Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用

doi:10.16085/j.issn.1000-6613.2018-0742

化工进展 ›› 2019, Vol. 38 ›› Issue (02): 1038-1044.DOI: 10.16085/j.issn.1000-6613.2018-0742

Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用

李琦^1,²(),蒋雯怡¹(),孙雨薇¹,吴涛^1,²,赵林果^1,²()

1. 南京林业大学化学工程学院，江苏南京 210037
2. 江苏省农林生物质化学与利用国家重点实验室培育点，江苏南京 210037

收稿日期:2018-04-11 修回日期:2018-05-17 出版日期:2019-02-05 发布日期:2019-02-05
通讯作者: 赵林果
作者简介:中文作者简介:<named-content content-type="corresp-name">李琦</named-content>（1988—），女，实验师，研究方向为酶工程。E-mail：<email>amanadahere@sina.com</email>；<named-content content-type="corresp-name">蒋雯怡</named-content>为共同第一作者。|中文作者简介:<named-content content-type="corresp-name">李琦</named-content>（1988—），女，实验师，研究方向为酶工程。E-mail：<email>amanadahere@sina.com</email>；<named-content content-type="corresp-name">蒋雯怡</named-content>为共同第一作者。|赵林果，教授，研究方向为酶工程及生物催化转化。E-mail：<email>lg.zhao@163.com</email>。
基金资助:
国家重点研发计划(2017YFD0601001);江苏高校优势学科建设工程项目(PAPD);南京林业大学大学生创新训练计划(2016NFUSPITP031)

Thermostability modification of the GH11 xylanase MxynB from Aspergillus niger NL-1 and its application in xylooligosaccharide production

Qi LI^1,²(),Wenyi JIANG¹(),Yuwei SUN¹,Tao WU^1,²,Linguo ZHAO^1,²()

1. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
2. Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest, Nanjing 210037, Jiangsu, China

Received:2018-04-11 Revised:2018-05-17 Online:2019-02-05 Published:2019-02-05
Contact: Linguo ZHAO

摘要/Abstract

摘要：

为提高来源于Aspergillus niger NL-1的木聚糖酶MxynB的热稳定性，通过定点突变技术在木聚糖酶MxynB的相应位置引入二硫键（Cys¹¹⁶-Cys¹³⁵），获得突变酶MynxB-116-135；通过基因融合技术在木聚糖酶MxynB的N端融合了具有较高热稳定性的来源于嗜热菌Thermotoga thermarum DSM5069的纤维素结合结构域（CBD），获得突变酶CBD-MxynB和CBD-MxynB-116-135。分别将原酶及突变酶在E.coli BL21（DE3）中表达，经纯化后对比其酶学性质。结果显示，突变酶MxynB-116-135的最适温度为50℃，较原酶MxynB提高了5℃；突变酶CBD-MxynB和CBD-MxynB-116-135的温度稳定性明显提高，其中CBD-MxynB-116-135尤为突出，在70℃下保温2h仍能保持50%以上的酶活力，而原酶基本失活。研究表明，二硫键的引入和CBD的融合对提高MxynB的热稳定性具有重要的作用。此外，研究了突变酶CBD-MxynB-116-135水解玉米芯木聚糖的产物，结果显示玉米芯木聚糖质量浓度为2.5mg/mL，酶用量为40U/g，在50℃、pH 5.0条件下水解10h后，水解得率为39.6%，水解产物XOS_2-3含量占87.9%。结果表明，利用该突变木聚糖酶酶解碱提玉米芯木聚糖可产生以木二糖及木三糖为主要成分的低聚木糖。

关键词: 酶, 分子生物学, 温度稳定性, 水解, 低聚木糖

Abstract:

To improve the thermostability of family GH11 xylanase MxynB from Aspergillus niger NL-1, a disulfide bridge (Cys¹¹⁶-Cys¹³⁵) was introduced into the corresponding region of MxynB by site-directed mutagenesis. Then, xylanases MxynB and MxynB-116-135 were fused with the thermophilic cellulose binding domain (CBD) from Thermotoga thermarum DSM5069 at N-terminal of xylanases using PCR. The wild-type enzyme MxynB and the mutated enzymes MxynB-116-135, CBD-MxynB and CBD-MxynB-116-135 were expressed in Escherichia coli BL21(DE3), separately. After purification, their enzymatic properties were analyzed and compared. The optimal temperature of the recombinant MxynB-116-135 was 50℃, which was 5℃ higher than that of MxynB. Moreover, the thermostabilities of two recombinant mutants CBD-MxynB and CBD-MxynB-116-135 were clearly increased as compared with the wild-type enzyme under 70℃. Between them, CBD-MxynB-116-135 increased notably. After 2h, the relative activity of the mutant CBD-MxynB-116-135 was over 50%, and the wild-type MxynB was basically inactivated. All the results indicated that the introduction of a disulfide bridge and cellulose binding domain (CBD) could improve the thermostability of the MxynB notably. In addition, 2.5mg/mL corncob xylan was treated with 40U/g mutant xylanase CBD-MxynB-116-135 for hydrolysis. After 10h, the xylooligosaccharides yield was 39.6%, and the content of XOS_2-3 reached 87.9%. All these results indicated that this mutant xylanase could be suitable for potential applications in the xylobiose and xylotriose production.

Key words: enzyme, molecular biology, thermostability, hydrolyzation, xylooligosaccharide

中图分类号:

Q814

李琦, 蒋雯怡, 孙雨薇, 吴涛, 赵林果. Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用[J]. 化工进展, 2019, 38(02): 1038-1044.

Qi LI, Wenyi JIANG, Yuwei SUN, Tao WU, Linguo ZHAO. Thermostability modification of the GH11 xylanase MxynB from Aspergillus niger NL-1 and its application in xylooligosaccharide production[J]. Chemical Industry and Engineering Progress, 2019, 38(02): 1038-1044.

图/表 7

表1 定点突变引物序列

突变位点	引物序列（5’-3’）
116F	TGTTCTGGAGGTACTTACAAGGGA
116R	AGGATTATAGTCTCCGTAACTCTCAACG
135F	TGTACAGCCACCAGAACTAACGCT
135R	GATGTCGTAGACGGAACCATCAGA
CBD-F	CGCGGATCCGTTCCTCACGACT
CBD-R	CCGCTCGAGCTACTGAACTGTAAT

图1 木聚糖酶MxynB三维结构图及融合蛋白的构建

图2 重组木聚糖酶及突变酶的SDS-PAGE电泳图

图3 重组木聚糖酶及突变酶的最适温度

图4 重组木聚糖酶及突变酶的温度稳定性

图5 重组木聚糖酶和突变酶的最适pH及pH稳定性

图6 重组木聚糖酶水解玉米芯木聚糖产物的离子色谱分析图

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Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用

Thermostability modification of the GH11 xylanase MxynB from Aspergillus niger NL-1 and its application in xylooligosaccharide production

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