1. 安徽医科大学基础医学院,安徽,合肥,230032
2.
纸质出版日期:2018,
网络出版日期:2018-5-25,
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范新炯, 莫忠兴, 张曼曼, 等. 产菊酯降解酶重组菌的高密度发酵工艺优化[J]. 中山大学学报(自然科学版)(中英文), 2018,57(3):108-119.
FAN Xinjiong, MO Zhongxin, ZHANG Manman, et al. Enhancing production of a novel pyrethroid-hydrolyzing enzyme by high density fermentation of Escherichia coli[J]. Acta Scientiarum Naturalium Universitatis SunYatseni, 2018,57(3):108-119.
范新炯, 莫忠兴, 张曼曼, 等. 产菊酯降解酶重组菌的高密度发酵工艺优化[J]. 中山大学学报(自然科学版)(中英文), 2018,57(3):108-119. DOI:
FAN Xinjiong, MO Zhongxin, ZHANG Manman, et al. Enhancing production of a novel pyrethroid-hydrolyzing enzyme by high density fermentation of Escherichia coli[J]. Acta Scientiarum Naturalium Universitatis SunYatseni, 2018,57(3):108-119. DOI:
为提高菊酯降解酶的产量,利用单因素和正交实验法对重组菌Escherichia coli BL21 (DE3)/pET-28a (+)-〖STBX〗sys410的发酵培养基组成和发酵条件进行优化。结果显示最优培养基含有20.0 g·L
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甘油、20.0 g·L
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酵母粉、8.0 g·L
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硫酸铵、100.0 mmol·L
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磷酸盐、5.0 mmol·L
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硫酸镁,以及1.5 mL·L
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的微量元素;发酵条件为装液量50 mL/250 mL、初始培养基pH 7.0、接种量2%,接种培养8 h后乳糖诱导6 h。在7.5 L发酵罐上,使用低浓度碳氮源进行培养,前期恒速补料,对数中后期进行乳糖诱导,后期恒溶氧补料,最终菌体密度和酶活力分别为142.6和3 105.1 U·mL
-1
较摇瓶发酵分别提高13.7倍和31.9倍。发酵液经破碎和冷冻干燥后酶活力达到119 426.9 U·g
-1
且该酶对菊酯的降解率高于95%。
We previously screened and obtained a novel pyrethroid-hydrolyzing gene sys 410 from Turban Basin metagenomic library
which was expressed in E coli BL21 (DE3) in soluble form. Here
to enhance the production and activity of the enzyme
flaskshaking medium and fermentation condition were investigated. The optimum medium was as follows: glycerol 20.0 g·L
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yeast extract 20.0 g·L
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ammonium sulfate 8.0 g·L
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phosphate 100.0 mmol·L
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magnesium sulfate 5.0 mmol·L
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trace element solution 1.5 mL·L
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. The optimum conditions of flaskshaking fermentation were as follows: loading volume 50/250 (V/V)
initial medium pH 7.0
inoculum size 2%
lactose induction for 6 h. We also studied highdensity fermentation of the recombinant strain in 75 L fermenter. The results showed that dispersed oxygen supply
low concentration of the initial medium
and carbon and nitrogen fedbatch culture could stimulate the growth of the recombinant strain. Inducible expression started at the middle and later stages of logarithmic growth with 6 g·L
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lactose at 37 ℃. After 10 h
cell density reached 142.6 and the enzymatic activity was 3 105.1 U·mL
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13.7 and 31.9 times better than those of the flaskshaking fermentation
respectively. In addition
crude enzyme powder was obtained after sonication and freeze drying with the amount of 68.4 g·L
-1
of fermentation broth. The specific enzyme activity was 119 426.9 U·g
-1
Remarkably
the enzyme was able to efficiently degrade all tested pyrethroids within 15 min
reaching over 95% conversion.
农药降解酶高密度发酵拟除虫菊酯生物降解
pesticide degradation enzymehigh density fermentationpyrethroidsbiodegradation
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