| 苏平安,王莹,李尚勇,等.κ-卡拉胶酶CgkX高效重组表达菌株的构建和发酵优化?[J].中国海洋药物,2015,34(4):1-7. |
| κ-卡拉胶酶CgkX高效重组表达菌株的构建和发酵优化? |
| Recombinant expression and fermentation optimization of κ-carrageenase CgkX |
| 投稿时间：2015-01-07 修订日期：2015-02-04 |
| DOI： |
| 中文关键词: κ-卡拉胶酶 重组表达 发酵优化 寡糖制备 |
| English Keywords:κ-carrageenase recombinant expression fermentation optimization oligosaccharide preparation |
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| 中文摘要: |
| 目的 CgkX是一种高活性、高稳定性的κ-卡拉胶酶,但产量低。本文旨在构建多种重组表达菌株,并进行发酵条件优化,以提高κ-卡拉胶酶CgkX的产量。方法 在分析了κ-卡拉胶酶CgkX基因序列中稀有密码子和氨基酸序列中含硫氨基酸的基础上,构建多种CgkX表达载体,在大肠杆菌BL21(DE3)中进行重组表达和酶活测定,筛选其中最高效的表达菌株,并对发酵起始pH、诱导温度、IPTG浓度、装液量以及甘氨酸浓度等因素进行优化。结果 构建了4种重组表达载体,转入大肠杆菌BL21(DE3),发现E. coli BL21(DE3)/pET-22b-CgkX酶产量最高,是以前菌株的8倍。通过优化确定了最佳发酵条件为：发酵起始pH 7.5,IPTG浓度为0.05 mmol?L-1,诱导温度20 oC,装液量为75 mL,甘氨酸浓度为1 g?L-1。优化后酶产量达到32 U/mL,是优化前的7倍。结论 经过重组表达载体筛选和发酵优化,CgkX产量提高到最初的56倍,为酶解制备κ-卡拉胶寡糖提供了足量的工具酶。 |
| English Summary: |
| Objective κ-Carrageenase CgkX from Pseudoalteromonas sp. QY203 is more stable than most of previously reported κ-carrageenases, however, its production is low. The purpose of this study is to construct and screen a variety of recombinant expression strains and optimize the fermentation to get a higher yield of CgkX. Methods A variety of expression vectors were constructed on the basis of analyzing the rare codon and sulfur-containing amino acid residues in κ-carrageenase gene cgkX. The expression vectors were transformed in E. coli BL21(DE3) to screen the most efficient strain. Initial pH, induction temperature, concentration of IPTG, culture volume and concentration of glycine were optimized. Results Four recombinant expression vectors were constructed and screened, and E. coli BL21(DE3)/pET-22b-CgkX exhibited the highest enzymatic activity, which was 8 times of that of other strain. The optimal conditions of fermentation were conformed as follows: initial pH of 7.5, induction temperature of 20 oC, concentration of IPTG of 0.05 mmol?L-1, culture volume of 75 mL and concentration of glycine of 1 g?L-1. The optimized enzyme activity is 6 times higher than before and reached 32 U/mL. Conclusions The production of CgkX increased 55-fold through recombinant expression vectors construction and fermentation optimization. CgkX can be expressed more for κ-carrageenan oligosaccharide preparation. |
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