【摘要】：將新阿波羅棲熱袍菌(Thermotoga neapolitana)的耐熱α-半乳糖苷酶基因經(jīng)PCR擴(kuò)增,以p ET-28a為表達(dá)載體,轉(zhuǎn)化至大腸桿菌(Escherichia coli)BL21(DE3)中進(jìn)行表達(dá),用0.8 mmol/L的異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo),采用超聲波細(xì)胞破碎菌體得到α-半乳糖苷酶。通過(guò)十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)的方法獲得α-半乳糖苷酶的分子質(zhì)量約為35 ku。并對(duì)α-半乳糖苷酶的酶學(xué)性質(zhì)進(jìn)行研究,結(jié)果表明:該α-半乳糖苷酶的最適反應(yīng)溫度為85℃;最適反應(yīng)p H值為5.0;在金屬離子對(duì)酶活影響的研究中發(fā)現(xiàn)Al~(3+)、Na~+、K~+、Ca~(2+)、Mg~(2+)、Zn~(2+)、Co~(2+)對(duì)酶活起到促進(jìn)作用,Cu~(2+)、Fe~(2+)、Ag~(2+)、Mn~(2+)、Ni~(2+)的終濃度達(dá)到100 mmol/L時(shí)對(duì)酶活有較強(qiáng)的抑制作用;具有較好的熱穩(wěn)定性,在95℃處理38 h后仍具有50%的活性。
[Abstract]:The heat-resistant 偽 -galactosidase gene of (Thermotoga neapolitana) was amplified by PCR and transformed into E. coli (Escherichia coli) BL21 (DE3) with p ET-28a as expression vector. It was induced by isopropyl- 尾 -Dthiogalactoside (IPTG) of 0.8 mmol/L. 偽-galactosidase was obtained by supersonic cell fragmentation. The molecular weight of 偽 -galactosidase was about 35 ku. by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties of 偽 -galactosidase were studied. The results showed that the optimum reaction temperature of 偽 -galactosidase was 85 鈩，

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