| 张兰 苏平安 杨雪梅 于文功 韩峰△.k-卡拉胶酶CgkX催化区的重组表达菌株筛选和发酵优化[J].现代生物医学进展英文版,2016,16(6):1008-1011. |
| k-卡拉胶酶CgkX催化区的重组表达菌株筛选和发酵优化 |
| Recombinant Expression and Fermentation Optimization of the CatalyticModule of k-carrageenase CgkX |
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| DOI： |
| 中文关键词: k-卡拉胶酶 催化结构域 重组表达 发酵优化 寡糖制备 |
| 英文关键词: k-carrageenase Catalytic module Recombinant expression Fermentation optimization Oligosaccharide preparation |
| 基金项目:海洋公益性行业科研专项（201105027-3，201005024）；国家自然科学基金项目（31070712，41376144）；
国家科技支撑计划项目（2013BAB01B02） |
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| 中文摘要: |
| 目的：CgkX 是来自QY203 的一种高活性、高稳定性的k-卡拉胶酶，本文旨在构建CgkX 催化结构域
（CgkX-CM）重组表达菌株，并对发酵条件进行优化，提高CgkX-CM 的产量。方法：在分析k- 卡拉胶酶CgkX-CM基因序列的基
础上，构建多种CgkX-CM 表达载体，在大肠杆菌BL21（DE3）中进行重组表达，通过酶活测定筛选其中产量最高的表达菌株，并
优化发酵起始pH、诱导温度、IPTG浓度、装液量以及甘氨酸浓度等因素。结果：构建了5 种重组表达菌株，其中BL21
(DE3)/pET22b-CgkX-CM 酶产量是其他重组菌株的7.4-11.6 倍。确定了该菌株最佳发酵条件为：培养基含1 g·L-1甘氨酸（起始
pH 7.5），装液量为75 mL，0.1 mmol·L-1 IPTG 20 ℃诱导28 h，酶产量是优化前的7.3 倍。结论：经过重组表达载体筛选和发酵优
化，CgkX-CM产量大幅度提高，为今后比较CgkX 全长及其催化区域的酶学性质，确定C 端Big_2 结构域对CgkX的作用奠定了
基础。 |
| 英文摘要: |
| Objective:CgkX-CM is the catalytic module of k-carrageenase CgkX from Pseudoalteromonas sp. QY203. The
purpose of this study was to construct and screen several recombinant expression strains followed by the optimization of the fermentation
to get a high yield of CgkX-CM.Methods:A variety of expression vectors were constructed and transformed into E.coliBL21(DE3).
These strains were induced by IPTG to screen the most efficient strain. Initial pH, induction temperature, concentration of IPTG, culture
volume and concentration of glycine were optimized.Results:Five recombinant expression vectors were constructed and screened, and
BL21 (DE3)/pET22b-CgkX-CM exhibited the highest enzymatic activity, which was 7.4-11.6 times of other strains. The optimal
fermentation conditions of this strain were conformed as follows: initial pH of 7.5, induction temperature of 20℃, concentration of IPTG
of 0.1 mmol·L-1, culture volume of 75 mL and concentration of glycine of 1 g·L-1. The optimized enzyme production was 7.3-fold
compared to the initial condition and reached 42.6 U/mL.Conclusion:The production of CgkX-CM reached 42.6 U/mL through
recombinant expression vectors construction and fermentation optimization. CgkX-CM could be used for the structure-function
relationship study of k-carrageenase CgkX and k-carrageenan oligosaccharide preparation. |
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