【摘要】 在Vc生产的第2步发酵中,普通生酮基古龙酸菌S2能利用醇醛脱氢酶,将L-山梨糖转化为Vc的前体2-酮基-L-古龙酸(2-KLG).对普通生酮基古龙酸菌S2基因组DNA进行部分酶切,与黏粒载体pKC505连接后,用包装蛋白进行包装,转染大肠杆菌DH5α,构建成普通生酮基古龙酸菌S2基因组文库,得到12 000余个转化子.再利用纯化的醇醛脱氢酶免疫家兔制备出合格抗血清,应用免疫酶斑点技术(Dot-ELISA)对文库进行筛选,获得1个阳性克隆K719#.通过检测此基因工程菌的活性,表明在添加辅酶PQQ后,K719#具有使L-山梨糖转化为2-KLG的功能,从而使醇醛脱氢酶在大肠杆菌中得到了表达,这为简化Vc的生产工艺奠定了基础.更多还原

【Abstract】 Ketogulonigenium vulgare S2 is widely used in production of Vitamin C,which can transform L sorbose to 2ketoL-gulonic acid(2 KLG) because of L sorbose/Lsorbosne dehydrogenase.In order to obtain the engineering strain to simplify the fermentation technology,its chromatosomal DNA was partially digested with Sau3 AⅠ,then,collected fragments about 23～30 kb and connected them to cosmid pKC505 vector digested by Hpa Ⅰ and Pst Ⅰ.The genomic library of Ketogulonigenium vulgare was constructed by packing in vitro with λ phage package protein and transfecting E.coli DH5α.At the same time,antibody was acquired from abbit which had been injected purified Lsorbose/Lsorbosne on it.Finally,a positive strain K719# was selected from more than 12 000 clones via Dot ELISA method.The K719# strain was tested by SDSPAGE and thin layer chromatography,the results showed that the K719# strain has the same function of transforming L sorbose to 2 KLG as Ketogulonigenium vulgare S2 after adding coenzyme PQQ.更多还原