戊二酰－7－氨基头孢烷酸酰化酶

摘要

一种从假单胞菌中克隆的并经基因工程方法改造的,长为2.3kb的GL－7ACA酰化酶基因,并测得其全序列。构建了该基因的高表达重组质粒pKKCAl,该质粒转化至大肠杆菌菌株JM109,得到的基因工程菌株JM109/pKKCAl在普通的LB培养基中,勿需添加诱导剂即能高效表达GL－7ACA酰化酶,粗抽液中的酶比活可达3.5单位每毫克蛋白。

主权项

1.一种从假单胞菌中克隆的戊二酰-7-氨基头孢烷酸酰化酶基因，其特征在于该基因经基因工程方法改造后得到的23kb的DNA片段，它编码GL-7ACA酰化酶的核苷酸序列及相应的氨基酸序列如下： 1 ATGGGAATTCCGACCTCGACGCCGCAGGCGCCGATTGCGGCCTATAAACCGAGAAGCAAT 60 1 MetGlyIleProThrSerThrProGlnAlaProIleAlaAlaTyrLysProArgSerAsn 20 61 GAGATCCTGTGGGACGGCTACGGCGTCCCGCACATCTACGGCGTCGACGCGCCCTCAGCC 120 21 GluIleLeuTrpAspGlyTyrGlyValProHisIleTyrGlyValAapAlaProSerAla 40 121 TTCTACGGCTATGGCTGGGCCCAGGCGCGCAGCCACGGCGACAATATCCTGCGCCTGTAT 180 41 PheTyrGlyTyrGlyTrpAlaGlnAlaArgSerHisGlyAspAsnIleLeuArgLeuTyr 60 181 GGAGAAGCGCGGGGCAAGGGGGCCGAATACTGGGGCCCGGATTACGAACAGACGACCGTC 240 61 GlyGluAlaArgGlyLyaGlyAlaGluTyrTrpGlyProAspTyrGluGlnThrThrVal 80 241 TGGCTGCTGACCAACGGCGTGCCGGAGCGCGCTCAGCAGTGGTATGCGCAGCAGTCGCCT 300 81 TrpLeuLeuThrAsnGlyValProGluArgAlaGlnGlnTrpTyrAlaGlnGlnSerPro 100 301 GATTTCCGCGCCAACCTCGACGCCTTCGCGGCGGGCATCAACGCCTATGCGCAGCAGAAC 360 101 AspPheArgAlaAsnLeuAspAlaPhaAlaAlaGlyIlaAsnAlaTyrAlaGlnGlnAsn 120 361 CCCGACGACATCTCGCCCGACGTGCGGCAGGTGCTGCCGGTTTCCGGCGCCGACGTGGTG 420 121 ProAspAsPIleSerProAspValArgGlnValLeuProValSerGlyAlaAspValVal 140 421 GCCCACGCCCACCGCCTGATGAACTTCCTCTATGTCGCGTCGCCCGGCCGCACCCTGGGC 480 141 AlaHisAlaHisArgLeuMetAsnPheLeuTyrValAlaSerProGlyArgThrLeuGly 160 481 GAGGGCGACCCGCCGGACCTGGCCGATCAAGGATCCAACTCCTGGGCGGTGGCGCCGGGA 540 161 GluGlyAspProProAspLeuAlaAspGlnGlySerAsnSerTrpAlaValAlaProGly 180 541 AAGACGGCGAACGGGAACGCCCTGCTGCTGCAGAACCCGCACCTGTCCTGGACGACGGAC 600 181 LysThrAlaAsnGlyAsnAlaLeuLeuLeuGlnAsnProHisLeuSerTrpThrThrAsp 200 601 TACTTCACCTACTACGAGGCGCATCTCGTCACGCCGGACTTCGAAATCTATGGCGCGACC 660 201 TyrPheThrTyrTyrGluAlaHisLeuValThrProAspPheGluIleTyrGlyAlaThr 220 661 CAGATCGGCCTGCCGGTCATCCGCTTCGCCTTCAACCAGCGGATGGGCATCACCAATACC 720 221 GlnIleGlyLeuProValIleArgPheAlaPheAsnGlnArgMetGlyIleThrAsnThr 240 721 GTCAACGGCATGGTGGGGGCCACCAACTATCGGCTGACGCTTCAGGACGGCGGCTATCTG 780 241 ValAsnGlyMetValGlyAlaThrAsnTyrArgLeuThrLeuGlnAspGlyGlyTyrLeu 260 781 TATGACGGTCAGGTGCGGCCGTTCGAGCGGCCTCAGGCCTCGTATCGCCTGCGTCAGGCG 840 261 TyrAspGlyGlnValArgProPheGluArgProGlnAlaSerTyrArgLeuArgGlnAla 280 841 GACGGGACGACGGTCGACAAGCCGTTGGAGTACCGCTCCAGCGTCCATGGCCCGGTCTTC 900 281 AspGlyThrThrValAspLyeProLeuGluTyrArgSerSerValHisGlyProValPhe 300 901 GAGCGCGCGGACGGCACGGCCGTCGCCGTTCGGGTCGCCGGTCTGGACCGGCCGGGCATG 960 301 GluArgAlaAspGlyThrAlaValAlaValArgValAlaGlyLeuAapArgProGlyMet 320 961 CTCGAGCAGTATTTCGACATGATCACGGCGGACAGCTTCGACGACTACGAAGCCGCTTTG 1020 321 LeuGluGlnTyrPheAspMetIleThrAlaAspSerPheAspAspTyrGluAlaAlaLeu 340 1021 GCGCGGATGCAGGTGCCGACCTTCAACATCGTCTACGCCGACCGCGAAGGGACCATCAAC 1080 341 AlaArgMetGlnValProThrPheAsnIleValTyrAlaAspArgGluGlyThrIleAsn 3601081 TACAGCTTCACGGCGTGGCGCCCAAACGGGCCGAGGGCGACATCGCCTTCTGGCAGGGGC 1140 361 TyrSerPheThrAlaTrpArgProAsnGlyProArgAlaThrSerProSerGlyArgGly 3801141 TCGGCCGGGCCATTCCTCGCGTTACTGTGGACTGAGACACACCCCTGGACGATCTGCCGC 1200 381 8erAlaGlyProPheLeuAlaLeuLeuTrpThrGluThrHisProTrpThrIleCysArg 4001201 GCGTCACCAATCCGCCGGGCGGCTTCGTGCAGAACTCCAATGATCCGCCGTGGACGCCGA 1260 401 AlaSerProIleArgArgAlaAlaSerCysArgThrProMetIleArgArgGlyArgArg 4201261 CCTGGCCCGTCACCTACACGCCCAAGGACTTCCCCTCCTATCTGGCGCCCCAGACGCGCA 1320 421 ProGlyProSerProThrArgProArgThrSerProProIleTrpArgProArgArgAla 4401321 CTCCCTGCGCGCTCAGCAAAGCGTGCGTCTGATGTCGAGAACGACGACCTGAGGCTGGAA 1380 441 LeuProAlaArgSerAlaLysArgAlaSerAspValGluAsnAspAspLeuArgLeuGlu 4601381 CGCTTCATGGCGCTGCAGTTGAGCCATCGCGCCGTCATGGCCGACCGCACCTTGCCGGAC 1440 461 ArgPheMetAlaLeuGlnLeuSerHisArgAlaValMetAlaAspArgThrLeuProAsp 4801441 CTGATCCCGGCCGCCCTCATCGACCCCGATCCCGAGGTCCAGGCGGCGGCGCGCCTGCTG 1500 481 LeuIleProAlaAlaLeuIleAspProAspProGluValGlnAlaAlaAlaArgLeuLeu 5001501 GCGGCGTGGGATCGCGAGTTCACCAGCGACAGCCGCGCCGCCCTGCTGTTCGAGGAATGG 1560 501 AlaAlaTrpAspArgGluPheThrSerAspSerArgAlaAlaLeuLeuPheGluGluTrp 5201561 GCGCGTCTGTTCGCCGGCCAGAATTTCGCAGGCCAGGCCGGCTTCGCCACGCCCTGGTCG 1620 521 AlaArgLeuPheAlaGlyGlnAsnPheAlaGlyGlnAlaGlyPheAlaThrProTrpSer 5401621 CTGGATAAGCCGGTCAGCACGCCTTACGGCGTCCGCGACCCCAAGGCCGCCGTCGATCAA 1680 541 LeuAspLysProValSerThrProTyrGlyValArgAspProLysAlaAlaValAspGln 5601681 CTGCGGACCGCCATCGCCAACACCAAGCGCAAATACGGCGCGATCGACCGGCCGTTCGGC 1740 561 LeuArgThrAlaIleAlaAsnThrLysArgLysTyrGlyAlaIleAspArgProPheGly 5801741 GACGCCTCGCGCATGATCCTGAACGACGTGAATGTTCCGGGCGCCGCCGGCTACGGCAAC 1800 581 AspAlaSerArgMetIleLeuAsnAspValAsnValProGlyAlaAlaGlyTyrGlyAsn 6001801 CTGGGTTCCTTCCGGGTCTTCACCTGGTCCGATCCTGACGAAAACGGGGTTCGCACGCCC 1860 601 LeuGlySerPheArgValPheThrTrpSerAspProAapGluAsnGlyValArgThrPro 6201861 GTCCACGGCGAGACGTGGGTGGCGATGATCGAGTTCTCCACGCCGGTGCGGGCCTATGGC 1920 621 ValHisGlyGluThrTrpValAlaMetIleGluPheSerThrProValArgAlaTyrGly 6401921 CTGATGAGCTACGGCAACTCTCGCCAGCCGGGCACGACGCACTACAGCGATCAGATCGAA 1980 641 LeuMetSerTyrGlyAsnSerArgGlnProGlyThrThrHisTyrSerAspGlnIleGlu 6601981 CGCGTGTCGCGGGCCGACTTCCGCGAGCTGTTGCTGCGGCGAGAGCAGGTCGAGGCCGCC 2040 661 ArgValSerArgAlaAspPheArgGluLeuLeuLeuArgArgGluGlnValGluAlaAla 6802041 GTCCAGGAACGCACGCCCTTCAACTTCAAGCCATGAAAGGCCTGACCATGACACGACGGA 2100 681 ValGlnGluAugThrProPheAsnPheLysPro*** 6922101 TTGGGTATTCGGCGGGCGCGGCGTCTCTGGCGCTGATGGTCGCCGCCTCTGGAGCGGCGG 21602161 CGGGCGAGCCGGCCTTCACTTCGGTTCAGGTCGAGGGCTTTTCGGTTCCGGGCGCTCTGT 22202221 CGAACGCCTGGGCCGACTTCGACAACGACGGCGACCTGGACCTGGCCGTCTCCTGGAAGA 22802281 GCGGCGAAGCTT 2292