醇醛脱氢酶的分离纯化及其基因文库的构建和筛选

Purification of L-sorbose/L-sorbosne Dehydrogenase from Ketogulonigenium vulgare and Construction and Selection of Genomic Library

摘要:

在维生素C的发酵生产过程中，普通生酮基古龙酸菌S₂（Ketogulonigenium vulgare)能产生醇醛脱氢酶，将L-山梨糖转化为VC的前体2-酮基-L-古龙酸（2-KLG）。通过超声波破碎菌体、硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析，Q Sepharose High Performance柱层析等过程，从普通生酮基古龙酸菌S₂发酵液中分离纯化了醇醛脱氢酶，并用该纯化酶免疫新西兰兔制备出了合格抗血清。同时，普通生酮基古龙酸菌S₂基因组DNA经Sau3AⅠ部分酶切后，与黏粒载体pKC505连接，用包装蛋白进行包装，转染大肠杆菌DH5ɑ，构建了基因组文库。最后应用免疫酶斑点技术（Dot-ELISA）从12 000个克隆子中筛选得到一个阳性克隆K719^#。通过检测该基因工程菌的活性，表明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获得了高效表达，这为简化VC的生产工艺奠定了基础。

Abstract:

L-sorbose/L-sorbosone dehydrogenase from Ketogulonigenium vulgare ₂ can transform L-sorbose to 2-KLG, which is widely used in production of Vitamin C. In order to obtain the engineering strain producing L-sorbose/L-sorbosowas purified by the methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Q Sepharose High Performance.Then, the purified L-sorbose/L-sorbosone dehydrogenase was injected to rabbit to obtain antibody. Next, the genomic library of Ketogulonigenium vulgare₂was constructed by inserting the restriction fragments of chromatosomal DNA digested with Sau3AⅠ into cosmid pKC505 vector digested by HpaⅠ and PstⅠ,which were packed with λ phage package protein and transferred into E.coli DH5α in vitro. Finally, the positive strain K719^#was selected from more than 12 000 clones via Dot-ELISA. Through the test of SDS-PAGE and thin layer chromatography, the results showed that the engineering strain K719^#had the same biological activity as Ketogulonigenium vulgare ₂ after adding coenzyme PQQ.