耐底物腈水合酶融合子的产酶条件优化

中国生物工程杂志

2012, Vol. 32

Issue (08): 75-80

技术与方法

耐底物腈水合酶融合子的产酶条件优化

张丽铃^1,2, 王筱兰^1,2

1. 江西师范大学生命科学学院南昌 330022;
2. 功能有机小分子教育部重点实验室南昌 330022

Optimization Fermentation Conditions of the Nitrile Hydratase Fusant Strain Tolerance to the Substrate

ZHANG Li-ling^1,2, WANG Xiao-lan ^1,2

1. School of life Science ,Jiangxi Normal University, Nanchang 330022,China;
2. Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Nanchang 330022,China

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摘要： 采用正交设计法对耐底物腈水合酶融合子的发酵条件进行优化,以发酵液起始pH,发酵周期,接种量,装料系数作为考察因素,最终确定最佳发酵条件为:起始pH8.0、发酵周期54h、接种量12%、装液系数12%。在此优化条件下融合子腈水合酶的活力达到1100万U/ml, 较优化前提高了83.3%。通过响应面法对发酵培养基配方进行优化研究,采用Plackett-Burman 法对8 个因素进行了筛选, 结果表明, 葡萄糖、尿素、磷酸氢二钾、磷酸二氢钾是影响发酵液腈水合酶产量的主效应因子。用最陡爬坡试验及Central composite design 设计进一步优化, 利用Design-Expert 软件进行二次回归分析, 得到各因素的最佳浓度为: 葡萄糖22.62g/L、尿素9.76g/L、K₂HPO₄ 1.22g/L、KH₂PO₄ 1.268g/L。在此培养基优化配方下融合子腈水合酶的活力达到1280万U/ml, 较原配方的酶活提高了16.4%。

关键词： 腈水合酶; 产酶条件; 优化

Abstract: Orthogonal design was used to optimized the fermentation conditions of the nitrile hydratase(NHase) fusant strain tolerance to the substrate, Fermented liquid starting pH, fermentation period, inoculation quantity, pack liquid coefficient as the investigation factors,Finally determined the best fermentation conditions for: start pH8.0, fermentation period 54 h, Inoculation quantity 12%, pack fluid coefficient 12%. Under the optimized conditions, activity of nitriles hydratase of the fusant strain increase to 11 millionU/ml, is up 83.3% before optimization. Response surface methodology was used to optimize the fermentation medium. A Plackett-Burman design was used to evaluate the influence of eight factors firstly. Results showed that the concentration of glucose, urea, K₂HPO₄ and KH₂PO₄ played important roles in influencing the activity of nitrile hydratase. Then, the path of steepest ascent and the Central composite design were adopted for further optimization, and the optimum concentration levels and the relationships among these factors was found out by quadratic regression model equation with Design-Expert statistic methods, the optimal concentration of the variables were determined as: glucose 22.62 g/L, urea 9.76g/L, K₂HPO₄ 1.22g/L,KH₂PO₄ 1.268g/L. Under such conditions, the activity of nitrile hydratase was increased to 12.80 millionU/ml, which was 16.4% higher than old medium composition.

Key words: Nitrile hydratase Nitrile hydratase producing conditions Optimization

收稿日期: 2012-03-19 出版日期: 2012-08-25

ZTFLH:

Q935

基金资助: 江西省科技支撑计划资助项目(2009JX00564)

通讯作者: 王筱兰 E-mail: xlwang08@yahoo.com.cn

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引用本文:

张丽铃, 王筱兰. 耐底物腈水合酶融合子的产酶条件优化[J]. 中国生物工程杂志, 2012, 32(08): 75-80.

ZHANG Li-ling, WANG Xiao-lan. Optimization Fermentation Conditions of the Nitrile Hydratase Fusant Strain Tolerance to the Substrate. China Biotechnology, 2012, 32(08): 75-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/75

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