一株高产纳豆激酶菌株的ARTP诱变育种及培养基的优化

doi:10.11924/j.issn.1000-6850.casb2022-0358

摘要/Abstract

摘要：

旨在提高纳豆激酶发酵水平，扩大其作为新型的纤溶药物的生产应用。利用常温室压等离子体(ARTP)技术对一株产纳豆激酶的野生菌株P进行诱变；筛选获得一株具有稳定遗传性能的纳豆激酶优势突变株P501，纳豆激酶活力比野生菌株提高了1.02倍，通过单因素实验、响应面实验对P501产纳豆激酶的发酵培养基进行优化，得到最佳发酵培养基为：葡萄糖30 g/L、玉米粉40 g/L、豆粕粉30 g/L、Na₂HPO₄ 3 g/L、K₂HPO₄ 0.3 g/L、MgSO₄ 0.6 g/L、CaCl₂ 0.63 g/L、丝氨酸0.07 g/L、豆油0.61 g/L，经过验证实验，纳豆激酶活力最高为9691 Fu/mL，较基础发酵培养基提高了6.68倍。

关键词: 纳豆激酶, ARTP诱变, 单因素实验, 响应面实验, 培养基优化

Abstract:

The aim of this paper is to improve the fermentation level of nattokinase and expand its application as a new fibrinolytic drug. A wild strain P producing nattokinase was mutagenized by atmospheric and room temperature plasma (ARTP). A high-yield nattokinase mutant P501 with stable genetic performance was selected and the activity of nattokinase was 1.02 times higher than that of the wild strain. The fermentation medium for nattokinase production was optimized by single factor experiment and response surface experiment. The optimal fermentation medium was as follows: glucose 30 g/L, corn flour 40 g/L, soybean meal 30 g/L, Na₂HPO₄ 3 g/L, K₂HPO₄ 0.3 g/L, MgSO₄ 0.6 g/L, CaCl₂ 0.63 g/L, serine 0.07 g/L and soybean oil 0.61 g/L. After verification experiment, the activity of nattokinase reached 9691 Fu/mL, which was 6.68 times higher than that of basic fermentation medium.

Key words: nattokinase, ARTP mutation, single factor experiment, response surface experiment, medium optimization

潘冬梅, 张心青, 杨传伦, 田杰伟, 王红霞, 杨丹丹, 郭南南, 于帅帅, 韩立霞, 马春峰, 部丽群, 蔡倩倩, 郭中瑞. 一株高产纳豆激酶菌株的ARTP诱变育种及培养基的优化[J]. 中国农学通报, 2023, 39(9): 115-122.

PAN Dongmei, ZHANG Xinqing, YANG Chuanlun, TIAN Jiewei, WANG Hongxia, YANG Dandan, GUO Nannan, YU Shuaishuai, HAN Lixia, MA Chunfeng, BU Liqun, CAI Qianqian, GUO Zhongrui. A High-yield Nattokinase Strain: ARTP Mutation Breeding and Medium Optimization[J]. Chinese Agricultural Science Bulletin, 2023, 39(9): 115-122.

图/表 16

参考文献 20

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因素	平方和	自由度	均方	F值	P值	显著性
模型	6.878E+006	9	7.642E+005	31.28	0.0314	显著
A	2.987E+005	1	2.987E+005	12.23	0.0730	不显著
B	1.892E+005	1	1.892E+005	7.74	0.1085	不显著
C	3.392E+005	1	3.392E+005	13.88	0.0651	不显著
D	86790.12	1	86790.12	3.55	0.2001	不显著
E	8953.86	1	8953.86	0.37	0.6065	不显著
F	15045.71	1	15045.71	0.62	0.5148	不显著
G	1.285E+006	1	1.285E+006	52.58	0.0185	显著
H	2.475E+006	1	2.475E+006	101.31	0.0097	显著
J	2.180E+006	1	2.180E+006	89.20	0.0110	显著

因素	平方和	自由度	均方	F值	P值	显著性
模型	6.878E+006	9	7.642E+005	31.28	0.0314	显著
A	2.987E+005	1	2.987E+005	12.23	0.0730	不显著
B	1.892E+005	1	1.892E+005	7.74	0.1085	不显著
C	3.392E+005	1	3.392E+005	13.88	0.0651	不显著
D	86790.12	1	86790.12	3.55	0.2001	不显著
E	8953.86	1	8953.86	0.37	0.6065	不显著
F	15045.71	1	15045.71	0.62	0.5148	不显著
G	1.285E+006	1	1.285E+006	52.58	0.0185	显著
H	2.475E+006	1	2.475E+006	101.31	0.0097	显著
J	2.180E+006	1	2.180E+006	89.20	0.0110	显著

实验号	CaCl₂/(g/L)	丝氨酸/(g/L)	豆油/(g/L)	纳豆激酶活力/(Fu/mL)
1	0.8	0.09	0.4	5631
2	0.7	0.08	0.5	7349
3	0.6	0.07	0.6	9476
4	0.5	0.06	0.7	8389
5	0.4	0.05	0.8	7528
6	0.3	0.03	0.9	5092

实验号	CaCl₂/(g/L)	丝氨酸/(g/L)	豆油/(g/L)	纳豆激酶活力/(Fu/mL)
1	0.8	0.09	0.4	5631
2	0.7	0.08	0.5	7349
3	0.6	0.07	0.6	9476
4	0.5	0.06	0.7	8389
5	0.4	0.05	0.8	7528
6	0.3	0.03	0.9	5092

符号	因素	水平(g/L)
符号	因素	-1.68	-1	0	1	1.68
A	CaCl₂	0.432	0.5	0.6	0.7	0.768
B	丝氨酸	0.0532	0.06	0.07	0.08	0.0868
C	豆油	0.432	0.5	0.6	0.7	0.768