Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase
Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase
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DOI：
10.1007/s10295-008-0373-2
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发表时间：
2008-09-01
影响因子：
3.4
通讯作者：
Chu, Wen-Shen
中科院分区：
文献类型：
--
作者：
Lin, Shie-Jea;Hsieh, Yi-Fang;Chu, Wen-Shen
由变青链霉菌转化体25-2产生的重组钝顶链霉菌转谷氨酰胺酶(MtgA)通过硫酸铵分级纯化，随后通过CM-Sepharose CL-6B快速流层析和blue-Sepharose快速流层析纯化。纯化倍数约为33.2倍，收率为65%。通过 SDS-PAGE 估计纯化的重组 MtgA 的分子量为 40.0 KDa。酶活性的最适pH和温度分别为6.0和55℃，酶在pH 5.0-6.0和温度45-55℃下稳定。酶活性不受Ca2+、Li+、Mn2+、Na+、Fe3+、K+、Mg2+、Al3+、Ba2+、Co2+、EDTA或IAA的影响，但受到Fe2+、 Pb2+、Zn2+、Cu2+、Hg2+、PCMB、NEM 和 PMSF。发酵培养基的优化使两个烧瓶 (5.78 U/ml) 和 5-1 发酵罐 (5.39 U/ml) 中的重组 MtgA 活性增加了一倍。重组MtgA在30-1气升式发酵罐和250-1搅拌罐发酵罐中实现了大规模生产，最大活性分别为5.36和2.54 U/ml。
Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was similar to 33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca2+, Li+, Mn2+, Na+, Fe3+, K+, Mg2+, Al3+, Ba2+, Co2+, EDTA, or IAA but was inhibited by Fe2+, Pb2+, Zn2+, Cu2+, Hg2+, PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-1 fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-1 air-lift fermenter and a 250-1 stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.