【摘要】：本試驗旨在構(gòu)建不同纖維素酶的融合表達(dá)系統(tǒng)及探討融合纖維素酶的酶學(xué)性質(zhì)。利用PCR技術(shù)從實驗室前期分離的枯草芽孢桿菌中分別擴(kuò)增2個纖維素酶基因Cel42和Cel22,設(shè)計一段柔性接頭(GSGGGS),通過酶切連接將2個纖維素酶基因構(gòu)建在一個開放閱讀框(ORF)內(nèi),插入到pET32a(+)中構(gòu)建重組表達(dá)載體pET32a(+)-Cel42-Cel22,轉(zhuǎn)化大腸桿菌BL21(DE3)進(jìn)行誘導(dǎo)表達(dá),并對其酶學(xué)性質(zhì)進(jìn)行研究。結(jié)果表明:本試驗成功克隆了2個纖維素酶基因Cel42和Cel22,并構(gòu)建了重組表達(dá)系統(tǒng)BL21(DE3)/pET32a(+)-Cel42-Cel22,十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)估計其分子質(zhì)量約為101 ku,粗酶液中葡聚糖內(nèi)切酶活性為57.62 U/mL,葡聚糖外切酶活性為32.57 U/mL。試驗所得融合纖維素酶Cel42-Cel22的最適反應(yīng)溫度為50℃,最適反應(yīng)pH為6.0,溫度在30~70℃范圍內(nèi)時可維持70%以上的纖維素酶活性,pH在4.0~9.0范圍內(nèi)時可保持75%以上的纖維素酶活性,除Mn~(2+)外的其他金屬離子對纖維素酶的活性均具有一定的抑制作用,其中Hg~(2+)和Cu~(2+)對的抑制作用較明顯。由此可見,本試驗在大腸桿菌BL21(DE3)中成功表達(dá)出了融合纖維素酶Cel42-Cel22,且該酶具有一定的活性,可適應(yīng)較寬廣的溫度和pH范圍,對金屬離子敏感。
[Abstract]:The purpose of this study was to construct fusion expression systems of different cellulases and to investigate the enzymatic properties of the fusion cellulases. Two cellulase genes Cel42 and Cel22 were amplified by PCR from Bacillus subtilis isolated in early laboratory, and a flexible joint (GSGGGS) was designed. The two cellulase genes were constructed into an open reading frame (ORF) by enzyme ligation. The recombinant expression vector pET32a () -Cel42-Cel22 was inserted into pET32a (), and transformed into E. coli BL21 (DE3) to induce the expression, and its enzymatic properties were studied. The results showed that two cellulase genes Cel42 and Cel22 were cloned successfully, and the recombinant expression system BL21 (DE3) / pET32a () -Cel42-Cel22 was constructed. SDS-PAGE estimated its molecular weight to be about 101 ku. The activity of endoglucinase was 57.62 U / mL, and that of dextran was 32.57 U / mL. The optimum reaction temperature and pH of the fusion cellulase Cel42-Cel22 were 50 鈩，

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