CN112322604A - 一种高比酶活木聚糖酶突变体及其应用 - Google Patents
一种高比酶活木聚糖酶突变体及其应用 Download PDFInfo
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- CN112322604A CN112322604A CN202011206842.4A CN202011206842A CN112322604A CN 112322604 A CN112322604 A CN 112322604A CN 202011206842 A CN202011206842 A CN 202011206842A CN 112322604 A CN112322604 A CN 112322604A
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- xylanase
- ser
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Abstract
本发明涉及基因工程领域，公开了一种高比酶活木聚糖酶突变体及其应用。本发明以来源于Bacillus subtilis Lucky9的GH11家族的木聚糖酶XYN为母本，采用分子生物技术进行氨基酸突变后并表达。与原始木聚糖酶XYN相比，突变体N36Y比酶活提高了180％，达150U/mg，取得了意料不到的技术效果。本发明所述木聚糖酶突变体比活力的提高，有利于降低木聚糖酶的生产成本，进一步拓展了该酶在功能性糖领域的广泛应用，具有广阔市场前景。
Description
技术领域
本发明属于基因工程领域，具体涉及一种高比酶活木聚糖酶突变体及其应用
背景技术
木聚糖是半纤维素的主要成分，是继纤维素之后自然界含量第二丰富的异质多聚体。木聚糖广泛存在于玉米芯、甘蔗渣、麦麸、秸秆等农业废弃物。木聚糖酶是木聚糖降解过程中起关键作用的一类酶，起最主要作用的是内切β-1,4-D-木聚糖酶(EC 3.2.1.8)。其主要水解木聚糖碳骨架中β-1,4-木糖苷键。木聚糖酶的分子结构复杂，按照其功能区域划分可分为：催化区域、纤维素结合区域、木聚糖结合区域、热稳定区域、连接序列、重复序列和其他未知功能的非催化区域。其中不同种类木聚糖酶的结构区域会存在一定差别，有的仅含有一个催化区域，有的则含有一个催化区域和多个非催化区域。
木聚糖酶在造纸、饲料、食品和能源等领域应用广泛。在食品工业中，木聚糖酶主要用于烘焙和酿造工艺。饲料中添加木聚糖酶，可显著降低木聚糖分子大小，将其分解成低聚木糖，从而改善饲料性能，消除或降低因粘度增加而引起的抗营养作用。目前，多数木聚糖酶的最适温度大都低于55℃，且稳定性差，不能满足工业化的应用。随着基因工程和分子生物学的发展，利用外源蛋白高效表达系统表达目的蛋白，或是利用木聚糖酶蛋白结构进行人工改良提高其比活性，是进一步提高木聚糖酶工业应用的有效途径。到目前为止，已有许多报道对蛋白活性成功的改造。
发明内容
本发明的目的是一种高比酶活木聚糖酶突变体及其应用，通过定点突变的方法改造木聚糖酶，使改造后的木聚糖酶突变体在活性上更加优良，有利于其在工业范围内的应用。
本发明具体技术方案如下：
一种木聚糖酶突变体，所述的木聚糖酶突变体的氨基酸序列如SEQ ID NO.3～4所述。
本发明对来源于Bacillus subtilis Lucky9的GH11家族的木聚糖酶XYN(氨基酸序列如SEQ ID NO.1，核酸序列：SEQ ID NO.2所示)的N末端突变，经过大量筛选发现，第36位氨基酸由Asn变为Tyr或Phe能引起木聚糖酶水解活性的提高。
SEQ ID NO.3～4所述的序列为SEQ ID NO.1所述的木聚糖酶XYN第36位氨基酸Asn突变为Tyr或Phe的木聚糖酶突变体。本发明所述木聚糖酶突变体的第1～28位氨基酸为信号肽，信号肽负责把木聚糖引导到细胞周质空间内进行进一步加工。SEQ ID NO.5～6所述序列为SEQ ID NO.3～4所述的序列去掉信号肽后的序列。
本发明的另一个目的是提供编码上述木聚糖酶突变体的基因，所述的基因的核酸序列如SEQ ID NO.7-10所示。
本发明的另一个目的是提供一种木聚糖酶突变体的表达载体，表达本发明所述的木聚糖酶突变体。具体的，所述的表达载体包含如SEQ ID NO.7-10所示的基因。本发明所述载体可以为质粒、噬菌体、病毒或宿主细胞。
优选的，所述宿主细胞选自大肠杆菌、酵母、芽孢杆菌、乳酸杆菌、曲霉或木霉。
本发明的另一个目的是提供本发明所述木聚糖酶突变体、所述的木聚糖酶突变体的基因或者所述的木聚糖酶突变体的表达载体在木聚糖生物降解中的应用。
具体的，本发明所述的木聚糖酶突变体、基因或者其表达载体可以用于制备具有木聚糖酶解能力的酶类产品。可用于制备木二糖、木三糖、木四糖等。
具体的，所述木聚糖包括榉木木聚糖、可溶小麦阿拉伯木聚糖、桦木木聚糖、不可溶小麦阿拉伯木聚糖。
本发明的木聚糖突变体的最适pH为7.0，在碱性、中性范围内具有较高的活性。木聚糖突变体的最适作用温度为60℃。本发明所述木聚糖突变体与野生型相比，对底物的亲和力显著提高，比活力提高。
附图说明
图1.木聚糖酶XYN与突变体的SDS-PAGE电泳结果。
图2.本发明所述木聚糖酶突变体的相对水解活性。
图3.木聚糖酶XYN及其突变体N36Y和N36F的最适温度和最适pH以及温度和pH稳定性考察结果。(图3a为木聚糖酶XYN及其突变体N36Y和N36F的最适pH，图3b为木聚糖酶XYN及其突变体N36Y和N36F的pH稳定性，图3c为木聚糖酶XYN及其突变体N36Y和N36F的最适温度，图3d为木聚糖酶XYN及其突变体N36Y和N36F的温度稳定性)。
图4.木聚糖酶XYN及其突变体N36Y和N36F对于榉木木聚糖底物的水解产物TLC分析结果。
具体实施方式
本发明采用遗传工程和分子生物学领域使用的常规技术和方法，例如，Molecularcloning:a laboratory manual,3nd ED.(Sambrook,2001)和Current protocols inmolecular biology(Ausubel,2003)中所记载的方法。这些一般性的参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其他常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。
下面结合具体实施方式对本发明进行详述描述
实施例一 木聚糖酶突变体基因的突变
为提高来源于Bacillus subtilis Lucky9的GH11家族的木聚糖酶XYN(氨基酸序列如SEQ ID NO.1，核酸序列：SEQ ID NO.2所示)的活性，通过底物对接选择了4个与木聚糖链结合相关的位点，结果发现36N对酶活力影响最为显著。因而，进一步通过定向进化技术对该位点进行大量的突变筛选，设计如下突变引物：
以木聚糖酶XYN基因为模版，参照Vazyme生物产品及操作手册，全质粒扩增出定点突变序列。经Dpn I进行酶切处理后，消化结束后，将PCR产物采用热激法转化至大肠杆菌感受态细胞E.coli BL21(DE3)，并涂布于含有100μg/ml硫酸卡那霉素LB琼脂平板上，37℃培养14-16h。经序列测定(由安徽通用生物公司完成)验证突变结果，获得相应突变体。
实施例二 木聚糖酶及突变体酶的获取
分别将实施例一构建的突变体以及原始酶XYN接种至50mL的LB液体培养基中，加入硫酸卡那霉素使其终浓度为100μg/mL，180rpm，37℃过夜培养；以2％的接种量将过夜培养的种子液，接种至新鲜的50mL的LB液体培养基中，180rpm，37℃恒温培养至OD600为0.6～1.0时，加入诱导剂IPTG(异丙基-β-D硫代半乳糖苷)(终浓度0.1mmol/L)，25℃诱导表达24h。
取诱导表达的发酵液，12000rpm离心20min，弃去上清，然后用50mM Na2HPO4-KH2PO4(pH 7.0)缓冲重悬菌体，然后超声破碎，进行SDS-PAGE电泳检测，浓缩胶浓度为4％，分离胶浓度为12.5％，样品与上样缓冲按照3:1的比例混合，沸水浴反应5min进行上样电泳。电泳仪设定初始电压为120V，当样品移动至分离胶时提高电压至230V，直至样品移动到电泳槽底部时结束电泳。
结果如图1所示，木聚糖酶XYN的分子量为20kDa，诱导后木聚糖酶XYN和突变体都在在20kDa处都有明显的条带，这与木聚糖酶XYN的分子量一致。表明木聚糖酶XYN和突变体成功诱导表达。
由于木聚糖酶XYN的C端融合了六个组氨酸(His)标签，因此其突变体的C端同样有组氨酸标签。Ni柱中的氯化镍可以与含有His标签的蛋白结合，也可以与咪唑结合，因此使用镍柱分别对目的蛋白进行纯化，通过增加咪唑浓度达到蛋白洗脱的目的，从而得到纯化的目的蛋白，发酵诱导表达的菌液经离心破碎后，所得上清液经0.22μm滤膜过滤后即得粗酶液，然后使用镍柱进行目的蛋白的分离纯化。步骤如下：
a.用0.22μm滤膜过滤的蒸馏水以2mL/min的流速冲洗镍柱，洗去填料保护液同时压实填料；
b.用至少5倍体积的Buffer A(20mM Tris-HCl，pH 7.5)以2mL/min的流速冲洗镍柱，平衡镍柱内pH；
c.用注射器将预处理的蛋白样品打入进样环中，流速为0.5mL/min，收集样品的穿透峰蛋白；
d.再次用至少5-10倍体积的Buffer A(20mM Tris-HCl，pH 7.5)以2mL/min的流速冲洗镍柱，平衡镍柱内pH，直至无蛋白被洗脱出来；
e.使用梯度洗脱法，每个梯度用至少5倍体积的Buffer B(20mM Tris-HCl，500mM咪唑，pH 7.5)以2mL/min的流速冲洗镍柱，收集每个梯度的吸收峰蛋白，直至无蛋白被洗脱出来；
f.用至少5-10倍体积的已过滤蒸馏水以2mL/min的流速冲洗镍柱，直至纯化仪显示无离子曲线和蛋白吸收峰；
g.加入20％乙醇保存镍柱；
h.使用GE公司预装的脱盐柱，以50mM Na2HPO4-KH2PO4(pH 7.0)缓冲置换含有咪唑的Buffer，去除蛋白液中的咪唑；
i.将收集得到的不同梯度的蛋白溶液进行SDS-PAGE验证，即得不同突变体得纯化蛋白。
实施例三 木聚糖酶XYN最优突变体的筛选程序
木聚糖酶XYN突变体水解活力测定方法
酶活力单位定义：一个酶活力单位即为在60℃，pH 7.0条件下，每分钟由底物产1mmol还原糖所需的酶量定义为一个活力单位。
木聚糖酶(Endo-glucanase)：准确称取1g榉木木聚糖溶于100mL的Na2HPO4-KH2PO4缓冲中(50mM，pH 7.0)，搅拌混匀，准确吸取1.0mL加入试管作为酶反应底物，60℃预热5min后加入已适当稀释的蛋白酶液0.5mL，放入60℃的水浴摇床反应10min，加入3mL的DNS(3,5-二硝基水杨酸)试剂，沸水浴反应5min后迅速冷却至室温，以灭活的酶反应液为对照测定540nm波长下的吸光度值。
比酶活X＝(还原糖含量/150/10)/n
其中：X---比酶活，U/mg
150----------还原糖从毫克转换为微摩尔
10--反应时间
n-------------反应蛋白含量，mg
将实施例二中获得的18个突变体，以榉木木聚糖为底物测定酶活变化，突变体的相对水解活性结果如图2所示。大多数氨基酸突变后对于榉木木聚糖的底物活性均显著下降。当野生型木聚糖酶第36位氨基酸由Asn突变为Tyr和Phe时，突变体N36F和N36Y相对于野生型木聚糖酶XYN的酶活力显著上升。
实施例四 木聚糖酶突变体N36F和N36Y的酶学性质分析
1.最适pH和pH稳定性
配制不同pH的缓冲和底物：柠檬酸-柠檬酸钠(pH 3.0-6.0)、Na2HPO4-KH2PO4(pH 6.0-8.0)、甘氨酸-氢氧化钠(pH 8.0-9.0)，分别配制不同pH条件下的1％的榉木木聚糖底物反应液。
将实施例三得到的纯化得到的酶液按照一定的浓度稀释并吸取500ul加入装有1.0mL榉木木聚糖不同pH缓冲配制底物的试管中，60℃反应10min，反应结束后加入3mLDNS溶液煮沸5min，测定其酶活。以最高的酶活为100％，依次计算相对酶活，绘制酶活随着底物pH变化而变化的曲线。
将纯化得到的酶液用不同pH的缓冲进行稀释，置于冰上4℃孵育2h，然后按照测定木聚糖酶活力的方式测定其酶活力。以最高的酶活为100％，依次计算相对酶活，绘制不同孵育条件下酶活的变化曲线。
结果如图3a所示，木聚糖酶XYN及其突变体N36Y和N36F最适反应pH均为pH7.0，木聚糖酶XYN及其突变体在pH6.0条件下，具有最高水解活性，随着pH的升高或降低，其酶活性均减少。木聚糖酶XYN及其突变体N36Y和N36F在pH 6.0-9.0之间的稳定性较为平稳，在各个条件下孵育2h后均能保留70％以上的酶活力，而在pH 3.0-5.0之间，木聚糖酶XYN及其突变体的稳定性均下降，在各个pH条件下，表明突变体的pH稳定性与原酶一致(图3b)。
2.最适反应温度和温度稳定性。
将实施例三得到的纯化酶液按照一定浓度稀释并吸取500μl加入装有1.0mL榉木木聚糖底物的试管中，并分别在30℃、40℃、50℃、60℃、70℃、80℃条件下反应10min，反应结束后加入3mL DNS溶液煮沸5min，测定其酶活。以最高的酶活为100％，依次计算相对酶活，绘制酶活随着温度变化的曲线。
为了测定突变体和原始酶的温度稳定性，将纯化得到的酶液分别放入30℃、40℃、50℃、60℃、70℃水浴锅中孵育2h，然后按照测定木聚糖酶XYN活力的方式测定其剩余的酶活力。以最高的酶活为100％，依次计算相对酶活，绘制不同孵育条件下酶活的变化曲线。
木聚糖酶XYN与突变体N36Y和N36F的最适反应温度均为60℃，当温度在30℃-60℃时，木聚糖酶XYN及突变体的酶活逐渐升高，当温度高于60℃时，木聚糖酶XYN及其突变体的酶活均显著下降(图3c)。木聚糖酶XYN及其突变体N36Y和N36F在低于50℃条件下保温2h，仍然保留了80％以上的酶活力，而在高于60℃条件下保温6h后，木聚糖酶XYN活力还保留30％，而突变体N36Y活力仍有55％，表明突变体的热稳定性显然高于木聚糖酶XYN(图3d)。
实施例五 木聚糖酶XYN及其突变体N36Y和N36F的水解产物分析
根据实施例二获得纯化酶液体，以榉木木聚糖为底物进行产物水解分析，具体步骤如下：
(1)吸取适当稀释的纯酶液与底物榉木木聚糖，按照1:1的比例混合均匀；
(2)反应液置于60℃下反应6h后，煮沸反应液使酶灭活；
(3)将反应液离心，12000rpm，10min，弃去沉淀收集上清酶解产物；
(4)水解产物中加入3倍体积预冷的乙醇，过夜沉淀除去杂质后烘干；
(5)加入适当的蒸馏水使产物溶解，即得水解产物。
薄层层析法(TLC)检测
(1)将薄板置于120℃烘箱(除去挥发性杂质和水分，提高分离效率)中1h活化并置于干燥器中备用；
(2)配制展开剂(约60mL)：按正丁醇：乙酸：水＝3：2：1混合，实验前加入展层缸中约15-30min，防止边缘效应；
(3)点样：标准品点样量为1μL(木糖、木二糖、木三糖、木四糖、木五糖)，各个样品点样量为2μL；
(4)将展层缸置于通风橱中，室温下展层两次；
(5)样品显色：配制显色剂(约20mL)：按浓硫酸：乙醇＝1：9混合，将显色剂均匀的喷洒在薄板表面并用吹风机烘干，然后放入120℃烘箱中显色5min；
(6)根据标准品判断木聚糖酶XYN及其突变体N36Y和N36F的水解产物情况。
如图4所示，木聚糖酶XYN及其突变体N36Y和N36F对于榉木木聚糖底物的水解产物主要以木二糖和木三糖为主。
实施例六 木聚糖酶XYN及其突变体N36Y和N36F比活力、Km和Vmax测定用不同浓度的木聚糖酶(0.5，0.75，1.0，2.0，4.0,5.0，6.0，8.0，10.0mg/mL)为底物，在最适条件下测定酶活力，利用双倒数作图法计算出相应的反应速度，Km和Vmax值。
按照Bio-Rad试剂盒的方法，绘制标准曲线。选用蛋白浓度分别为2.0、1.5、1.0、0.75、0.5、0.25、0.125mg/mL，采用5ul(蛋白)和250ul(显色液)的反应体系，室温下反应10-60min，在OD595下测定其吸收值绘制标准曲线。比活力的测定方法，首先通过标准曲线计算目的蛋白的含量，其次在最适条件下测定木聚糖酶XYN及其突变体N36Y和N36F的酶活力，用酶活数与蛋白浓度的比值即为比活力。比活力的定义为：每毫克酶蛋白所具有的酶活力单位数。
表1木聚糖酶XYN及其突变体N36Y和N36F的反应动力学
| Enzyme | Km(mg/mL) | Vmax(μmol/mg min,) |
| 木聚糖酶XYN | 3.1 | 318.9 |
| 突变体N36Y | 1.2 | 605.2 |
| 突变体N36F | 1.9 | 440.3 |
结果显示，相对于木聚糖酶XYN，突变体N36Y和N36F对底物的亲和力大大提高，酶促反应速率得到显著提高，表明突变体N36Y和N36F的酶活力有显著的提高。
序列表
<110> 南京工业大学
<120> 一种高比酶活木聚糖酶突变体及其应用
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<212> DNA
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<212> DNA
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 20
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
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<210> 25
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 27
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 28
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ccgcccccat cagtccatgg ttgccagtag tctgtgctag 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ctagcacaga ctactggcaa ccatggactg atgggggcgg 40
<210> 35
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gcccccatca gtccaccttt gccagtagtc tgtgct 36
<210> 36
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
agcacagact actggcaaag gtggactgat gggggc 36
<210> 37
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ccgcccccat cagtccataa ttgccagtag tctgtgctag 40
<210> 38
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ctagcacaga ctactggcaa ttatggactg atgggggcgg 40
<210> 39
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gcccccatca gtccattctt gccagtagtc tgtgct 36
<210> 40
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
agcacagact actggcaaga atggactgat gggggc 36
<210> 41
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
cccccatcag tccaatgttg ccagtagtct gtgc 34
<210> 42
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gcacagacta ctggcaacat tggactgatg gggg 34
<210> 43
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gcccccatca gtccacattt gccagtagtc tgtgct 36
<210> 44
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
agcacagact actggcaaat gtggactgat gggggc 36
<210> 45
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gcccccatca gtccaacatt gccagtagtc tgtgct 36
<210> 46
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
agcacagact actggcaatg ttggactgat gggggc 36
Claims (7)
1.一种木聚糖酶突变体，其特征在于，所述的木聚糖酶突变体的氨基酸序列如SEQ IDNO.3～6所述。
2.编码权利要求1所述的木聚糖酶突变体的基因，其特征在于，所述的基因的核酸序列如SEQ ID NO.7-10所示。
3.一种木聚糖酶突变体的表达载体，其特征在于表达如权利要求1所述的木聚糖酶突变体。
4.如权利要求3所述的表达载体，其特征在于包含如SEQ ID NO.7-10所示的基因。
5.如权利要求3所述的表达载体，其特征在于所述载体为质粒、噬菌体、病毒或宿主细胞。
6.如权利要求5所述的表达载体，其特征在于所述宿主细胞选自大肠杆菌、酵母、芽孢杆菌、乳酸杆菌、曲霉或木霉。
7.根据权利要求1所述的木聚糖酶突变体，权利要求2所述的木聚糖酶突变体的基因或权利要求3-6任一项所述的木聚糖酶突变体的表达载体在木聚糖生物降解中的应用。
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| CN115725550A (zh) * | 2022-08-10 | 2023-03-03 | 中国海洋大学 | 一种木聚糖酶突变体及其应用 |
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| CN115725550A (zh) * | 2022-08-10 | 2023-03-03 | 中国海洋大学 | 一种木聚糖酶突变体及其应用 |
| CN115725550B (zh) * | 2022-08-10 | 2024-07-12 | 中国海洋大学 | 一种木聚糖酶突变体及其应用 |
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