摘要
目的:在成功构建咖啡豆α半乳糖苷酶毕赤酵母工程菌株及完成基因重组α半乳糖苷酶发酵与纯化研究的基础上,对基因重组α半乳糖苷酶的生物化学性质进行研究。方法:测定了基因重组α半乳糖苷酶的N端序列、相对分子质量、氨基酸组成、三批发酵产物的紫外吸收光谱和肽图以及Vmax、Km、最适反应温度、最适反应pH值等酶的生化性质。结果与结论:基因重组α半乳糖苷酶的N端序列与理论序列完全一致;氨基酸组成与理论值相符;相对分子质量为39.4×103。三批发酵产物的紫外光谱吸收和肽图基本一致,表明发酵纯化后的产物稳定;Km=0.275mmol/L,最大酶促反应速度Vmax=0.014mmol/min;最适反应温度为37℃,最适反应pH值为5.6。
Objective:To study the structure and biochemical properties of recombinant α-galactosidase on basis of construction of expression vector which was transfused into yeast Pichia pastoris as well as on the studies of fermentation and purification of recombinant α-galactosidase. Methods:The N-terminal sequence, relative molecular mass, amino acid components, UV light spectrum,peptide identification and biochemical characterization including K m, V max, optional temperature and pH of recombinant α-galactosidase were studied.Results and conclusion: The results showed that the N-terminal amino acids sequence of recombinant α-galactosidase were identical with theoretical sequence completely; amino acid components were largely agree with theoretical value; M r=39.4×10 3; the UV light spectrum and peptide identification of purified products were same for three barches of preparation; K m and V max of recombinant α-galactosidase were 0.275 mmol/L and 0.014 mmol/min, respectively; optional temperature and pH of recombinant α-galactosidase reaction were 37℃ and 5.6. It has no difference to natural enzyme.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第2期139-141,155,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家"973"计划资助项目(2002CB713804)
全军重点课题资助(2000252910)
