CAJ | 학술논문

褐色高温单胞菌编码内切纤维素酶E4的催化域（CAD）DNA片段与丙酮丁醇梭菌编码对接蛋白的基因片段通过装配PCR方法，在大肠杆菌BL21（DE3）重组表达形成融合蛋白E4SD，然后在钙离子介导下与在大肠杆菌BL21（DE3）表达的来源于丙酮丁醇梭菌的含有纤维素结合单元和2个粘连蛋白的脚手架蛋白CipA互相作用，在体外组装成微纤维小体。结果显示，E4SD通过C-端的对接蛋白成功地与脚手架蛋白上的粘连蛋白互相作用，结合到脚手架蛋白上，且E4SD在微纤维小体中的羧甲基纤维素和微晶纤维素酶活性分别提高到游离状态下的130％与300％，提示非纤维小体酶系的纤维素酶，特别是远缘物种的具有优良酶特性的纤维素酶可以通过将其与脚手架蛋白同样种属的对接蛋白添加至末端，从而参与到丙酮丁醇梭菌的纤维小体中来，并与其他纤维素酶在脚手架蛋白的参与下协调作用，高效降解纤维素。
갈색고온단포균편마내절섬유소매E4적최화역（CAD）DNA편단여병동정순사균편마대접단백적기인편단통과장배PCR방법，재대장간균BL21（DE3）중조표체형성융합단백E4SD，연후재개리자개도하여재대장간균BL21（DE3）표체적래원우병동정순사균적함유섬유소결합단원화2개점련단백적각수가단백CipA호상작용，재체외조장성미섬유소체。결과현시，E4SD통과C-단적대접단백성공지여각수가단백상적점련단백호상작용，결합도각수가단백상，차E4SD재미섬유소체중적최갑기섬유소화미정섬유소매활성분별제고도유리상태하적130％여300％，제시비섬유소체매계적섬유소매，특별시원연물충적구유우량매특성적섬유소매가이통과장기여각수가단백동양충속적대접단백첨가지말단，종이삼여도병동정순사균적섬유소체중래，병여기타섬유소매재각수가단백적삼여하협조작용，고효강해섬유소。
Discussed in this paper are the heterologous expression of a truncated scaffolding protein from Clostri-dium acetobutylicum containing a cellulose-binding module and two cohesin modules,and a recombinant endoglu-canase E4SD,a family of 9 cellulase Cel9A catalysis domain from Thermomonospora fusca appended with C.aceto-butylicum dockerin in C-terminal,which are both produced by E.coli BL21 (DE3),and subsequently purified, mixed,and then assembled with minicellulosomes by calcium-mediated cohesin-dockerin interaction ex-vivo.The results show that the truncated scaffoldin CipA is successfully assembled with recombinant endoglucanase E4SD to form minicellulosome via cohesin-dockerin interaction,and that,as compared with those in enzyme-free state,the enzyme activities of E4SD in carboxymethyl cellulose hydrolysis and microcrystal cellulose hydrolysis respectively improve by 30% and 200%.Thus,it is demonstrated that noncellulosomal cellulase,especially the cellulase with excellent enzymatic characteristics from other species,can incorporate into C.acetobutylicum cellulosome by ap-pending a C.acetobutylicum dockerin and can synergize with other cellulase to hydrolyze cellulose more efficiently.