CN106190935A - 一种产木聚糖酶的重组菌及其应用 - Google Patents

一种产木聚糖酶的重组菌及其应用 Download PDF

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CN106190935A
CN106190935A CN201610551416.1A CN201610551416A CN106190935A CN 106190935 A CN106190935 A CN 106190935A CN 201610551416 A CN201610551416 A CN 201610551416A CN 106190935 A CN106190935 A CN 106190935A
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xylanase
recombinant bacterium
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coli
pet28a
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唐蕾
胡方觊
张建华
张宏建
毛忠贵
杜建辉
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Jiangnan University
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Abstract

本发明公开了一种产木聚糖酶的重组菌及其应用，属于酶工程领域。本发明成功地实现了Clostridium clariflavum来源的木聚糖酶的异源表达，并通过IPTG诱导的方法提高重组菌产木聚糖酶的能力。采用本发明的诱导方法可以使重组菌生产的木聚糖酶是未诱导酶活的73倍，比酶活最高可达2.84U·mg‑1。该酶具有良好的pH稳定性，在pH3.5‑9.5酶活性保持在80％以上，并可将木聚糖降解为木糖。

Description

一种产木聚糖酶的重组菌及其应用
技术领域
本发明涉及一种产木聚糖酶的重组菌及其应用，属于酶工程领域。
背景技术
木聚糖是植物细胞壁半纤维素的主要成分，是一种丰富的可再生生物资源。木聚糖酶是一类可以将木聚糖降解成低聚木糖或木糖的酶的总称。由于木聚糖主链的聚合度不同，支链上残基在主链上的结合位点也不同，化学性质比较复杂，所以多种酶协同作用才能完全降解木聚糖。
木聚糖酶在自然界中来源十分广泛，陆地和海洋的细菌、真菌和酵母、原生动物、甲壳动物中都存在木聚糖酶。微生物中厌氧菌、需氧菌、嗜温微生物、嗜热微生物和极端微生物都可产生木聚糖酶。目前研究和应用得最多的木聚糖酶主要来自曲霉、木霉和细菌，而商品酶应用最多的是丝状真菌来源的木聚糖酶。
木聚糖酶应用非常广泛，目前已经在食品、饲料、造纸、纺织等工业中成功应用，许多来源于木霉、曲霉以及芽孢杆菌等的木聚糖酶被商业化生产，但适用于不同需求，具有优良性质的木聚糖酶仍在不断开发之中。
Clostridium clariflavum是一种嗜热厌氧木质纤维素降解菌，对木质纤维素的降解率非常高，可降解42～59％的无预处理仅经高压灭菌的植物材料柳枝稷，而同属的C.thermocellum仅能降解33％的柳枝稷，但是对来源于Clostridium clariflavum的木聚糖酶的具体性质及相关应用鲜有报道。
发明内容
本发明的第一个目的在于提供一种产木聚糖酶的重组菌，所述重组菌是以pET28a(+)为载体，以E.coli BL21(DE3)为表达宿主，表达如SEQ ID NO.1所示基因。
本发明的第二个目的在于提供构建木聚糖酶重组菌的方法，所述方法包括如下步骤：PCR扩增SEQ ID NO.1所示基因，与pET28a(+)连接并转化到E.coli BL21(DE3)，获得重组菌。
本发明的第三个目的是提供一种诱导表达木聚糖酶的方法，所述方法是将所述重组大肠杆菌接种到培养基中，当OD600为1.0～1.9时加入IPTG诱导木聚糖酶的表达。
在本发明的一种实施方式中，所述诱导温度为20～30℃。
在本发明的一种实施方式中，所述诱导时间为4～12h。
在本发明的一种实施方式中，所述诱导剂IPTG的终浓度为0.1～0.9mmol·L-1。
在本发明的一种实施方式中，所述接种是以2～5％接种量进行接种。
在本发明的一种实施方式中，所述培养基配方为氯化钠10g/L，胰蛋白胨10g/L，酵母提取物5g/L，pH 7。
本发明的第四个目的是提供所述的重组菌在生产木聚糖酶中的应用。
有益效果：本发明成功地实现了Clostridium clariflavum来源的木聚糖酶的异源表达，并通过IPTG诱导的方法提高了木聚糖酶的表达量，使木聚糖酶的酶活是未诱导的73倍。此外，本发明提供的木聚糖酶具有良好的pH稳定性，在pH3.5-9.5酶活性保持在80％以上，并可将木聚糖降解为木糖。
附图说明
图1为本发明的木聚糖酶的pH稳定性；
图2为诱导前后木聚糖酶酶活；
图3为本发明的HPLC检测木聚糖酶降解木聚糖的产物，峰7为木糖。
具体实施方式
酶液制备：将发酵液离心，上清液用于胞外酶活测定；沉淀用20mmol·L-1的pH 6.0磷酸钠缓冲液重悬细胞，超声波破碎10min，功率为390W，工作3s，间歇5s，破碎以后离心，上清液即为胞内酶液。
酶活测定方法：采用3，5-二硝基水杨酸比色法(DNS法)，取500uL稀释后的酶液、500uL木聚糖底物于比色管中混匀(每种样品平行做3个样)，在65℃水浴中保温10min，加入1mL DNS显色剂，沸水浴反应5min。取出，迅速冷却，用水定容至10mL，混匀，使用分光光度计，在波长540nm处测量吸光度，使测得的吸光值在0.2-0.5之间。根据标准曲线计算木聚糖酶酶活。酶活定义为每分钟降解木聚糖底物生成1μmol木糖所需的酶量为1U。
蛋白浓度的测定：Super-Bradford蛋白定量试剂盒与稀释好的蛋白质反应，用酶标仪测定595nm处的吸光值，根据蛋白标曲计算蛋白浓度。比酶活(U·mg-1)＝酶活(U·mL-1)×[蛋白浓度(mg·mL-1)]-1。
实施例1
PCR扩增xyn基因，其序列如SEQ ID NO.1所示，将xyn基因和质粒载体pET28a(+)用限制性内切酶BamHⅠ和NotⅠ双酶切并连接转化至E.coli JM109感受态细胞中。提取转化子中重组质粒pET28a(+)-xyn，将此重组质粒转化至E.coli BL21(DE3)感受态细胞，获得重组E.coli BL21(DE3)/pET28a(+)-xyn。
实施例2
将重组大肠杆菌接种于含有100μg·mL-1硫酸卡那霉素的LB培养基(氯化钠10，胰蛋白胨10，Yeast Extract 5，pH 7)中，200r·min-137℃培养10h，然后以2％(v·v-1)接种量转接至50mL新鲜培养基中，添加诱导剂IPTG至0.1mmol·L-1，诱导5h后取样10mL，8000r·min-1离心10min收获细胞。测定细胞内外酶活情况，此时细胞内酶活达1.05U·mL-1，胞外酶活为0.01U·mL-1。
实施例3
在pH 6的磷酸氢二钠-柠檬酸缓冲液中，45～85℃范围内，每隔5℃，测定木聚糖酶酶活，确定最适反应温度为65℃。在最适反应温度条件下，pH 5～7.5范围内，每隔0.5，测定酶活，确定最佳反应pH为6～6.5。
温度稳定性：将酶液在pH为6.5时分别在50、55、60、65、70℃中处理不同时间，冷却后在最佳反应温度下测定剩余酶活，以未处理的酶活为100％。
pH稳定性：将酶液浓缩后以相同的稀释倍数分别稀释到pH 3.5、4.5、5.5、6.5、7.5、8.5、9.5，4℃放置5h，然后在最佳反应条件下测定剩余酶活，以未处理的酶活为100％。
结果显示，木聚糖酶在pH3.5-9.5酶活性保持在80％以上，具有良好的pH稳定性。
实施例4
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600达到1.1时添加诱导剂IPTG至0.1mmol·L-1，并于25℃诱导6h，测定细胞内外酶活情况，此时细胞内酶活达0.89U·mL-1，胞外酶活为0.04U·mL-1。
实施例5
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600达到1.5时添加诱导剂IPTG至0.1mmol·L-1，并于25℃诱导6h，测定细胞内外酶活情况，此时细胞内酶活达0.81U·mL-1，胞外酶活为0.02U·mL-1。
实施例6
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600达到0.8时添加诱导剂IPTG至0.1mmol·L-1，并于25℃诱导6h，测定细胞内外酶活情况，此时胞内酶活达0.65U·mL-1，胞外酶活为0.08U·mL-1。
实施例7
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时添加诱导剂IPTG至0.1mmol·L-1，并于20℃诱导6h，测定细胞内外酶活情况，此时，胞内酶活为1.27U·mL-1，胞外酶活为0.02U·mL-1。
实施例8
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时添加诱导剂IPTG至0.1mmol·L-1，并于20℃诱导4h，测定细胞内外酶活情况，此时，胞内酶活为0.93U·mL-1，胞外酶活为0.01U·mL-1。
实施例9
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时添加诱导剂IPTG至0.1mmol·L-1，并于20℃诱导12h，测定细胞内外酶活情况，此时，胞内酶活为1.22U·mL-1，胞外酶活为0.12U·mL-1。
实施例10
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时分别添加诱导剂IPTG0.1mmol·L-1，于20℃诱导9h，测定细胞内外酶活变化情况，结果显示，胞内酶活为1.31U·mL-1，胞外酶活为0.02U·mL-1。
实施例11
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时分别添加诱导剂IPTG0.7mmol·L-1，于20℃诱导9h，测定细胞内外酶活变化情况，结果显示，胞内酶活为1.36U·mL-1，胞外酶活为0.05U·mL-1。
实施例12
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时分别添加诱导剂IPTG0.9mmol·L-1，于20℃诱导9h，测定细胞内外酶活变化情况，结果显示，胞内酶活为1.25U·mL-1，胞外酶活为0.08U·mL-1。
实施例13
在重组菌E.coli BL21(DE3)/pET28a(+)-xyn菌体量OD600约为1.1时分别添加诱导剂IPTG0.4mmol·L-1，于20℃诱导9h，测定细胞内外酶活变化情况，并以未诱导的重组菌为对照。结果显示，诱导后的重组菌胞内酶活为1.46U·mL-1，比酶活为2.84U·mg-1，胞外酶活为0.04U·mL-1。不添加诱导剂IPTG时重组菌具有微量木聚糖酶酶活，胞内酶活0.02U·mL-1，胞外酶活0.01U·mL-1；添加诱导剂IPTG后重组菌木聚糖酶胞内酶活是未诱导的73倍(图2)。
实施例14
取1mL的1％(w·v-1)山毛榉木聚糖(溶解于20mmol·L-1磷酸钠缓冲液)分别与1mL初始酶活1U或10U的重组木聚糖酶反应不同时间，立即煮沸灭活10min，8000r·min-1离心10min，0.22μm滤膜过滤，采用HPLC法检测生成的木糖、木二糖、木三糖、木四糖、木五糖含量(图3)，结果显示，重组木聚糖酶可以降解山毛榉木聚糖为寡聚糖和单糖木糖，从反应开始就有较多单糖生成，且反应初始产物以木糖和木三糖为主，随着反应的进行木三糖逐渐被降解，产物最终以木糖和木二糖为主，反应3h后分别占总还原糖含量的52.7％和43.2％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

1.一种产木聚糖酶的重组菌，其特征在于，以pET28a(+)为载体，以E.coli BL21(DE3)为宿主，表达如SEQ ID NO.1所示基因。
2.一种构建权利要求1所述重组菌的方法，其特征在于，包括如下步骤：PCR扩增SEQ IDNO.1所示基因，与pET28a(+)连接并转化到E.coli BL21(DE3)，获得重组菌。
3.一种诱导表达木聚糖酶的方法，所述方法是将权利要求1所述重组大肠杆菌接种到培养基中，当OD600为1.0～1.9时加入IPTG诱导木聚糖酶的表达。
4.根据权利要求3所述的方法，其特征在于，诱导温度为20～30℃。
5.根据权利要求3所述的方法，其特征在于，诱导时间为4～12h。
6.根据权利要求3所述的方法，其特征在于，所述诱导剂IPTG的终浓度为0.1～0.9mmol·L-1。
7.根据权利要求3所述的方法，其特征在于，所述接种是以2～5％接种量进行接种。
8.根据权利要求3所述的方法，其特征在于，所述培养基为发酵培养基，其配方为氯化钠10g/L，胰蛋白胨10g/L，酵母提取物5g/L，pH 7。
9.权利要求1所述的重组菌在生产木聚糖酶中的应用。
10.权利要求1所述重组菌生产的木聚糖酶在降解山毛榉木聚糖中的应用。
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