A High-yield Nattokinase Strain: ARTP Mutation Breeding and Medium Optimization

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

Abstract

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

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.

Figures/Tables 16

References 20

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符号	因子	水平/(g/L)
符号	因子	-1	+1
A	葡萄糖	30	45
B	玉米粉	40	60
C	豆粕粉	30	45
D	Na₂HPO₄	3	4.5
E	K₂HPO₄	0.3	0.45
F	MgSO₄	0.6	0.9
G	CaCl₂	0.8	1.2
H	丝氨酸	0.09	0.13
J	豆油	0.4	0.6

符号	因子	水平/(g/L)
符号	因子	-1	+1
A	葡萄糖	30	45
B	玉米粉	40	60
C	豆粕粉	30	45
D	Na₂HPO₄	3	4.5
E	K₂HPO₄	0.3	0.45
F	MgSO₄	0.6	0.9
G	CaCl₂	0.8	1.2
H	丝氨酸	0.09	0.13
J	豆油	0.4	0.6

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