[发明专利]能催化吲哚生成靛蓝的质粒pET28a(+)－P450BM3－gdh0310、制备方法及其用途

摘要

本发明公开了一种能共表达P450BM3和葡萄糖脱氢酶，并能催化吲哚合成靛蓝的质粒pET28a(+)－P450BM3－gdh0310、制备方法及其用途。它是将P450BM3基因和葡萄糖脱氢酶基因共同连接在同一个表达载体pET28a(+)上，将此质粒转入E.coliBL21，得到的菌株能同时表达细胞色素P450BM3单加氧酶和葡萄糖脱氢酶，并能协同作用催化吲哚生成靛蓝，与以往菌株相比，其催化活性提高了22－27倍，为生物转化生产染料靛蓝提供了广阔的应用前景。

主权项

1.一种能催化吲哚生成靛蓝的质粒pET28a(+)-P450 BM3-gdh0310，由P450 BM3基因和葡萄糖脱氢酶基因共同连接在同一个表达载体pET28a(+)上构建，从P450 BM3基因的启动子开始，到葡萄糖脱氢酶基因的终止子结束的基因序列为：ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAAAAATTTACCGTTATTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGAATTAGGAGAAATCTTTAAATTCGAGGCGCCTGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAGCATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGGTCTTAAATTTGTACGTGATTTTGCAGGAGACGGGTTGGTTACAAGCTGGACGCATGAAAAAAATTGGAAAAAAGCGCATAATATCTTACTTCCAAGCTTCAGTCAGCAGGCAATGAAAGGCTATCATGCGATGATGGTCGATATCGCCGTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATTGAAGTACCGGAAGACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTAACTATCGCTTTAACAGCTTTTACCGAGATCAGCCTCATCCATTTATTACAAGTATGGTCCGTGCACTGGATGAAGCAATGAACAAGCAGCAGCGAGCAAATCCAGACGACCCAGCTTATGATGAAAACAAGCGCCAGTTTCAAGAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATTATTGCAGATCGCAAAGCAAGCGGTGAACAAAGCGATGATTTATTAACGCATATGCTAAACGGAAAAGATCCAGAAACGGGTGAGCCGCTTGATGACGAGAACATTCGCTATCAAATTATTACATTCTTAATTGCGGGACACGAAACAACAAGTGGTCTTTTATCATTTGCGCTGTATTTCTTAGTGAAAAATCCACATGTATTACAAAAAGCAGCAGAAGAAGCAGCACGAGTTCTAGTAGATCCTGTTCCAAGCTACAAACAAGTCAAACAGCTTAAATATGTCGGCATGGTCTTAAACGAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGTTTTCCCTATATGCAAAAGAAGATACGGTGCTTGGAGGAGAATATCCTTTAGAAAAAGGCGACGAACTAATGGTTCTGATTCCTCAGCTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGAAGAGTTCCGTCCAGAGCGTTTTGAAAATCCAAGTGCGATTCCGCAGCATGCGTTTAAACCGTTTGGAAACGGTCAGCGTGCGTGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGTACTTGGTATGATGCTAAAACACTTTGACTTTGAAGATCATACAAACTACGAGCTGGATATTAAAGAAACTTTAACGTTAAAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAAAAATTCCGCTTGGCGGTATTCCTTCACCTAGCACTGAACAGTCTGCTAAAAAAGTATGCAAAAAGGCAGAAAACGCTCATAATACGCCGCTGCTTGTGCTATACGGATCCAATATGGGAACAGCTGAAGGAACGGCGCGTGATTTAGCAGATATTGCAATGAGCAAAGGATTTGCACCGCAGGTCGCAACGCTTGATTCACACGCCGGAAATCTTCCGCGCGAAGGAGCTGTATTAATTGTAACGGCGTCTTATAACGGTCATCCGCCTGATAACGCAAAGCAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGATGAAGTAAAAGGCGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAACTGGGCTACTACGTATCAAAAAGTGCCTGCTTTTATCGATGAAACGCTTGCCGCTAAAGGGGCAGAAAACATCGCTGACCGCGGTGAAGCAGATGCAAGCGACGACTTTGAAGGCACATATGAAGAATGGCGTGAACATATGTGGAGTGACGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAAGATAATAAATCTACTCTTTCACTTCAATTTGTCGACAGCGCCGCGGATATGCCGCTTGCGAAAATGCACGGTGCGTTTTCAACGAACGTCGTAGCAAGCAAAGAACTTCAACAGCCAGGCAGTGCACGAAGCACGCGACATCTTGAAATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCATTTAGGTGTTATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGCAAGGTTCGGCCTAGATGCATCACAGCAAATCCGTCTGGAAGCAGAAGAAGAAAAATTAGCTCATTTGCCACTCGCTAAAACAGTATCCGTAGAAGAGCTTCTGCAATACGTGGAGCTTCAAGATCCTGTTACGCGCACGCAGCTTCGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGAGCTTGAAGCCTTGCTTGAAAGCAAGCCTACAAGACAAGTGCTGGCAAACGTTTAACAATGCTTGAACTGCTTGAAAAATACCCGGCGTGTGAATGAAATTCAGCGAATTTATCGCCATTCTGCCAAGCATACGCCCGCGCTATTACTCGATTTCTTCATCACCTCGTGTCGATGAAAAACAAGCAAGCATCACGGTCAGCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAGAATATAAAGGAATTGCGTCGAACTATCTTGCCGAGCTGCAAGAAGGAGATACGATTACGTGCTTTATTTCCACACCGCAGTCAGAATTTACGCTGCCAAAAGACCCTGAAACGCCGCTTATCATGGTCGGACCGGGAACAGGCGTCGCGCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAGCTAAAAGAACAAGGACAGTCACTTGGAGAAGCACATTTATACTTCGGCTGCCGTTCACCTCATGAAGACTATCTGTATCAAGAAGAGCTTGAAAACGCCCAAAGCGAAGGCATCATTACGCTTCATACCGCTTTTTCTCGCATGCCAAATCAGCCGAAAACATACGTTCAGCACGTAATGGAACAAGACGGCAAGAAATTGATTGAACTTCTTGATCAAGGAGCGCACTTCTATATTTGCGGAGACGGAAGCCAAATGGCACCTGCCGTTGAAGCAACGCTTATGAAAAGCTATGCTGACGTTCACCAAGTGAGTGAAGCAGACGCTCGCTTATGGCTGCAGCAGCTAGAAGAAAAAGGCCGATACGCAAAAGACGTGTGGGCTGGGTAAGAATTCGGATCCATGTATACAGATTTAAAAGATAAAGTAGTAGTTGTAACAGGCGGATCAAAAGGATTGGGTCGCGCAATGGCCGTTCGTTTTGGTCAAGAGCAGTCAAAAGTGGTTGTAAACTACCGCAGCAATGAAGAAGAAGCGCTAGAAGTAAAAAAAGAAATTGAACAAGCTGGCGGCCAAGCAATTATTGTTCGAGGCGACGTAACAAAAGAGGAAGACGTTGTGAATCTTGTAGAGACAGCTGTTAAAGAGTTTGGCACATTAGACGTTATGATTAACAATGCTGGTGTTGAAAACCCGGTTCCTTCACATGAATTATCGTTAGAAAACTGGAATCAAGTAATCGATACAAACTTAACAGGCGCGTTTTTAGGAAGCCGCGAAGCGATTAAATATTTTGTTGAAAATGATATTAAAGGAAACGTTATTAACATGTCCAGCGTTCACGAGATGATTCCTTGGCCACTATTTGTTCACTATGCAGCAAGTAAAGGCGGTATGAAACTAATGACAGAAACATTGGCTCTTGAATATGCGCCAAAAGGTATCCGCGTAAATAACATTGGACCAGGCGCGATCGATACGCCAATCAACGCTGAAAAATTCGCAGATCCGGAACAGCGTGCAGACGTAGAAAGCATGATTCCAATGGGCTACATCGGCAACCCGGAAGAAATTGCATCAGTTGCAGCATTCTTAGCATCGTCACAAGCAAGCTACGTAACAGGTATTACACTATTTGCTGATGGCGGTATGACAAAATATCCTTCTTTCCAAGCGGGAAGAGGTTAATAA。