微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究

为研究纤维素酶纤维素结合结构域的结构与功能 ,进而深入了解天然纤维素的生物降解机制和提高纤维素酶的生物工艺学价值 ,采用 PCR技术体外扩增了携带微紫青霉外切葡聚糖纤维二糖水解酶 ( CBH ) CBD编码区的 DNA片段 ,将 CBD编码区 DNA片段插入带有 Erwiniacarotovora pe1 B前导肽序列的大肠杆菌质粒 p KK- tac- new上进行了表达 .携带微紫青霉 CBDCBH 编码区的大肠杆菌重组菌株 DH5α( p KK- tac- new- 8)产生有活性的分泌型 CBDCBH 蛋白 .SDS-PAGE检测显示所产生的 CBDCBH 蛋白分子量约 1 0 .8k D.在 IPTG诱导下 ,该菌株所产生的CBDCBH 蛋白含量达 45.2 mg/L,且 90 %以上的 CBD蛋白分泌到培养物上清液中 .结晶纤维素 CF-1 1溶液经 CBDCBH 处理后 ,浊度比对照提高了 1 2 8.9% ,天然棉花纤维结构经 CBDCBH 处理后产生一定程度的非水解性降解作用 ,表明微紫青霉 CBDCBH 具有解聚天然结晶纤维素的作用 .

Expression and Characteristic of Secretary CBDCBH1 from Penicillium janthinellum in E.coli

In order to investigate the function and acting model of cellulose binding domain(CBD),further understand degradation mechanism of native cellulose and enhance the biotechnological value of cellulolytic enzymes,the CBD encoding region of cellobiohydrolase I gene from Penicillium janthinellum was amplified in vitro by PCR.After being treated with Xba Ⅰ and Hin dⅢ,the CBD CBHⅠ encoding region was inserted into the downstream of the pelB signal sequence at E.coli vector pKK tac new.The plasmid with inserted CBD CBHⅠ encoding region DNA fragment was selected and designed pKK tac new 8.Induced by IPTG,DH5 α(pKKtac new 8)secreted 45.2 mg of CBD CBHⅠ per liter of culture.By microcrystalline absorption and desorption,the CBD CBHⅠ secreted into the culture supernatant by DH5 α(pKKtac new 8)was purified and concentrated.The CBD CBHⅠ protein had a molecular mass of 10.8 kD and more than 90% of the CBD CBHⅠ produced by DH5α(pKKtac new 8)were secreted into the culture supernatant.Treated by CBD CBHⅠ ,the increment of turbidity of crystalline cellulose CF 11 was 128.9%,and the disruption effect on native cotton fibrils structure occurred after being treated with CBD CBHⅠ .Both of the experiments proved the role of CBD CBHⅠ in penetrating the fibers of crystalline cellulose.