高产纤维素酶工程菌株产酶条件优化

doi:10.19586/j.2095-2341.2024.0108

生物技术进展 ›› 2024, Vol. 14 ›› Issue (6): 1032-1041.DOI: 10.19586/j.2095-2341.2024.0108

高产纤维素酶工程菌株产酶条件优化

孔蒙蒙(), 金静静, 卢鹏, 顾梦丽, 陈千思, 曹培健, 张剑锋, 陶界锰()

中国烟草总公司郑州烟草研究院，郑州 450001

收稿日期:2024-05-27 接受日期:2024-07-08 出版日期:2024-11-25 发布日期:2024-12-27
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(110202102050)

Optimization of Enzyme Production Conditions of High-yielding Cellulase Engineering Strains

Mengmeng KONG(), Jingjing JIN, Peng LU, Mengli GU, Qiansi CHEN, Peijian CAO, Jianfeng ZHANG, Jiemeng TAO()

Zhengzhou Tobacco Research Institute，China National Tobacco Corporation，Zhengzhou 450001，China

Received:2024-05-27 Accepted:2024-07-08 Online:2024-11-25 Published:2024-12-27
Contact: Jiemeng TAO

摘要/Abstract

摘要：

为提高工程菌株纤维素酶产酶效率，采用单因素试验和正交试验法对高产纤维素酶枯草芽孢杆菌（Bacillus subtilis）工程菌株C36、CM、KF、GH5的发酵培养基和培养条件进行优化。结果表明，工程菌株C36、CM、KF和GH5在最优发酵条件下的产酶效率显著提高。发酵条件优化后，工程菌株CM的纤维素酶活最高，在醇化烟叶添加量7%、蛋白胨添加量0.8%、pH 6.1、发酵温度37 ℃、转速220 r·min^-1、装液量40 mL及发酵时间96 h的条件下，其羧甲基纤维素（carboxymethylcellulose, CMC）酶活和滤纸酶活（filter paper activity, FPA）分别达到504.95和222.11 U·mL^-1。发酵条件优化后，工程菌株GH5的纤维素酶活提升倍数最高，在醇化烟叶添加量6%、蛋白胨添加量0.9%、pH 6.1、发酵温度37 ℃、转速220 r·min^-1、装液量40 mL及发酵时间96 h的条件下，其CMC和FPA酶活分别是优化前的28.55和9.68倍。

关键词: 纤维素酶, 工程菌株, 发酵条件, 正交设计

Abstract:

In order to improve the efficiency of cellulase production， the fermentation medium and culture conditions of Bacillus subtilis engineered strains C36， CM， KF and GH5 were optimized by single factor test and orthogonal test. The results showed that the enzyme production efficiency of engineered strains C36， CM， KF and GH5 was significantly improved under the optimal fermentation conditions. After optimizing the fermentation conditions， the cellulase activity of the engineered strain CM was the highest. Under the conditions of 7% of aged tobacco leaves， 0.8% of peptone， pH 6.1， fermentation temperature 37 ℃， rotation speed 220 r·min^-1， liquid medium volume 40 mL and fermentation for 96 h， the enzyme activities of carboxymethylcellulose （CMC） and filter paper activity （FPA） of the engineered strain CM were 504.95 and 222.11 U·mL^-1， respectively. After optimizing the fermentation conditions， the increase of cellulase activity of the engineered strain GH5 was the highest. Under the conditions of 6% of aged tobacco leaves， 0.9% of peptone， pH 6.1， fermentation temperature 37 ℃， rotation speed 220 r·min^-1， liquid medium volume 40 mL and fermentation for 96 h， the enzyme activities of CMC and FPA was 28.55 and 9.68 times higher than that before optimization.

Key words: cellulase, engineered strains, fermentation conditions, orthogonal design

中图分类号:

Q814.9

孔蒙蒙, 金静静, 卢鹏, 顾梦丽, 陈千思, 曹培健, 张剑锋, 陶界锰. 高产纤维素酶工程菌株产酶条件优化[J]. 生物技术进展, 2024, 14(6): 1032-1041.

Mengmeng KONG, Jingjing JIN, Peng LU, Mengli GU, Qiansi CHEN, Peijian CAO, Jianfeng ZHANG, Jiemeng TAO. Optimization of Enzyme Production Conditions of High-yielding Cellulase Engineering Strains[J]. Current Biotechnology, 2024, 14(6): 1032-1041.

图/表 10

表1 发酵培养基条件正交试验因素与水平

Table 1 Factors and levels of orthogonal experiments for fermentation medium conditions

水平	A:碳源/%	B:氮源/%	C:初始pH
1	5	0.8	5.5
2	6	0.9	6.1
3	7	1.0	6.6

表2 发酵培养条件正交试验因素与水平

Table 2 Factors and levels of orthogonal experiments for fermentation culture conditions

水平	D：发酵温度/℃	E：转速/（r·min^-1）	F：装液量/mL	G：发酵时间/h
1	34	180	40	72
2	37	200	80	96
3	40	220	100	120

图1 不同碳源和氮源对工程菌株纤维素酶活的影响A~B：不同碳源对工程菌株CMC和FPA酶活的影响；C~D：不同氮源对工程菌株CMC酶活和FPA酶活的影响；同一菌株不同处理上的不同小写字母表示在P<0.05水平上具有统计学意义。

Fig. 1 Effects of different carbon and nitrogen sources on cellulase activity of engineered strains

图2 不同碳源添加量、氮源添加量、初始pH对工程菌株纤维素酶活的影响A、B:不同醇化烟叶添加量对工程菌株CMC酶活和FPA酶活的影响；C、D:不同蛋白胨添加量对工程菌株CMC酶活和FPA酶活的影响；E、F:不同初始pH对工程菌株CMC酶活和FPA酶活的影响

Fig. 2 Effects of different additions of carbon source and nitrogen source and different initial pH values on cellulase activities of engineered strains.

表3 C36、CM、KF和GH5发酵培养基正交试验表

Table 3 Orthogonal test of fermentation medium conditions for C36, CM, KF and GH5

正交编号	A：醇化烟叶	B：蛋白胨	C：pH
1	1	1	1
2	1	2	3
3	1	3	2
4	2	1	3
5	2	2	2
6	2	3	1
7	3	1	2
8	3	2	1
9	3	3	3

图3 菌株发酵培养基优化正交实验结果A：C36正交实验结果；B：CM正交实验结果；C：KF正交实验结果；D：GH5正交实验结果

Fig. 3 Results of orthogonaltests on the optimization of fermentation medium for strains

图4 不同培养条件对工程菌株纤维素酶酶活的影响A、B:不同温度对工程菌株CMC酶活和FPA酶活的影响；C、D:不同摇床转速对工程菌株CMC酶活和FPA酶活的影响；E、F:不同装液量对工程菌株CMC酶活和FPA酶活的影响；G、H:不同发酵时间工程菌株CMC酶活和FPA酶活

Fig. 4 Effects of different culture conditions on cellulase activities of engineered strains

表4 C36、CM、KF和GH5发酵培养条件正交实验表

Table 4 Orthogonal test of fermentation culture conditions for C36, CM, KF and GH5

编号	D：发酵温度	E：转速	F：装液量	G：发酵时间
1	1	1	1	1
2	1	2	2	2
3	1	3	3	3
4	2	1	2	3
5	2	2	3	1
6	2	3	1	2
7	3	1	3	2
8	3	2	1	3
9	3	3	2	1

图5 菌株发酵培养条件优化正交实验结果A：C36正交实验结果；B：CM正交实验结果；C：KF正交实验结果；D：GH5正交实验结果

Fig. 5 Results of orthogonal tests on the optimization of fermentation conditions for strains

表5 C36、CM、KF和GH5的最优发酵条件下的酶活

Table 5 Cellulase activities under the optimal combinations of C36, CM, KF and GH5

菌株	醇化烟叶/%	pH	蛋白胨/%	发酵温度/℃	转速/(r·min^-1)	装液量/mL	发酵时间/h	CMC酶活/（U·mL^-1）	FPA酶活/（U·mL^-1）
C36	7	5.5	0.9	37	220	40	96	542.95	191.03
CM	7	6.1	0.8	37	220	40	96	504.95	222.11
KF	7	6.1	0.8	37	220	40	96	545.57	193.98
GH5	6	6.1	0.9	37	220	40	96	530.78	193.76

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高产纤维素酶工程菌株产酶条件优化

Optimization of Enzyme Production Conditions of High-yielding Cellulase Engineering Strains

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