【摘要】 通过分子伴侣蛋白共表达的方式,对6种商业化的分子伴侣蛋白进行筛选以提高β-木糖苷酶(Xln-DT)的可溶性表达量。研究结果表明:构建诱导型质粒pG-KJE8和含有xln-DT基因的重组表达质粒pET-28a-xln-DT共表达系统后,Xln-DT的可溶性表达量为原酶初始表达量的1.31倍;以质量分数2.0%甘油为碳源,质量分数1.2%蛋白胨为氮源,添加0.01 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)、 5μg/L四环素、 1.5 g/L L-阿拉伯糖,32℃下诱导60 h,重组酶的表达量可达11.9 U/mL,较原酶提高了4.22倍。此外,利用内切木聚糖酶(XynB-DT)和Xln-DT协同酶解木聚糖,在添加Xln-DT后,榉木、桦木、玉米芯、燕麦和甘蔗渣5种木聚糖的酶解率分别为84.4%、 90.2%、 79.6%、 77.3%和64.7%,相较于仅添加XynB-DT的酶解率有了显著的提高。更多还原

【Abstract】 Based on the purpose of improving the soluble expression of β-xylosidase(Xln-DT) from Dictyoglomus thermophilum, six commercial chaperone proteins were screened by co-expression of chaperone proteins. The results showed that the soluble expression of Xln-DT was increased to 1.31 times of initial expression of proenzyme after the co-expression system of inducible plasmid pG-KJE8 and recombinant expression plasmid pet-28 a-xln-DT containing xln-DT gene were constructed. The terrific-broth medium was induced by adding 0.01 mmol/L isopropylthio-β-D-galactoside(IPTG), 5 μg/L tetracycline, 1.5 g/L L-arabinose with 2.0% glycerol and 1.2% peptone as carbon and nitrogen sources, respectively. Finally, enzyme activity of Xln-DT was 11.9 U/mL at 32 ℃ for 60 h, which was 4.22 times higher than that of the original enzyme in LB medium. In addition, the process and enzymatic mode of 5 kinds of xylan with endoxylanase XynB-DT and β-xylosidase Xln-DT were investigated. After adding Xln-DT, the enzymatic hydrolysis yield of five kinds of xylans(beechwood xylan, birchwood xylan, corncob xylan, oat xylan and bagasse xylan) were 84.4%, 90.2%, 79.6%, 77.3% and 64.7%, respectively, which was significantly improved compared with that only adding XynB-DT. All the results could not only significantly improve the soluble expression of Xln-DT, but also had a good application prospect in the production of xylooligosaccharides and xylose by enzymatic hydrolysis of xylan.更多还原