海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化<sup>*</sup>

doi:10.13523/j.cb.2207056

中国生物工程杂志

2022, Vol. 42

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

技术与方法

海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化^*

庞光武,梁智群(

)

广西大学生命科学与技术学院南宁 530004

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

全文: PDF(1109 KB) HTML

摘要：

目的:对海洋来源的具有产纤溶酶能力的枯草芽孢杆菌(Bacillus subtilis)LC6-1进行紫外诱变,得到高产且稳定的突变株PW6-3,对该突变株发酵产酶的条件进行优化。方法:采用单因素和正交试验进行发酵培养基组分和培养条件的优化。结果:突变株PW6-3的酶活力为(6 960.21 ± 85.51)U/mL,较原始菌株提高了30.48%。以PW6-3为出发菌株,采用单因素及正交试验的方法对菌株进行发酵培养基组分与培养条件优化,最终得到的最佳培养基组分是:玉米淀粉30 g/L,玉米浆干粉40 g/L,CaCl₂ 3 g/L;最佳发酵培养条件是:32℃,转速200 r/min,接种量3%,pH 6.5,种龄18 h,发酵培养时间66 h,最终菌株的酶活力稳定在(9 203.63 ± 67.85)U/mL。结论:发酵工艺优化后,菌株PW6-3纤溶酶产量较诱变之前的菌株LC6-1提高72.53%,且发酵工艺成本较低,具有较好的经济效益。

关键词： 枯草芽孢杆菌; 纤溶酶; 诱变; 发酵优化

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

收稿日期: 2022-07-25 出版日期: 2023-01-05

ZTFLH:

Q939

基金资助: *国家自然科学基金资助项目(21062001)

通讯作者: 梁智群 E-mail: zqliang@gxu.edu.cn

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引用本文:

庞光武,梁智群. 海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化^*[J]. 中国生物工程杂志, 2022, 42(12): 27-36.

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.

链接本文:

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

表1 正交试验L9(34)的因素和水平

图1 尿激酶标准曲线

图2 紫外诱变致死率曲线

表2 紫外诱变的平板初筛和摇瓶复筛结果

图3 紫外诱变菌株PW6-3的遗传稳定性

图4 碳源(a)与玉米淀粉浓度(b)对酶活力的影响

图5 氮源种类(a)与玉米浆干粉浓度(b)对酶活力的影响

图6 金属离子(a)与CaCl2的添加量(b)对酶活力的影响

表3 正交试验结果

表4 正交试验结果方差分析

图7 温度对酶活力的影响

图8 转速(a)与装液量(b)对酶活力的影响

图9 接种量对酶活力的影响

图10 初始pH对酶活力的影响

图11 菌株PW6-3的生长曲线(a)与种龄对产酶的影响(b)

图12 发酵时间对酶活力的影响

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