产纤维素酶工程菌株的构建及其在醇化烟叶中的应用

doi:10.19586/j.2095-2341.2023.0159

生物技术进展 ›› 2024, Vol. 14 ›› Issue (2): 263-270.DOI: 10.19586/j.2095-2341.2023.0159

• 研究论文 • 上一篇

产纤维素酶工程菌株的构建及其在醇化烟叶中的应用

孔蒙蒙¹(), 卢鹏¹, 陈千思¹, 乔学义¹, 陈善义², 金静静¹, 郑雪坳¹, 曹培健¹, 陶界锰¹()

^1.中国烟草总公司郑州烟草研究院，郑州 450001
^2.福建中烟工业有限责任公司技术中心，福建厦门 361021

收稿日期:2023-12-12 接受日期:2024-01-05 出版日期:2024-03-25 发布日期:2024-04-17
通讯作者: 陶界锰
作者简介:孔蒙蒙 E-mail: 2477658055@qq.com；
基金资助:
烟草行业烟草工艺重点实验室引领计划专项项目(202022AWHZ08);中国烟草总公司重点研发项目(110202102017)

Construction of Cellulase Producing Engineering Strains and the Application in Aged Tobacco Leaves

Mengmeng KONG¹(), Peng LU¹, Qiansi CHEN¹, Xueyi QIAO¹, Shanyi CHEN², Jingjing JIN¹, Xueao ZHENG¹, Peijian CAO¹, Jiemeng TAO¹()

^1.Zhengzhou Tobacco Research Institute，China National Tobacco Corporation，Zhengzhou 450001，China
^2.Technology Center，China Tobacco Fujian Industrial Co. ，Ltd. ，Fujian Xiamen 361021，China

Received:2023-12-12 Accepted:2024-01-05 Online:2024-03-25 Published:2024-04-17
Contact: Jiemeng TAO

摘要/Abstract

摘要：

烟叶中过高的纤维素含量使烟叶组织容易破碎，影响加工过程中烟叶的可塑性，使烟叶杂气变重等。为了获得产纤维素酶的优良菌株，实现醇化烟叶纤维素的有效降解，利用同源重组法成功构建了10株纤维素降解的枯草芽孢杆菌（Bacillus subtilis）工程菌株。通过刚果红平板筛选、羧甲基纤维素钠酶活、滤纸酶活及滤纸崩解率等检测，共筛选出C36、CM、KF和GH5 4株产纤维素酶能力较强的重组菌株。将醇化烟叶作为底物进行纤维素降解，发现重组菌株CM的产纤维酶效率最高，其羧甲基纤维素钠酶活和滤纸酶活分别为39.55和23.52 U·mL^-1。结果表明，构建的重组菌株能够利用醇化烟叶中的纤维素产生纤维素酶，可为工业生产中醇化烟叶纤维素降解提供理论支撑。

关键词: 工程菌株, 醇化烟叶, 纤维素降解, 枯草芽孢杆菌

Abstract:

High cellulose content make the tissue of tobacco leaves broken easily， affect the plasticity of tobacco leaves during processing， and make the heavy impurity of tobacco leaves. In order to obtain excellent cellulase producing strains and achieve effective degradation of cellulose in aged tobacco leaves， ten Bacillus subtilis engineering strains of cellulose-degrading were successfully constructed by homologous recombination method. Four recombinant strains C36， CM， KF and GH5 with strong cellulase production capacity were screened by the Congo red plate method， carboxymethyl cellulose sodium enzyme activity assay， filter paper enzyme activity assay and filter paper disintegration rate detection. When aged tobacco leaves were used as substarate， the recombinant strain CM showed the highest cellulase production efficiency， and its carboxymethyl cellulose sodium enzyme activity was 39.55 U·mL^-1 and filter paper enzyme activity was 23.52 U·mL^-1， respectively. The results indicated that the recombinant strains could utilize cellulose in aged tobacco leaves to produce cellulase， which could provide theoretical support for cellulose degradation of aged tobacco leaves in industrial production.

Key words: engineered strains, aged tobacco leaves, cellulose degradation, Bacillus subtilis

中图分类号:

Q814

孔蒙蒙, 卢鹏, 陈千思, 乔学义, 陈善义, 金静静, 郑雪坳, 曹培健, 陶界锰. 产纤维素酶工程菌株的构建及其在醇化烟叶中的应用[J]. 生物技术进展, 2024, 14(2): 263-270.

Mengmeng KONG, Peng LU, Qiansi CHEN, Xueyi QIAO, Shanyi CHEN, Jingjing JIN, Xueao ZHENG, Peijian CAO, Jiemeng TAO. Construction of Cellulase Producing Engineering Strains and the Application in Aged Tobacco Leaves[J]. Current Biotechnology, 2024, 14(2): 263-270.

图/表 11

表1 纤维素降解相关基因的基本信息

Table 1 Basic information of genes related to cellulase degradation

基因名称	基因编号	长度/bp	所编码的酶
Y11	Orf_116303	996	糖基水解酶
Y12	Orf_54032	1 042	糖苷水解酶
Y22	Orf_70371	1 590	纤维二糖水解酶
C36	DQ782954	1 500	内切-1,4-β-葡聚糖酶
CM	KM377648.1	1 500	纤维素酶
KF	KF240848.1	1 523	内切-1,4-β-葡聚糖酶
GH5	KJ869438.1	1 248	糖苷水解酶
KM	KM009051.1	741	内切-1,3-1,4-β-葡聚糖酶
CBH	XM_745951.1	1 599	纤维二糖水解酶
BGL	MH553379.1	729	β-葡聚糖酶

表2 本研究所使用的引物序列

Table 2 Primer sequences used in this study

引物名称	引物序列(5′→3′)
Y11-F	5′-ggagcagccggggcaggatccATGGCCGTCAAACAACTTTTCG-3′
Y11-R	5′-atgatgatgatgatgaagcttCTAGCGCTGCTGCGAGATCC-3′
Y12-F	5′-ggagcagccggggcaggatccATGGCCACGCTACGCCTG-3′
Y12-R	5′-atgatgatgatgatgaagcttCTAGCCGGGACCCAGGGT-3′
Y22-F	5′-ggagcagccggggcaggatccATGGCCTACAAGAAGTTCGCC-3′
Y22-R	5′-atgatgatgatgatgaagcttCTACAGGCACTGGCTGTACCAG-3′
C36-F	5′-ggagcagccggggcaggatccATGAAACGGTCAATCTCTATTTTTATTAC-3′
C36-R	5′-atgatgatgatgatgaagcttCTAATTTGGTTCTGTTCCCCAAA-3′
CM-F	5′-ggagcagccggggcaggatccATGAAACGGTCAATCTCTATTTTTATTAC-3′
CM-R	5′-atgatgatgatgatgaagcttCTAATTTGGTTCTGTTCCCCAAA-3′
KF-F	5′-ggagcagccggggcaggatccATGAAACGGTCAATCTCTATTTTTATTAC-3′
KF-R	5′-atgatgatgatgatgaagcttCTAATTTGGTTCTGTTCCCCAAA-3′
GH5-F	5′-ggagcagccggggcaggatccATGAAGAAATTATCAGCATTATTTGTTG-3′
GH5-R	5′-atgatgatgatgatgaagcttTTATTTGCGAGAGGATTCGGG-3′
KM-F	5′-ggagcagccggggcaggatccATGCCTTATCTGAAACGAGTGTTG-3′
KM-R	5′-atgatgatgatgatgaagcttTTATTTTTTTGTATAGCGCACCCA-3′
CBH-F	5′-ggagcagccggggcaggatccATGCTGGCCTCCACCTTCTC-3′
CBH-R	5′-atgatgatgatgatgaagcttCTACAGGCACTGAGAGTAATAATCATTC-3′
BGL-F	5′-ggagcagccggggcaggatccATGTACCGTATGAAACGAATGTTGA-3′
BGL-R	5′-atgatgatgatgatgaagcttTTATCTTTTTGTGTAACGCACCCA-3′

图1 重组表达质粒构建流程

Fig. 1 Construction process of recombinant expression plasmid

图2 纤维素降解相关基因扩增后的琼脂糖凝胶电泳结果注：M1—DL2 000 maker；1—GH5；2—CBH；3—BGL；4—CM；5—C36；M2—DL5 000 maker；6—KF；7—Y22；8—Y12；9—Y11；10—KM。

Fig. 2 Agarose gel electrophoresis results of amplified genes related to cellulose degradation

图3 质粒酶切产物图注：M—DL7 000 maker；1—未酶切pMB402-1；2—酶切pMB402-1。

Fig. 3 Enzyme-digested products of plasmid

图4 枯草芽孢杆菌WB600转化结果

Fig. 4 Transformation results of Bacillus subtilis WB600

图5 刚果红染色后重组菌株的水解圈及HC值A：重组菌株的水解圈；B：重组菌株的HC值。pMB-BS—空载质粒，Y11、Y12、Y22、C36、CM、KF、GH5、KM、CBH、BGL—重组菌株，WB600—枯草芽孢杆菌。柱形图上标记的不同小写字母表示不同处理组在P<0.05水平上具有统计学意义。

Fig. 5 Hydrolytic rings and HC values of recombinant strains after Congo red staining

图6 葡萄糖标准曲线图

Fig. 6 Glucose standard curve

图7 重组菌株的CMC和FPA酶活A: CMC酶活；B： FPA酶活。柱形图上不同小写字母表示不同处理组在P<0.05水平上具有统计学意义。

Fig. 7 The CMC and FPA enzyme activities of recombinant strains

图8 各重组菌株作用下滤纸崩解率注：KB—空白；PMB-BS—空载质粒；WB600—枯草芽孢杆菌。柱形图上不同小写字母表示不同处理组在P<0.05水平上具有统计学意义。

Fig. 8 Disintegration rate of filter paper under the action of each recombinant strains

图9 各重组菌株的酶活（以醇化烟叶为底物）A：CMC酶活；B：FPA酶活。PMB-BS—空载质粒，柱形图上不同小写字母表示不同处理在P<0.05水平上具有统计学意义。

Fig. 9 Enzyme activity of each recombinant strain using aged tobacco leaf as substrate

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产纤维素酶工程菌株的构建及其在醇化烟叶中的应用

Construction of Cellulase Producing Engineering Strains and the Application in Aged Tobacco Leaves

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