摘要
将新阿波罗栖热袍菌(Thermotoga neapolitana)的耐热α-半乳糖苷酶基因经PCR扩增,以p ET-28a为表达载体,转化至大肠杆菌(Escherichia coli)BL21(DE3)中进行表达,用0.8 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)诱导,采用超声波细胞破碎菌体得到α-半乳糖苷酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)的方法获得α-半乳糖苷酶的分子质量约为35 ku。并对α-半乳糖苷酶的酶学性质进行研究,结果表明:该α-半乳糖苷酶的最适反应温度为85℃;最适反应p H值为5.0;在金属离子对酶活影响的研究中发现Al^(3+)、Na^+、K^+、Ca^(2+)、Mg^(2+)、Zn^(2+)、Co^(2+)对酶活起到促进作用,Cu^(2+)、Fe^(2+)、Ag^(2+)、Mn^(2+)、Ni^(2+)的终浓度达到100 mmol/L时对酶活有较强的抑制作用;具有较好的热稳定性,在95℃处理38 h后仍具有50%的活性。
The heat resistant α-galactosidase gene from Therrnotoga neapolitana was amplified by PCR. Using the pET-28a as expression vector, the gene was converted into （Eschedchia colt） BL21 （DE3） and expressed. After 0.8 mmol/L isopropylthio-β-D-galactoside （IPTG） inducing, the modi- fied E. coil cell was broken by ultrasonic wave and α-galactosidase was gotten. The molecular mass of α-galactosidase was about 35 ku by SDS-PAGE. The α-galactosidase property was researched. The results showed that the optimal reaction temperature of α-galaetosidase was 85℃, the optimal reaction pH was 5.0. Al^3＋, Na^＋, K^＋, Ca^2＋, Mg^2＋, Zn^2＋, and Co^2＋ had a promoting effect on α-galactosidase activity. Cu^2＋, Fe^2＋, Ag^2＋, Mn^2＋, and Ni^2＋ had a strong inhibiting effect on α-galactosidase activity when the final concentration reached 100 mmol/L. The α-galactosidase had good thermal stability and still had 50% activity at 95℃ for 38 h.
出处
《中国酿造》
CAS
北大核心
2017年第1期61-65,共5页
China Brewing
基金
吉林省教育厅"十二五"科学技术研究项目(吉教科合字[2015]第207号)
吉林省科技厅科技创新人才培育计划项目(20140519011JH)
吉林省科技厅科技成果转化计划项目(20130302017NY)
关键词
Α-半乳糖苷酶
基因克隆
表达
酶学性质
α-galactosidase
gene cloning
expression
characterizations enzyme property
