Mutagenic Breeding and Optimization of Fermentation Conditions of Fibrinolytic Enzyme from Marine <i>Bacillus subtilis</i>

doi:10.13523/j.cb.2207056

China Biotechnology

2022, Vol. 42

Issue (12): 27-36 DOI: 10.13523/j.cb.2207056

Mutagenic Breeding and Optimization of Fermentation Conditions of Fibrinolytic Enzyme from Marine Bacillus subtilis

PANG Guang-wu,LIANG Zhi-qun(

)

College of Life Science and Technology, Guangxi University, Nanning 530004, China

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Abstract

Objective: Cardiovascular diseases caused by thrombus have severely impaired human health. However, there are most of defects on traditional thrombolytic agents, like high price, low fibrin specificity and side effect. Enzyme from marine environment are rarely reported and expected to be efficient and economical thrombolytic drugs. Methods: In this experiment, a mutant strain PW6-3 with high fibrinolytic activity was obtained by UV mutagenesis using the marine-derived Bacillus subtilis LC6-1. The acquire mutant PW6-3 was subjected to process optimization of fibrinolytic enzyme production. Results: The UV-mutagenized high-yielding mutant strain PW6-3 was (6 960.21 ± 85.51) U/mL, which was 30.48% higher than the initial strain. The medium components and culture conditions of strain PW6-3 in shake-flask were optimized by single factor and orthogonal experiments. The optimal medium components were: Corn starch 30 g/L, corn pulp dry powder 40 g/L, CaCl₂ 3g/L. The optimal culture conditions were: Temperature of 32℃, speed of 200 r/min, loading volume of 50 mL in 250 mL flask, inoculation volume of 3% (v/v), initial pH of 6.5, seed age of 18 h, fermentation time of 66 h. Conclusion: After fermentation optimization, the enzyme activity of strain PW6-3 reached (9 203.63 ± 67.85) U/mL, which was 72.53% higher than initial strain LC6-1. The optimized yield is at a high level in the world at present.

Key words： Bacillus subtilis Fibrinolytic enzyme Mutagenesis Fermentation optimization

Received: 25 July 2022 Published: 05 January 2023

ZTFLH:

Q939

Corresponding Authors: Zhi-qun LIANG E-mail: zqliang@gxu.edu.cn

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Cite this article:

PANG Guang-wu,LIANG Zhi-qun. Mutagenic Breeding and Optimization of Fermentation Conditions of Fibrinolytic Enzyme from Marine Bacillus subtilis. China Biotechnology, 2022, 42(12): 27-36.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2207056 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I12/27

Table 1 The factors and levels in the orthogonal test L9 (3⁴)

Fig.1 Standard curve of urokinase

Fig.2 UV-mutagenesis lethality curve

Table 2 Results of plate primary screening and shake flask re-screening of UV mutagenesis

Fig.3 The genetic stability of UV mutagenized strain PW6-3

Fig.4 Effect of carbon sources (a) and corn starch concentration (b) on enzyme activity

Fig.5 Effect of nitrogen sources (a) and concentration of corn pulp dry powder (b) on enzyme activity

Fig.6 Effect of metal ions (a) and ratio of CaCl₂ (b) on enzyme activitivity

Table 3 The result of orthogonal test

Table 4 Analysis of variance for the results of orthogonal test

Fig.7 Effect of temperature on enzyme activity

Fig.8 Effect of speed (a) and liquid loading (b) on enzyme activity

Fig.9 Effect of volume on enzyme activity

Fig.10 Effect of initial pH on enzyme activity

Fig.11 Growth curve of strain PW6-3 (a) and effect of seed age on enzyme activity (b)

Fig.12 Effect of fermentation time on enzyme activity


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