn8A全长1293bp。
SEQ ID NO.4中信号肽的碱基序列如SEQ ID NO.6所示。
去掉信号肽后成熟的木聚糖酶编码基因BgXyn8A的基因序列如SEQ ID NO.5所示，编码402个氨基酸和一个终止密码子。
上述木聚糖酶基因BgXyn8A编码的木聚糖酶BgXyn8A的氨基酸序列如SEQ ID NO.1所示：
其中该酶基因编码430个氨基酸，N端28个氨基酸为其信号肽序列“MRKSLKWIMAVFIGLTCFCAAYSQTAMA”(SEQ ID NO.3)。
因此，成熟木聚糖酶BgXyn8A的理论分子量约为45kDa，其氨基酸序列如 SEQ IDNO.2所示。
本发明成熟木聚糖酶BgXyn8A蛋白理论分子量约为45kDa，通过与 GenBank中的酶进行BLAST比对发现，在已详尽分析生化特性的酶中，BgXyn8A 与来自Paenibacillusbarcinonensis BP-23的同为8家族木聚糖酶PbRex8A的序列一致性最高，仅为25％，且PbRex8A是一个从还原末端释放木糖的外切木聚糖酶。此外，BgXyn8A在PDBsum中最高只有30％的一致性。与其他第8家族木聚糖酶相比，本发明的木聚糖酶具有更好的pH稳定性和热稳定，且其能高效特异性产生木寡糖，因此本发明的木聚糖酶BgXyn8A很适合在食品、饲料等领域使用。
另外还通过定点突变对上述新型木聚糖酶进行热稳定性改造，获得了一个热稳定性提高的阳性突变子BgXyn8A-K29M，将所述木聚糖酶BgXyn8A的N端第29位的赖氨酸(Lys,K)突变为甲硫氨酸(Met,M)后，改造后的木聚糖酶和野生型木聚糖酶相比，酶的热稳定性得到显著提升。因此，突变后的木聚糖酶 BgXyn8A/K29M也应当在本发明保护范围之内。研究结果显示，所述木聚糖酶 BgXyn8A能够酶解木聚糖产生木寡糖，包括酶解含木聚糖的农林废弃物产生木寡糖。
因此，所述木聚糖酶BgXyn8A以及突变后的木聚糖酶BgXyn8A/K29M在酶解木聚糖产生木寡糖中的应用，酶解含木聚糖的农林废弃物产生木寡糖中的应用，制备相关酶制剂中的应用，均应在本发明保护范围之内。
基于此，本发明还提供一种酶解农林废弃物产生木寡糖的方法，所述方法是利用上所述木聚糖酶BgXyn8A酶解农林废弃物，以产生木寡糖。
优选地，所述农林废弃物需先进行碱预处理。
优选地作为一种可选择的实施方式，所述碱预处理方法如下：
S1.将5g玉米芯(50目)与80mL 1.25M NaOH混合15min；
S2.在150rpm的摇床上于37℃振荡3h；
S3.13,000g离心20min；
S4.上清液用浓盐酸酸化至pH 6.0，用苯酚硫酸法测得总糖含量为225g/kg。
本发明所提供的木聚糖酶BgXyn8A可以通过工业手段制备。
作为一种可选择的实施方式，本发明提供了一种制备木聚糖酶BgXyn8A的原核表达方法，包括如下步骤：
S1.将编码木聚糖酶的基因BgXyn8A序列插入表达载体，所得重组载体再转化宿主细胞得到得重组菌株；
S2.培养重组菌株，诱导重组木聚糖酶BgXyn8A编码基因表达；
S3.纯化所表达的木聚糖酶BgXyn8A。
优选地，步骤S1中所述宿主细胞为大肠杆菌BL21(DE3)。
优选地，步骤S1中所述表达载体为pET28a。
将本发明提供的木聚糖酶基因插入到表达载体合适的限制性酶切位点之间，使其核苷酸序列可控地与表达调控序列相连接。作为本发明的最优选的实验方案，优选为将本发明的新型木聚糖酶基因BgXyn8A插入到表达载体pET-28a的 EcoR I和Xho I限制性酶切位点之间，得到重组表达质粒pET28a-BgXyn8A。
基于此方法，其中所获得的含有木聚糖酶BgXyn8A编码基因的重组载体，以及所述重组菌株，也应在本发明保护范围之内。
本发明还对木聚糖酶BgXyn8A的性能做了许多的探索：
所述木聚糖酶BgXyn8A最适pH为5.0–6.0，同时具有好的pH稳定性，在 pH3.0–10.0范围内于25℃放置12h后仍能保留80％以上的酶活性；最适反应温度为60℃。
而且，所述木聚糖酶BgXyn8A以1％榉木木聚糖(Beechwood xylan,BeeWX) 为底物，在离子浓度为较低时(1mM)，Fe2+、Al3+和Fe3+能将BgXyn8A的酶活力分别提高至142％、153％和210％；在离子浓度较高时(10mM)，Al3+、Ni2+和Fe3+及EDTA将BgXyn8A的酶活力提高至131％、128％、115％和114％， BgXyn8A对其他金属离子也有较高的耐受能力。
此外，所述木聚糖酶BgXyn8A水解木聚糖能特异性产生低聚木糖，水解碱预处理玉米芯的低聚木糖产物中除了含有木二糖至木六糖外，还含有阿拉伯糖基木寡糖和大量DP为7–10的长链木寡糖。
本发明还通过定点突变确证了上述新型木聚糖酶的关键催化氨基酸残基为Glu62、Asp283和Asp125。
本发明具有以下有益效果：
(1)本发明的BgXyn8A是一个新型木聚糖酶，最适温度60℃，最适pH值为5.0–6.0，常温下，在酸性、中性和碱性范围内均具有较高活性；在55℃和50℃下的半衰期分别为20min和3.5h，酶的pH稳定性较好，在pH3.0–10.0范围内于25℃放置12h后仍能保留80％以上的酶活性；对金属离子耐受能力强。
(2)本发明的BgXyn8A水解木聚糖能特异性产生低聚糖，包括木二糖至木六糖及长链寡糖，不产生木糖。在农林废弃物的资源化利用上拥有独特的优势，水解碱预处理玉米芯不仅特异性产生了木二糖至木六糖，还产生了大量阿拉伯糖基木寡糖和聚合度大于6的长链木寡糖。
附图说明
图1为木聚糖酶的鉴定及SDS-PAGE分析。(A)BgXyn8A结构域结构示意图。(B)SDS-PAGE分析。M：蛋白Marker；1：粗酶液；2：镍柱纯化后的蛋白。
图2为pH和温度对重组木聚糖酶活性的影响。(A)最适pH；(B)最适温度；(C)pH稳定性；(D)温度稳定性。
图3为离子色谱分析重组木聚糖酶水解聚糖的产物(图3A显示BgXyn8A 能水解木四糖、木五糖和木六糖，但是不能水解木二糖和木三糖，图3B显示 BgXyn8A对纤维寡糖的水解特性与木寡糖相似)。
图4为离子色谱分析重组木聚糖酶水解聚糖的产物(显示BgXyn8A水解榉木木聚糖的产物中不含有木糖，低聚糖组分包括木二糖至木六糖及其他长链寡糖)。
图5为离子色谱分析重组木聚糖酶水解玉米芯的产物。
图6为AXOS、XOS和玉米芯水解产物PMP衍生物的HPLC-UV色谱图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例中所用芽孢杆菌Bacillus sp.KW1，保存于广东省广州市广州中医药大学中药资源科学与工程研究中心，并已在以下论文中披露过：Kui Wang, Ruoting Cao,Meiling Wang,Qibin Lin,Ruoting Zhan,Hui Xu,Sidi Wang.A novel thermostableGH10 xylanase with activities on a wide variety of cellulosic substrates froma xylanolytic Bacillus strain exhibiting significant synergy with commercialCelluclast 1.5L in pretreated corn stover hydrolysis.Biotechnology forBiofuels, 2019,12:48。
试验材料及试剂
1、菌株及载体：本发明从芽孢杆菌Bacillus sp.KW1中分离得到一种新型耐热木聚糖BgXyn8A，表达载体pET-28a购自德国Merck-Novagen公司，表达菌株大肠杆菌BL21(DE3)购自北京全式金生物科技有限公司。
2、酶类及其他生化试剂：限制性内切酶购自宝日医生物技术(北京)有限公司，连接酶购自日本Toyobo公司,其他普通生化试剂均可从普通生化试剂公司购买得到。
3、标准品：木寡糖[木二糖(X2)、木三糖(X3)、木四糖(X4)、木五糖(X5) 和木六糖(X6)]、纤维寡糖[纤维二糖(G2)、纤维三糖(G3)、纤维四糖(G4)、纤维五糖(G5)和纤维六糖(G6)]、阿拉伯呋喃糖基木寡糖[阿拉伯呋喃糖基木二糖(AX2)、阿拉伯呋喃糖基木三糖(AX3)和阿拉伯呋喃糖基木四糖(X4)]，购自爱尔兰Megazyme公司。
4、培养基：LB培养基(1％蛋白胨，0.5％酵母提取粉，1％NaCl)。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1：芽孢杆菌Bacillus sp.KW1木聚糖酶编码基因BgXyn8A的克隆
S1、提取来源于土壤样品的芽孢杆菌Bacillus sp.KW1基因组DNA；
S2、根据成熟蛋白BgXyn8A的编码序列设计合成了引物Xyn-F-EcoR I， Xyn-R-XhoI，引物序列：
Xyn-F-EcoR I(5’-CGCGGATCCgaattcGCCGTACATTCCAAGACTCC-3’)
Xyn-R-XhoI(5’-GAGGTGGTGctcgagTTAAGGAAGGTACATCGTAAA TTTGC-3’)，小写为酶切位点，小写序列的左侧序列为保护碱基
S3、以芽孢杆菌Bacillus sp.KW1总DNA为模板进行Touch-down PCR扩增获得序列如SEQ ID NO.5所示的成熟木聚糖酶基因BgXyn8A。
PCR反应参数为：
S3.1、94℃变性5min；
S3.2、94℃变性30sec；
S3.3、65-55℃退火30sec S3.每个循环降0.5度，
S3.4、72℃延伸60sec 10个循环，
S3.5、94℃变性30sec，
S3.6、60℃退火45sec,
S3.7、72℃延伸l min，
S3.8、30个循环后72℃保温10min。
S3.9、得到一约1239bp片段S3.含酶切位点和保护碱基)，编码402个氨基酸和一个终止密码子，该基因所编码的成熟蛋白理论分子量约为45kDa。
实施例2：重组木聚糖酶BgXyn8A的制备
S1、将表达载体pET-28a进行双酶切(EcoR I+Xho I)，同时将编码成熟木聚糖酶的基因BgXyn8A双酶切(EcoR I+Xho I)，切出编码成熟木聚糖酶的基因片段与表达载体pET-28a连接，获得含有木聚糖酶基因BgXyn8A的重组质粒 pET28a-BgXyn8A转化大肠杆菌DH5α，获得重组克隆菌株 DH5α/pET28a-BgXyn8A。
S2、提取pET28a-BgXyn8A质粒，通过化学转化法转入到表达菌株大肠杆菌 BL21(DE3)，并在含有卡那霉素(50μg/mL)的LB琼脂平板上37℃培养过夜。挑取单菌落接种于10mL含有相同浓度卡那霉素的新鲜LB培养基中，37℃剧烈振荡(220rpm)培养6h。随后，将10mL培养物转移至含500mL LB的三角瓶中，在37℃下继续培养至OD值至0.6–0.8，然后加入IPTG至终浓度为0.2 mM，并转移到16℃下200rpm进行诱导培养16h。培养结束后，将菌液在12,000 g离心30min，然后弃上清、收集菌体并置于-20℃保存备用。
S3、所有重组蛋白的N端都带有His标签，故选用TALON Metal Affinity Resins进行蛋白质纯化，SDS-PAGE结果表明(图1)，重组酶在大肠杆菌中得到了可溶性表达。
实施例3：木聚糖酶BgXyn8A的活性分析
4-羟基苯甲酰肼(pHBAH)法：具体方法如下：在设定的反应条件下酶解木聚糖底物(终浓度5mg/mL)，取50μL酶解反应液，加入150μL pHBAH溶液(0.4 M NaOH-0.1M柠檬酸钠缓冲液溶液配制，终浓度1mg/mL)，于100℃下反应10 min，然后取150μL反应液测OD410。一个酶活单位(1U)定义为在测定条件下每分钟释放1μmol产物所需的酶量。
1、木聚糖酶BgXyn8A最适pH和pH稳定性的测定方法如下：
将实施例2纯化的重组木聚糖酶在不同的pH下进行酶促反应以测定其最适 pH。以1％榉木木聚糖为底物。缓冲溶液的缓冲梯度不同：0.2M Na2HPO4-0.1M 柠檬酸缓冲液，pH3.0-6.0；0.1M Na2HPO4-NaH2PO4缓冲液，pH 6.0–7.5；0.2M Tris-盐酸缓冲液，pH 7.5–9.0；0.2M甘氨酸-氢氧化钠缓冲液，pH 9.0–10.0。在 25μL的pH 6.0的1％的BeeWX底物中加入50nM纯化酶液，分别用缓冲液补齐至50μL，在60℃反应15min后加入150μL pHBAH(1mg/mL)，100℃反应10min后取出150μL反应液测OD410的吸光值。结果表明，表明该酶的最适pH为5.0–6.0，在pH 6.0缓冲液(Na2HPO4-NaH2PO4)中酶的相对活性最高，在pH 为5.0和6.5时能够达到最大酶活的80％以上(图2A)。将重组酶于各种不同pH 值的缓冲液中25℃处理1h，并于pH 6.0、60℃的条件下测定残留酶活，以研究酶的pH稳定性。结果表明，测得其木聚糖酶活性在pH3.0–10.0之间都很稳定相对剩余酶活均在80％以上(图2C)。
2、木聚糖酶BgXyn8A最适温度和热稳定性的测定方法如下：
木聚糖酶的最适温度的测定为在Na2HPO4-NaH2PO4缓冲液(pH 6.0)缓冲液体系及不同温度下进行酶促反应。热稳定性测定为木聚糖酶在不同温度下处理不同时间，再在60℃下进行酶活性测定。酶反应最适温度测定结果表明酶活性最适温度为60℃(图2B)。酶的热稳定性性试验表明，BgXyn8A有良好的热稳定性，在50℃下温育4h，能保持40％以上的酶活(图2D)。
3、不同金属离子和化学试剂对BgXyn8A酶活的影响测定如下：
在酶促反应体系中加入不同的金属离子及化学试剂(表1)，研究其对酶活性的影响,各种物质终浓度为1mM和10mM。在60℃、pH 6.0条件下测定酶活性。结果表明，以1％榉木木聚糖为底物，在离子浓度为较低时(1mM)，Fe2+、Al3+、 Fe3+能将BgXyn8A的酶活力分别提高至142％、153％、210％；在离子浓度为较高时(10mM)，Al3+、Ni2+、Fe3+及EDTA将BgXyn8A的酶活力分别提高至131％、 128％、115％、114％；另外，BgXyn8A对其他金属离子也有较高的耐受能力。
4、木聚糖酶BgXyn8A的酶促反应动力学参数和比酶活的测定：
用不同浓度的榉木木聚糖为底物，在pH 6.0、60℃下测定酶活性，计算 BgXyn8A的酶促反应动力学参数。以榉木木聚糖为底物所测定的Km为2.88 mg/mL，Vmax为227.40μmol/min/mg，kcat为56.85s-1。另外，测得其对小麦阿拉伯木聚糖、榉木木聚糖、桦木木聚糖和燕麦木聚糖的比酶活分别为3700、1010、 551和368U/mg。
表1重组木聚糖酶的金属离子稳定性
实施例4：重组木聚糖酶BgXyn8A对聚糖的水解特性
分别用纯化的BgXyn8A在最适反应条件下水解聚糖[榉木木聚糖、木寡糖 (X2–X6)和纤维寡糖(G2–G6)]，然后用离子色谱仪(HPAEC-PAD)分析水解产物。离子色谱仪所用保护柱为CarboPac PA10 Guard(4×50mm)、分析柱为(4×250 mm)(Dionex，Sunnyvale，CA，USA)、检测器为脉冲安培检测器。使用淋洗液 A(100mM NaOH溶液)和B(100mM NaOH-500mMNaAc溶液)进行糖的洗脱。
榉木木聚糖及木寡糖水解产物的洗脱条件为：0–20min，0–30％淋洗液B 线性梯度洗脱，流速1.0mL/min；20–35min，100％淋洗液B等度洗脱，流速 1.0mL/min。将木糖(X1)和低聚木糖(X2–X6)混合用于水解产物中糖组分的鉴定。
纤维寡糖水解产物的洗脱条件为：0–40min，0–45％淋洗液B线性梯度洗脱，流速1.0mL/min；40–55min，100％淋洗液B等度洗脱，流速1.0mL/min。将葡萄糖(G1)和纤维寡糖(G2–G6)混合用于水解产物中糖组分的鉴定。
结果表明，BgXyn8A能水解木四糖、木五糖和木六糖，但是不能水解木二糖和木三糖(图3A)，其对纤维寡糖的水解特性与木寡糖相似(图3B)；BgXyn8A 水解榉木木聚糖的产物中不含有木糖，低聚糖组分包括木二糖至木六糖及其他长链寡糖(图4)，表明该酶是一个严谨的内切木聚糖酶，不具有木糖苷酶活性。
实施例5：BgXyn8A水解玉米芯生产功能性低聚糖
S1、玉米芯碱预处理生成总糖
将5g玉米芯(50目)与80mL 1.25M NaOH混合15min，在150rpm的摇床上于37℃振荡3h，13,000g离心20min，上清液用浓盐酸酸化至pH 6.0。用苯酚硫酸法测得总糖含量为225g/kg。
S2、酶解玉米芯提取物生产功能性低聚糖
在100mL锥形瓶中加入20mL玉米芯提取液，加入20U重组木聚糖酶，混匀后在60℃、220rpm摇床中反应12h，取25μL稀释20倍后进行离子色谱分析，见图5，酶解碱预处理玉米芯没有产生木糖，所产生的低聚糖除了DP为 2–6的低聚木糖外，还大量产生了其他长链木寡糖或取代低聚木糖。
实施例6：LC-MS分析玉米芯水解产物的组分
S1、玉米芯水解产物衍生化处理
取100μL玉米芯水解或XOS标准混合物(X1，X2–X6，各5mg/mL)与100 μL的0.3M氢氧化铵溶液混合。在离心管中加入120μL的0.5M PMP甲醇溶液，然后涡旋将其完全混合。整个混合物用金属浴加热到70℃，保温1h，然后冷却到室温，加入100μL的0.3M盐酸中和。用500μL氯仿萃取3次。去掉氯仿层，水层用0.22μm微孔滤膜过滤后进行色谱分析。
S2、玉米芯水解产物的LC-MS分析
LC-MS使用Thermo Surveyor光电二极管阵列检测器(PAD)，以及配备电喷雾电离(ESI)源的TSQ QuantumACCESS离子阱质谱仪。ESI用于记录在m/z 200–2000质量范围内的正离子模式和负离子模式。雾化电压为25psi，毛细管电压为4500V，N2温度设定为275℃，流速为6mL/min。分离柱为C18色谱柱(100 mm×4.6mm，5μm，Thermo)；流动相为乙腈(A)和10mM醋酸铵溶液(pH5.5) (B)。色谱条件为：0–12min，20％A等度洗脱；12–22min，20–35％A线性梯度洗脱；22–30min，35％A等度洗脱，整个过程流速均为0.3mL/min。进样量20μL。
分析结果表明，BgXyn8A水解碱预处理玉米芯不仅特异性产生了木二糖至木六糖，还产生了大量阿拉伯糖基低聚木糖(AXOS)和聚合度大于6的长链木寡糖(图6、表2)。
表2 PMP标记不同木寡糖的LC-ESI-MS表征
实施例7：定点突变确证关键催化氨基酸残基
S1、BgXyn8A与其他GH8家族酶蛋白序列多序列比对
使用CLUSTAL X软件进行基于结构的序列比对，根据比对结果，推测 Glu62、Asp283及Asp125为BgXyn8A的关键催化氨基酸残基。
S2、定点突变进一步确证关键催化氨基酸残基
突变后的木聚糖酶和野生型木聚糖酶相比，由原始序列62位的谷氨酸(Glu, E)和125、283位的天冬氨酸(Asp,D)突变为丙氨酸(Ala,A)。
根据突变位点设计引物E62A-F，E62A-R；D125A-F，D125A-R；D283A-F， D283A-R，引物序列：
E62A-F(5’-GTCAAAACGGCAGGCATGAGCTACGG-3’),
E62A-R(5’-CTCATGCCTGCCGTTTTGACGCTG-3’)；
D125A-F(5’-AACGTAGCATCTGCCGGCGAGGTCTGGTT-3’),
D125A-R(5’-CAGACCTCGCCGGCAGATGCTACGTTCT-3’)；
D283A-F(5’-AATTTCAGCTATGCTGCTTGGCGGACAG-3’), D283A-R(5’-TCCGCCAAGCAGCATAGCTGAAATTGC-3’)，下划线为突变位点
以已经连接了基因BgXyn8A的pET28a质粒作为突变模板，采用已设计好的各定点突变引物进行PCR以引入氨基酸定点突变，得到含突变基因的质粒pET28a-BgXyn8A/E62A、pET28a-BgXyn8A/D125A、pET28a-BgXyn8A/D283A。
突变后的质粒进行原核表达、活性测定，方法同野生型木聚糖酶。活性测定结果表明突变子完全失活，进一步确证了Glu62、Asp283及Asp125为BgXyn8A 的关键催化氨基酸残基，催化反应过程中谷氨酸(Glu62)的羧基起到酸催化作用，天冬氨酸(Asp283)的羧基作为碱性基团促使水分子提供的质子攻击异头碳原子，最终使得糖苷键断裂并导致异头碳的反转。此外，天冬氨酸(Asp125)可能在糖环变形和过渡态稳定中发挥重要作用。。
实施例8：定点突变改造木聚糖酶的热稳定性
突变后的木聚糖酶和野生型木聚糖酶相比，由原始序列第29位的赖氨酸 (Lys,K)突变为甲硫氨酸(Met,M)。
根据突变位点设计引物K29M-F，K29M-R，引物序列：
K29M-F(5’-ATTTTGATGGGATGGGCGGTTCCCTGTTTC-3’),
K29M-F(5’-GGAACCGCCCATCCCATCAAAATATTTTTTG-3’)，下划线为突变位点
以已经连接了基因BgXyn8A的pET28a质粒作为突变模板，采用定点突变引物进行PCR以引入氨基酸定点突变，得到含突变基因的质粒 pET28a-BgXyn8A/K29M。
突变后的质粒进行原核表达及酶学性质分子，方法同野生型木聚糖酶。热稳定性分析表明，BgXyn8A/K29M在55℃处理30min后，残留活性是原始木聚糖酶BgXyn8A的1.7倍，但是其最适反应温度没有改变。此外，BgXyn8A/K29M 的温度宽泛性得到增加：在温度为45–55℃时，原始木聚糖酶BgXyn8A的相对活性约为74–94％，而BgXyn8A/K29M的相对活性都在90％以上，约为91–97％。在65℃和70℃时，BgXyn8A/K29M的相对活性也比野生型木聚糖酶BgXyn8A 高。底物特异性分析表明突变体的底物谱和产物谱没有改变。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 广州中医药大学
<120> 一种特异性产生木寡糖的新型木聚糖酶BgXyn8A及其基因与应用
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 芽孢杆菌(Bacillus sp. KW1)
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atgcgaaaaa gtttaaaatg gatcatggct gtctttattg gtttaacatg tttttgtgcc 60
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ccggcaaaac aaatttgaac caggcttaca aaaaatattt tgatgggaaa ggcggttccc 180
tgtttcatta tatgagagac ggttcggctt tcatcgcttc gacgttggac gatgaccttg 240
gcaatggcta ttacagcgtc aaaacggaag gcatgagcta cgggatgatg atcgcgctgc 300
aaatgaatga tgagtataaa tttcaaaggc tgtgggattt cgtccgcaaa tatatgcgcc 360
ataccgacaa aaatgacagc ttgtacggct accatcgctg gcatatgaaa acgaacggct 420
ctgacgtgca aaccattgat cagaacgtag catctgacgg cgaggtctgg tttgcggcag 480
cgctcatgat ggcgtccggc cgctggggag ataaacaata cccgtatgat tacaaagcgc 540
gggcccaaga catgcttgat gccttggccg gtgacggtga atacgcaaac gccagcaagg 600
aatcaaggat ttttattaaa aacggcaatg atcagcgcta tgcgatggtt cgattcggcc 660
cttatgtcaa ttggacggat ccttcgtatc atgtgccggc ttttttcgag ctattcgcca 720
aaagtgccag aagcagccag cagtattttt ggaaagacgc ggcaaacaag tctcgaaaat 780
acttgtctga aacgacgttt aaaagcgtat tacctaatgg atcaaccgtt accaatgcag 840
ccaccggcct tttcccagat gaagccggct tcgatggtgt aagcgacgcg gcacattcct 900
caacgaaaac agaccgcaat ttcagctatg atgcttggcg gacagtgtcc catgtcgcca 960
tggactatac gctgtggtcc tcggcagata acccatatcg tgccagagag caaaaagcgg 1020
ttaacaagtt cttgacgttt atgaaacggg aaaactacgg gagaacggcc catgaatata 1080
tactgaatgg aacggccgtt aaaaaaggca gtgcgatggg tctgatcgcg gccaatgcag 1140
gcggtgccac agcagcaagc aatggctcat taaggacagg gtttgcgaat gcgtttaaca 1200
gcgcctatat tcctgacgat tattacggat cctgcctgta catgctgaac agcctcgcgg 1260
caaacggcaa atttacgatg taccttcctt aa 1292
<210> 5
<211> 1209
<212> DNA
<213> 芽孢杆菌(Bacillus sp. KW1)
<400> 5
gccgtacatt ccaagactcc ggatattctc ggaacaaccg gcaaaacaaa tttgaaccag 60
gcttacaaaa aatattttga tgggaaaggc ggttccctgt ttcattatat gagagacggt 120
tcggctttca tcgcttcgac gttggacgat gaccttggca atggctatta cagcgtcaaa 180
acggaaggca tgagctacgg gatgatgatc gcgctgcaaa tgaatgatga gtataaattt 240
caaaggctgt gggatttcgt ccgcaaatat atgcgccata ccgacaaaaa tgacagcttg 300
tacggctacc atcgctggca tatgaaaacg aacggctctg acgtgcaaac cattgatcag 360
aacgtagcat ctgacggcga ggtctggttt gcggcagcgc tcatgatggc gtccggccgc 420
tggggagata aacaataccc gtatgattac aaagcgcggg cccaagacat gcttgatgcc 480
ttggccggtg acggtgaata cgcaaacgcc agcaaggaat caaggatttt tattaaaaac 540
ggcaatgatc agcgctatgc gatggttcga ttcggccctt atgtcaattg gacggatcct 600
tcgtatcatg tgccggcttt tttcgagcta ttcgccaaaa gtgccagaag cagccagcag 660
tatttttgga aagacgcggc aaacaagtct cgaaaatact tgtctgaaac gacgtttaaa 720
agcgtattac ctaatggatc aaccgttacc aatgcagcca ccggcctttt cccagatgaa 780
gccggcttcg atggtgtaag cgacgcggca cattcctcaa cgaaaacaga ccgcaatttc 840
agctatgatg cttggcggac agtgtcccat gtcgccatgg actatacgct gtggtcctcg 900
gcagataacc catatcgtgc cagagagcaa aaagcggtta acaagttctt gacgtttatg 960
aaacgggaaa actacgggag aacggcccat gaatatatac tgaatggaac ggccgttaaa 1020
aaaggcagtg cgatgggtct gatcgcggcc aatgcaggcg gtgccacagc agcaagcaat 1080
ggctcattaa ggacagggtt tgcgaatgcg tttaacagcg cctatattcc tgacgattat 1140
tacggatcct gcctgtacat gctgaacagc ctcgcggcaa acggcaaatt tacgatgtac 1200
cttccttaa 1209
<210> 6
<211> 83
<212> DNA
<213> 芽孢杆菌(Bacillus sp. KW1)
<400> 6
atgcgaaaaa gtttaaaatg gatcatggct gtctttattg gtttaacatg tttttgtgcc 60
gcttattctc aaacagccat ggc 83
Claims (10)
1.一种木聚糖酶BgXyn8A，其特征在于，其氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.一种权利要求1所述木聚糖酶BgXyn8A的编码基因，其特征在于，其核苷酸序列如SEQID NO.4或SEQ ID NO.5所示。
3.一种突变的木聚糖酶BgXyn8A/K29M，其特征在于，其氨基酸序列与SEQ ID NO.2所示氨基酸序列的不同之处在于N端第29位的赖氨酸(Lys,K)突变为甲硫氨酸(Met,M)。
4.包含权利要求2所述木聚糖酶BgXyn8A编码基因的重组载体。
5.包含权利要求2所述木聚糖酶BgXyn8A编码基因的重组菌株。
6.权利要求1或3所述木聚糖酶BgXyn8A或BgXyn8A/K29M在酶解木聚糖产生木寡糖中的应用。
7.权利要求1或3所述木聚糖酶BgXyn8A或BgXyn8A/K29M在酶解含木聚糖的农林废弃物产生木寡糖中的应用。
8.权利要求1或3所述木聚糖酶BgXyn8A或BgXyn8A/K29M在制备酶制剂中的应用，所述酶制剂能够酶解木聚糖、或酶解含木聚糖的农林废弃物产生木寡糖。
9.一种制备权利要求1所述木聚糖酶BgXyn8A的方法，包括以下步骤:
S1.用权利要求4所述重组载体转化宿主细胞，得重组菌株；
S2.培养重组菌株，诱导重组木聚糖酶BgXyn8A编码基因表达；
S3.纯化所表达的木聚糖酶BgXyn8A。
10.一种酶解农林废弃物产生木寡糖的方法，其特征在于，利用权利要求1或3所述木聚糖酶BgXyn8A酶解农林废弃物。
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Citations (3)
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|---|---|---|---|---|
| CN102732496A (zh) * | 2012-05-17 | 2012-10-17 | 天津工业生物技术研究所 | 一种木聚糖酶及其应用 |
| CN107129976A (zh) * | 2017-06-02 | 2017-09-05 | 中国农业科学院饲料研究所 | 一种中性高温木聚糖酶及其编码基因和其应用 |
| CN111511771A (zh) * | 2017-12-22 | 2020-08-07 | 诺维信公司 | 小麦研磨方法和gh8木聚糖酶 |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732496A (zh) * | 2012-05-17 | 2012-10-17 | 天津工业生物技术研究所 | 一种木聚糖酶及其应用 |
| CN107129976A (zh) * | 2017-06-02 | 2017-09-05 | 中国农业科学院饲料研究所 | 一种中性高温木聚糖酶及其编码基因和其应用 |
| CN111511771A (zh) * | 2017-12-22 | 2020-08-07 | 诺维信公司 | 小麦研磨方法和gh8木聚糖酶 |
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| ANNICK POLLET: "Functional analysis of glycoside hydrolase family 8 xylanases shows narrow but distinct substrate specificities and biotechnological potential", APPL MICROBIOL BIOTECHNOL ., vol. 87, no. 6, 31 August 2010 (2010-08-31), pages 2125 - 2135, XP019841657 * |
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