苏氨酸醛缩酶、突变体及其在制备取代苯丝氨酸衍生物中的应用的制作方法

本发明涉及生物
技术领域：
，具体涉及苏氨酸醛缩酶、突变体及其在制备2-/3-/4-位取代的苯丝氨酸衍生物中的应用。
背景技术：
：2-/3-/4-位取代的苯丝氨酸衍生物是一种重要的药物中间体和化工中间体，特别是4-甲砜基苯丝氨酸，广泛用于人类抗生素，家禽家畜类抗生素制备等，其代表药物如氯霉素，甲砜霉素，氟苯尼考等等。2-/3-/4-位取代的苯丝氨酸衍生物合成方法主要包括化学合成法和酶催化法。其中，化学合成法主要是通过加成反应，酯化反应和化学拆分法来得到手性的2-/3-/4-位取代的苯丝氨酸衍生物，虽然化学法操作简单，但是反应过程中用到了大量的水，重金属和剧毒的硫化氢，并且有大量的光学异构体作为副产物，不符合目前和将来国家提倡和推进的原子经济学和环境保护政策。而酶催化法具有条件温和，用水量少(仅为化学法的十分之一)，没有重金属污染，不使用剧毒化学品，手性选择高等诸多优点，具有很好的应用前景。苏氨酸醛缩酶有着十分广泛的应用潜力，它可以催化合成手性的医药关键中间体β-羟基-α-氨基酸。但是由于苏氨酸醛缩酶的稳定性不好，手性选择性特别是β-羟基的手性选择性不足，限制了此酶在手性合成中的应用。技术实现要素：本发明目的是提供苏氨酸醛缩酶、突变体及其在制备2-/3-/4-位取代的苯丝氨酸衍生物中的应用，以解决现有技术的不足。本发明采用以下技术方案：第一方面，本发明提供了苏氨酸醛缩酶，选自假单胞菌(pseudomonasputida)，其氨基酸序列为seqidno：2所示，基因序列为seqidno：1所示；选自新月柄杆菌(caulobactercrescentus)，其氨基酸序列为seqidno：19所示，基因序列为seqidno：88所示；选自海栖热袍菌(thermotogamaritima)，其氨基酸序列为seqidno：23所示；选自单增李斯特菌(listeriamonocytogenes)，其氨基酸序列为seqidno：24所示；选自大肠杆菌(escherichiacoli)，其氨基酸序列为seqidno：25所示；选自简达气单胞菌(aeromonasjandaei)，其氨基酸序列为seqidno：26所示；选自酿酒酵母(saccharomycescerevisiae)，其氨基酸序列为seqidno：27所示；选自棉阿舒囊霉菌(ashbyagossypii)，其氨基酸序列为seqidno：28所示；选自鲍希瓦氏菌(shewanellaloihica)，其氨基酸序列为seqidno：29所示；选自维龙气单胞菌(aeromonasveronii)，其氨基酸序列为seqidno：30所示；选自硫还原地杆菌(geobactersulfurreducens)，其氨基酸序列为seqidno：31所示。第二方面，本发明提供了苏氨酸醛缩酶的突变体，是建立在不同菌株来源的苏氨酸醛缩酶的四个位点的突变，突变为单点突变、双点突变、三点突变或四点突变，其突变位点和原始位氨基酸列表如下：苏氨酸醛缩酶第一突变点第二突变点第三突变点第四突变点ps_ltan17h94s182t212cc_ltan14h91a178t206ec_ltat8h83a168s196lm_lta---h98a178y206tm_ltat8h83a170s198aj_ltat10h85a170s198sc_ltat21h97a184s212ag_ltat22h98a185s213sl_ltat8h83a168s196av_ltat10h85a170s198gs_ltat10h85a170s198其对应可更换的氨基酸如下：第一突变点n/t更换为q，第二突变点h更换为w/p/f/v/a/s，第三突变点s/a更换为y，第四突变点t/s/y更换为i；其中，不同菌株来源的苏氨酸醛缩酶定义如下：ps_lta-来源于假单胞菌(pseudomonasputida)的苏氨酸醛缩酶，cc_lta-来源于新月柄杆菌(caulobactercrescentus)的苏氨酸醛缩酶，ec_lta-来源于大肠杆菌(escherichiacoli)的苏氨酸醛缩酶，lm_lta-来源于单增李斯特菌(listeriamonocytogenes)的苏氨酸醛缩酶，tm_lta-来源于海栖热袍菌(thermotogamaritima)的苏氨酸醛缩酶，aj_lta-来源于简达气单胞菌(aeromonasjandaei)的苏氨酸醛缩酶，sc_lta-来源于酿酒酵母(saccharomycescerevisiae)的苏氨酸醛缩酶，ag_lta-来源于棉阿舒囊霉菌(ashbyagossypii)的苏氨酸醛缩酶，sl_lta-来源于鲍希瓦氏菌(shewanellaloihica)的苏氨酸醛缩酶，av_lta-来源于维龙气单胞菌(aeromonasveronii)的苏氨酸醛缩酶，gs_lta来源于硫还原地杆菌(geobactersulfurreducens)的苏氨酸醛缩酶；其中，氨基酸缩写含义如下：n-天冬酰胺，t-苏氨酸，q-谷氨酰胺，h-组氨酸，w-色氨酸，p-脯氨酸，f-苯丙氨酸，v-缬氨酸，a-丙氨酸，s-丝胺酸，y-酪氨酸，i-异亮氨酸。进一步地，苏氨酸醛缩酶的突变体，选自假单胞菌(pseudomonasputida)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：4，seqidno：6，seqidno：8，seqidno：10所示，基因序列为seqidno：3，seqidno：5，seqidno：7，seqidno：9所示，所述seqidno：4所示突变体是将seqidno：2所示氨基酸序列第182位丝氨酸突变成了酪氨酸，所述seqidno：6所示突变体是将seqidno：2所示氨基酸序列第94位组氨酸突变成了脯氨酸，所述seqidno：8所示突变体是将seqidno：2所示氨基酸序列第17位天冬酰胺突变成了谷氨酰胺，所述seqidno：10所示突变体是将seqidno：2所示氨基酸序列第212位苏氨酸突变成了异亮氨酸；选自新月柄杆菌(caulobactercrescentus)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：20，seqidno：21，seqidno：22，seqidno：32，seqidno：33，seqidno：34，seqidno：35，seqidno：36，seqidno：37，seqidno：38所示；选自海栖热袍菌(thermotogamaritima)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：39，seqidno：40，seqidno：41，seqidno：42，seqidno：43所示；选自单增李斯特菌(listeriamonocytogenes)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：44，seqidno：45，seqidno：46，seqidno：47，seqidno：48所示；选自大肠杆菌(escherichiacoli)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：49，seqidno：50，seqidno：51，seqidno：52，seqidno：53所示；选自简达气单胞菌(aeromonasjandaei)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：54，seqidno：55，seqidno：56，seqidno：57，seqidno：58所示；选自酿酒酵母(saccharomycescerevisiae)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：59，seqidno：60，seqidno：61，seqidno：62，seqidno：63所示；选自棉阿舒囊霉菌(ashbyagossypii)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：64，seqidno：65，seqidno：66，seqidno：67，seqidno：68所示；选自鲍希瓦氏菌(shewanellaloihica)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：69，seqidno：70，seqidno：71，seqidno：72，seqidno：73所示；选自维龙气单胞菌(aeromonasveronii)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：74，seqidno：75，seqidno：76，seqidno：77，seqidno：78所示；选自硫还原地杆菌(geobactersulfurreducens)的苏氨酸醛缩酶的突变体，其氨基酸序列为seqidno：79，seqidno：80，seqidno：81，seqidno：82，seqidno：83所示。第三方面，本发明提供了上述苏氨酸醛缩酶、苏氨酸醛缩酶的突变体的编码基因。第四方面，本发明提供了上述编码基因构建的重组载体，这些重组载体可通过本领域常规方法将本发明的苏氨酸醛缩酶、苏氨酸醛缩酶的突变体的核苷酸序列连接于各种载体上构建而成。所述载体可为本领域常规的各种载体，如各种质粒载体等。第五方面，本发明提供了上述重组载体转化获得的重组基因工程菌，可通过将本发明的重组表达载体转化至宿主微生物中获得。所述的宿主微生物可为本领域常规的各种宿主微生物，只要满足重组表达载体可以稳定自我复制且所携带的本发明的苏氨酸醛缩酶、苏氨酸醛缩酶的突变体的基因可以有效表达，如大肠杆菌，枯草芽孢杆菌，棒杆菌，酵母等。第六方面，本发明提供了上述苏氨酸醛缩酶、苏氨酸醛缩酶的突变体在制备2-/3-/4-位取代的苯丝氨酸衍生物中的应用。进一步地，以苏氨酸醛缩酶或苏氨酸醛缩酶的突变体为催化剂，以2-/3-/4-位取代的苯甲醛为底物，以5-磷酸吡哆醛为辅酶，以甘氨酸/甘氨酸酯为辅底物，控制温度和搅拌速度的条件下，在反应介质里进行酶催化，反应结束后，分离纯化，获得2-/3-/4-位取代的苯丝氨酸衍生物。进一步地，所述底物为下列之一：苯甲醛，2-氯苯甲醛，2-氟苯甲醛，2-溴苯甲醛，2-硝基苯甲醛，2-甲砜基苯甲醛，2-羟基苯甲醛，3-氯苯甲醛，3-氟苯甲醛，3-溴苯甲醛，3-硝基苯甲醛，3-甲砜基苯甲醛，3-羟基苯甲醛，4-氯苯甲醛，4-氟苯甲醛，4-溴苯甲醛，4-硝基苯甲醛，4-甲砜基苯甲醛，4-羟基苯甲醛；所述辅底物为下列之一：l-甘氨酸，l-甘氨酸盐酸盐，l-甘氨酸甲酯盐酸盐，l-甘氨酸乙酯盐酸盐，n-boc-l-甘氨酸，n-boc-l-甘氨酸乙酯，n-z-l-甘氨酸，n-z-l-甘氨酸乙酯，n-z-l-甘氨酸甲酯；反应介质为下列之一：ph8-9的磷酸缓冲体系，水-乙醇体系，水-甲醇体系，甲醇体系，乙醇体系。进一步地，酶的用量为50-1000mg/l，底物浓度为1-100g/l，甘氨酸/甘氨酸酯浓度为5-200g/l，5-磷酸吡哆醛浓度为0.01-0.2g/l，控制温度在25-45℃，搅拌速度70-300rpm。本发明的有益效果：1、本发明通过对不同菌株来源的苏氨酸醛缩酶进行定点突变，从而筛选出稳定性良好，手性选择性高的苏氨酸醛缩酶突变体并成功用于合成手性纯度高的2-/3-/4-位取代的苯丝氨酸衍生物，展现出了优秀的应用前景。2、本发明以苏氨酸醛缩酶、突变体为生物催化剂，以2-/3-/4-位取代的苯甲醛为底物，以5-磷酸吡哆醛为辅酶，以甘氨酸/甘氨酸酯为辅底物，在适当条件和介质内进行酶催化反应，分离制备出一系列不同取代基的苯丝氨酸衍生物，该方法总收率范围在76-99％，产物ee值大于99％，de值范围在70-99％。附图说明图1为实施例19hplc图谱。图2为实施例20核磁图谱。具体实施方式下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明，但并不用来限定本发明的实施范围。实施例1：苏氨酸醛缩酶基因psald的扩增根据genebank收录的来自假单胞菌(pseudomonasputida)的苏氨酸醛缩酶基因信息为依据(基因序列如seqidno：1所示)，用核酸快速提取仪提取菌体的总基因组dna，以该基因组dna为模板，在引物1(atgaatggtgaaaccagccg)、引物2(ttaacgttcctgggtgcgat)的作用下进行pcr扩增。pcr反应体系(总体积50μl):10×pfudnapolymerasebuffer5μl，10mmdntpmixture(datp、dctp、dgtp和dttp各2.5mm)1μl，浓度均为50μm的克隆引物1、引物2各1μl，基因组dnalμl，pfudnapolymerase1μl，无核酸水40μl。采用biorad的pcr仪，pcr反应条件:预变性95℃5min，95℃变性30s，65℃返火45s，72℃延伸1min，共30个循环，最后72℃延伸10min。pcr反应液用0.9％琼脂糖凝胶电泳检测并切胶回收纯化该片段，得到苏氨酸醛缩酶基因psald。实施例2：苏氨酸醛缩酶基因ccald的扩增根据genebank收录的来自新月柄杆菌(caulobactercrescentus)的苏氨酸醛缩酶基因信息为依据(基因序列如seqidno：88所示)，用核酸快速提取仪提取菌体的总基因组dna，以该基因组dna为模板，在引物3(atgacccafaccgcgccccgcta)、引物4(ctaagccactcgcttcagcgcc)的作用下进行pcr扩增。pcr反应体系(总体积50μl):10×pfudnapolymerasebuffer5μl，10mmdntpmixture(datp、dctp、dgtp和dttp各2.5mm)1μl，浓度均为50μm的克隆引物1、引物2各1μl，基因组dnalμl，pfudnapolymerase1μl，无核酸水40μl。采用biorad的pcr仪，pcr反应条件:预变性95℃5min，95℃变性30s，65℃返火45s，72℃延伸1min，共30个循环，最后72℃延伸10min。pcr反应液用0.9％琼脂糖凝胶电泳检测并切胶回收纯化该片段，得到苏氨酸醛缩酶基因ccald。实施例3：苏氨酸醛缩酶基因psald的重组大肠杆菌的构建一.重组质粒pet28a-psald的获得获得psald基因序列，测序后利用ndei和xhoi限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体pet28a进行连接，构建表达载体pet28a-psald。二.重组质粒转化大肠杆菌e.colibl21(de3)在每管的100μle.colibl21(de3)感受态细胞悬液中加入10μlpcr产物，轻轻混匀，冰浴中静置30min。转入42℃水浴中，热击90s。快速转移至冰浴中冷却3min。每管中加入700μllb液体培养基，37℃100rpm摇床温育培养1h。培养后菌液3,000rpm离心2min，弃上清700μl，剩余菌液混匀后涂布到含有50μg·ml-1硫酸卡那霉素的lb平板上，37℃倒置培养过夜。阳性克隆的选择：挑取4个克隆，转接装有5ml的含有50μg·ml-1硫酸卡那霉素的lb培养基中，37℃培养8h，利用质粒提取试剂盒mini-plasmidrapidisolationkit(北京博大泰克生物基因技术有限公司)提取质粒。取出20μl质粒交由上海生工测序。验证正确的e.colibl21(de3)/pet28a-psald。实施例4：苏氨酸醛缩酶基因ccald的重组大肠杆菌的构建一.重组质粒pet28a-ccald的获得获得ccald基因序列，测序后利用ndei和xhoi限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体pet28a进行连接，构建表达载体pet28a-ccald。二.重组质粒转化大肠杆菌e.colibl21(de3)在每管的100μle.colibl21(de3)感受态细胞悬液中加入10μlpcr产物，轻轻混匀，冰浴中静置30min。转入42℃水浴中，热击90s。快速转移至冰浴中冷却3min。每管中加入700μllb液体培养基，37℃100rpm摇床温育培养1h。培养后菌液3,000rpm离心2min，弃上清700μl，剩余菌液混匀后涂布到含有50μg·ml-1硫酸卡那霉素的lb平板上，37℃倒置培养过夜。阳性克隆的选择：挑取4个克隆，转接装有5ml的含有50μg·ml-1硫酸卡那霉素的lb培养基中，37℃培养8h，利用质粒提取试剂盒mini-plasmidrapidisolationkit(北京博大泰克生物基因技术有限公司)提取质粒。取出20μl质粒交由上海生工测序。验证正确的e.colibl21(de3)/pet28a-ccald。实施例5：表达突变体psald(s182y)、psald(h94p)、psald(n17q)、psald(t212i)基因的扩增合成两端引物，序列分别如seqidno：11、seqidno：12、seqidno：13、seqidno：14、seqidno：15、seqidno：16、seqidno：17、seqidno：18所示。：采用pcr的方法，将突变引入，具体方法如下：pcr反应体系：ddh2o22μl，premixprimestar25μl，上游引物(20μm)1μl，下游引物(20μm)1μl，质粒dna1μl。pcr反应：98℃预变性30s；98℃30s，55℃30s，72℃7min，进行30个循环；72℃延伸10min。以psald为模板，以引物进行pcr反应，获得突变的全长基因。利用3sspinagarosegeldnapurificationkit(上海申能博彩生物科技有限公司)纯化dna片断。纯化后的基因pcr产物用dpni酶消化模板：酶切体系：pcr产物30μl，10×buffer5μl，dpni1.5μl，ddh2o将体系补足50μl。酶切2h后利用3sspinagarosegeldnapurificationkit(上海申能博彩生物科技有限公司)纯化dna片断。pcr反应液用0.9％琼脂糖凝胶电泳检测并切胶回收纯化该片段，得到苏氨酸醛缩酶突变体基因psald(s182y)、psald(h94p)、psald(n17q)、psald(t212i)。实施例6：表达突变体ccald(a178y)、ccald(h91s)基因的扩增合成两端引物，序列分别如seqidno：84、seqidno：85、seqidno：86、seqidno：87所示。采用pcr的方法，将突变引入，具体方法如下：pcr反应体系：ddh2o22μl，premixprimestar25μl，上游引物(20μm)1μl，下游引物(20μm)1μl，质粒dna1μl。pcr反应：98℃预变性30s；98℃30s，55℃30s，72℃7min，进行30个循环；72℃延伸10min。以ccald为模板，以引物进行pcr反应，获得突变的全长基因。利用3sspinagarosegeldnapurificationkit(上海申能博彩生物科技有限公司)纯化dna片断。纯化后的基因pcr产物用dpni酶消化模板：酶切体系：pcr产物30μl，10×buffer5μl，dpni1.5μl，ddh2o将体系补足50μl。酶切2h后利用3sspinagarosegeldnapurificationkit(上海申能博彩生物科技有限公司)纯化dna片断。pcr反应液用0.9％琼脂糖凝胶电泳检测并切胶回收纯化该片段，得到苏氨酸醛缩酶突变体基因ccald(a178y)、ccald(h91s)。实施例7：表达突变体s182y、h94p、n17q、t212i的重组大肠杆菌的构建一.重组质粒pet28a-psald(s182y)、pet28a-psald(h94p)、pet28a-psald(n17q)、pet28a-psald(t212i)的获得获得突变体基因序列，测序后利用ndei和xhoi限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体pet28a进行连接，构建表达载体pet28a-psald(s182y)、pet28a-psald(h94p)、pet28a-psald(n17q)、pet28a-psald(t212i)。二.重组质粒转化大肠杆菌e.colibl21(de3)在每管的100μle.colibl21(de3)感受态细胞悬液中加入10μlpcr产物，轻轻混匀，冰浴中静置30min。转入42℃水浴中，热击90s。快速转移至冰浴中冷却3min。每管中加入700μllb液体培养基，37℃100rpm摇床温育培养1h。培养后菌液3，000rpm离心2min，弃上清700μl，剩余菌液混匀后涂布到含有50μg·ml-1硫酸卡那霉素的lb平板上，37℃倒置培养过夜。阳性克隆的选择：挑取4个克隆，转接装有5ml的含有50μg·ml-1硫酸卡那霉素的lb培养基中，37℃培养8h，利用质粒提取试剂盒mini-plasmidrapidisolationkit(北京博大泰克生物基因技术有限公司)提取质粒。取出20μl质粒交由上海生工测序。验证正确的e.colibl21(de3)/pet28a-psald(s182y)、e.colibl21(de3)/pet28a-psald(h94p)、e.colibl21(de3)/pet28a-psald(n17q)、e.colibl21(de3)/pet28a-psald(t212i)表达的苏氨酸醛缩酶psald的氨基酸序列，相对于seqidno:2所示的序列，第182位的丝氨酸突变成了酪氨酸、第94位的组氨酸突变成了脯氨酸、第17位的天冬酰胺突变成了谷氨酰胺、第212位的苏氨酸突变成了异亮氨酸。实施例8：表达突变体a178y、h91s的重组大肠杆菌的构建一.重组质粒pet28a-ccald(a178y)、pet28a-ccald(h91s)的获得获得突变体基因序列，测序后利用ndei和xhoi限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体pet28a进行连接，构建表达载体pet28a-ccald(a178y)、pet28a-ccald(h91s)。二.重组质粒转化大肠杆菌e.colibl21(de3)在每管的100μle.colibl21(de3)感受态细胞悬液中加入10μlpcr产物，轻轻混匀，冰浴中静置30min。转入42℃水浴中，热击90s。快速转移至冰浴中冷却3min。每管中加入700μllb液体培养基，37℃100rpm摇床温育培养1h。培养后菌液3，000rpm离心2min，弃上清700μl，剩余菌液混匀后涂布到含有50μg·ml-1硫酸卡那霉素的lb平板上，37℃倒置培养过夜。阳性克隆的选择：挑取4个克隆，转接装有5ml的含有50μg·ml-1硫酸卡那霉素的lb培养基中，37℃培养8h，利用质粒提取试剂盒mini-plasmidrapidisolationkit(北京博大泰克生物基因技术有限公司)提取质粒。取出20μl质粒交由上海生工测序。验证正确的e.colibl21(de3)/pet28a-ccald(a178y)、e.colibl21(de3)/pet28a-ccald(h91s)表达的苏氨酸醛缩酶ccald的氨基酸序列，相对于seqidno:19所示的序列，第178位的丙氨酸突变成了酪氨酸、第91位的组氨酸突变成了丝氨酸。实施例9：表达突变体s182y、h94p、n17q、t212i的重组枯草芽孢杆菌的构建一.重组质粒phcmc04-psald(s182y)、phcmc04-psald(h94p)、phcmc04-psald(n17q)、phcmc04-psald(t212i)的获得获得突变体基因序列，测序后利用kpni和smai限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体phcmc04进行连接，构建表达载体phcmc04-psald(s182y)、phcmc04-psald(h94p)、phcmc04-psald(n17q)、phcmc04-psald(t212i)。二.重组质粒转化枯草芽孢杆菌bacillussubtilissck6将枯草芽孢杆菌sck6划含有氨苄青霉素抗性的t2培养基固体平板，挑取单菌落接种于5mllb液体培养基中，37℃振荡培养过夜，取500μl培养物转接至10mllb液体培养基中(od600稀释为1左右)，加入终浓度1％(w/v)木糖，37℃振荡培养2小时，加入终浓度10％的甘油，按每管500μl分装后于-70℃冻存，所得即为感受态细胞。每500μl感受态细胞加入1μg质粒dna，37℃振荡培养1.5小时，涂氨苄青霉素抗性t2平板，37℃静置过夜后检查转化情况。阳性克隆的选择：从转化平板中挑取单菌落接种于5mllb液体培养基中，37℃振荡培养过夜，取3ml培养物离心收集菌体，用150μlte重悬，加入溶菌酶后于37℃温育1小时，加入终浓度0.5％(w/v)sds和蛋白酶k，颠倒混匀后37℃温育至混合液澄清，加入300μl溶液te，颠倒混匀后离心收集上清，使用omega小量质粒提取试剂盒中的硅基质柱吸附纯化，用50μlte洗脱得到质粒dna。取出20μl质粒交由上海生工测序。验证正确的表达的bacillussubtilissck6/phcmc04-psald(s182y)、bacillussubtilissck6/phcmc04-psald(h94p)、bacillussubtilissck6/phcmc04-psald(n17q)、bacillussubtilissck6/phcmc04-psald(t212i)苏氨酸醛缩酶psald的氨基酸序列，相对于seqidno:2所示的序列，第182位的丝氨酸突变成了酪氨酸、第94位的组氨酸突变成了脯氨酸、第17位的天冬酰胺突变成了谷氨酰胺、第212位的苏氨酸突变成了异亮氨酸。实施例10：表达突变体s182y、h94p、n17q、t212i的重组谷氨酸棒杆菌的构建一.重组质粒puc19-psald(s182y)、puc19-psald(h94p)、puc19-psald(n17q)、puc19-psald(t212i)的获得获得突变体基因序列，测序后利用hindi和bamhi限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体puc19进行连接，构建表达载体puc19-psald(s182y)、puc19-psald(h94p)、puc19-psald(n17q)、puc19-psald(t212i)。二.重组质粒转化谷氨酸棒杆菌c.glutamicumtk260251挑取新鲜的平皿的c.glutamicumtk260251单菌落接入0.5％葡萄糖的2ml液体lb培养基中，30℃，转速为250r/min的旋转式摇床上培养12-14h。按1％左右的接种量转接入含3％甘氨酸和0.1％tween-80的50ml液体lb培养基中，在30℃培养，直到细胞od600达到0.4。细胞培养结束后先将菌液冰浴15min，然后离心收集菌体(5500g，20min，4℃)。用250ml冰的缓冲液(10％w/vglycerol，8mmol/l，tris，ph7.4)悬浮菌体，然后离心收集菌体(4℃，5500r/min，20min)。用100ml预冷的10％的甘油洗涤菌体4次。最后用0.2ml10％甘油重悬细胞，用1.5ml离心管分装，每管50μl。处理好的细胞可直接用于电转化或于-70℃冰箱保存待用。电转化：取50μl谷氨酸棒杆菌感受态细胞冻融1次，加5μl质粒dna至50μl感受态细胞混匀，将混合液转移至0.2mm电转杯中，冰浴5min打开电转仪，设置参数:1.5-3.0kv，200q，25μf。擦干电转杯外的冷凝水，将电转杯放进电击仪，启动对细胞的电脉冲。脉冲结束后，尽可能快地取出电转杯，加入1mlsoc培养基。混匀后转入ep管中，于30℃静置培养1h。阳性克隆的选择：取100-300μl菌液涂布到含10μg/ml的氨苄青霉素抗性平板上30℃倒置培养，待长出菌落后，挑取单菌落接种于20μg/ml的3％甘氨酸的lb培养基，30℃培养过夜，提质粒酶切验证。取出20μl质粒交由上海生工测序。验证正确的c.glutamicumtk260251puc19-psald(s182y)、puc19-psald(h94p)、puc19-psald(n17q)、puc19-psald(t212i)表达的苏氨酸醛缩酶psald的氨基酸序列，相对于seqidno:2所示的序列，第182位的丝氨酸突变成了酪氨酸、第94位的组氨酸突变成了脯氨酸、第17位的天冬酰胺突变成了谷氨酰胺、第212位的苏氨酸突变成了异亮氨酸。实施例11：表达突变体s182y、h94p、n17q、t212i的重组酿酒酵母的构建一.重组质粒pesc-ura-psald(s182y)、pesc-ura-psald(h94p)、pesc-ura-psald(n17q)、pesc-ura-psald(t212i)的获得获得突变体基因序列，测序后利用bamhi和kpni限制性内切酶(takara)对扩增片段进行处理，并利用t4dna连接酶(takara)将该片段同用相同的限制性内切酶处理的商业化载体pesc-ura进行连接，构建表达载体pesc-ura-psald(s182y)、pesc-ura-psald(h94p)、pesc-ura-psald(n17q)、pesc-ura-psald(t212i)。二.重组质粒转化酿酒酵母菌saccharomycescerevisiaeyph501将酿酒酵母yph501感受态细胞冰浴解冻，放置备用；取1μg的重组载体pesc-ura-psald(s182y)、pesc-ura-psald(h94p)、pesc-ura-psald(n17q)、pesc-ura-psald(t212i)，与100μg的线性鲑鱼精加入至融化好的感受态细胞中；加入300μl的lte/peg-3350(40％)，上下颠倒混和均匀；30℃环境培养30min；42℃热击15min；加入500μl预热的ypd液体培养基，30℃培养2h；将上述菌液按梯度涂布到尿嘧啶缺陷型sd-u平板培养基上，30℃培养2～3d。转化后将菌液涂布到含有氨苄青霉素抗生素的ypd培养基上进行筛选，获得阳性转化子。将空载体paur123按同种方法转化至含有空载体pesc-ura的酵母感受态细胞。对转化子进行菌落pcr验证，体系配制如2.4.3，程序如下：94℃，5min；94℃，40s；58℃，50s；72℃，2min；②rep30；72℃，10min；hold，4℃。pcr结束后，用1％的琼脂糖凝胶电泳检测扩增结果。阳性克隆的选择：取100-300μl菌液涂布到含10μg/ml的cm抗性平板上30℃倒置培养，待长出菌落后，挑取单菌落接种于20μg/ml的3％甘氨酸的lb培养基，30℃培养过夜，提质粒酶切验证。取出20μl质粒交由上海生工测序。验证正确的yph501/pesc-psald(s182y)、yph501/pesc-psald(h94p)、yph501/pesc-psald(n17q)、yph501/pesc-psald(t212i)表达的苏氨酸醛缩酶psald的氨基酸序列，相对于seqidno:2所示的序列，第182位的丝氨酸突变成了酪氨酸、第94位的组氨酸突变成了脯氨酸、第17位的天冬酰胺突变成了谷氨酰胺、第212位的苏氨酸突变成了异亮氨酸。实施例12：重组大肠杆菌的诱导表达培养获得的e.colibl21(de3)/pet28a-psald(s182y)、e.colibl21(de3)/pet28a-psald(h94p)、e.colibl21(de3)/pet28a-psald(n17q)、e.colibl21(de3)/pet28a-psald(t212i)、e.colibl21(de3)/pet28a-ccald(a178y)、e.colibl21(de3)/pet28a-ccald(h91s)和对照组bl21(pet-28a空载体转化子)作为阴性对照，接种于5ml含50μg·ml-1硫酸卡那霉素的lb液体培养基中，于37℃，200rpm振荡培养过夜。取10ml培养液转接于1l含50μg·ml-1硫酸卡那霉素的lb液体培养基中，于37℃，200rpm振荡培养至od600约为0.6-0.8。向培养物中加入终浓度为0.5mm的异丙基-β-d-硫代半乳糖苷，在25℃下进行诱导培养12h。收集菌体，溶解于20mm磷酸缓冲液(ph7.0)中，超声破碎30min，工作时间2s，间歇时间3s。将破碎后的菌体12,000rpm，40min。分别取上清和沉淀，sds-page检测蛋白表达。实施例13：重组枯草芽孢杆菌的诱导表达培养获得的含4种基因的枯草芽孢杆菌和对照组sck6(phcmc04空载体转化子)作为阴性对照，接种于装有5mllb液体培养基的试管中，37℃振荡培养过夜。次日各培养物分别取2ml转接至装有50ml5yc液体培养基的250ml摇瓶中，33℃振荡培养8小时后取样离心收集上清并加入终浓度1mm的异丙基-β-d-硫代半乳糖苷诱导，继续培养至48小时，期间每8小时取样离心收集培养物上清。上述摇瓶实验中的培养基均含有25μg/ml氯霉素。取20μl培养物上清加入5μl4×上样缓冲液，混匀后沸水浴处理5min，上样量20μl，浓缩胶浓度5％，分离胶浓度17％，电泳时内槽为负极缓冲液，外槽为正极缓冲液。实施例14：重组谷氨酸棒杆菌的诱导表达培养获得的含4种基因的谷氨酸棒杆菌和对照组yph501(puc19空载体)接种于20ml种子培养基中，在30℃，200r/min的条件下在摇床上进行培养。当菌液的od600＝1.0时，按5％接种量将种子液接入发酵培养基中，在30℃，200r/min的条件下在摇床上进行培养。当od600＝3.5时，加入异丙基-β-d-硫代半乳糖苷，使培养基中的异丙基-β-d-硫代半乳糖苷浓度达到1mm，然后在28℃，200r/min的条件下继续培养36h。30ml培养液于小型离心，4℃下12000r/min离心10min。细胞重悬于30ml醋酸钠缓冲液(50mmol/l，ph6.0)中。细胞悬液超声处理30min。4℃，12000r/min离心30min除去细胞碎片。取40μl上清液加入9μl的5xsds-page电泳上样缓冲液，并进行充分混合。100℃下煮沸10min，样品保存于-20℃直到进行蛋白电泳分析。实施例15：重组酿酒酵母菌的诱导表达培养获得的含4种基因的酵母工程菌和对照组酿酒酵母yph501(pesc-ura空载体)同时接种于ypd液体培养基中，过夜培养后添加菌液至新培养基调节od600nm值为0.5，30℃振荡培养3d，加入半乳糖诱导表达1d；收集酵母培养液，6000rpm，4℃离心10min，弃上清液收集菌体沉淀；用液氮冻融菌体沉淀，将细胞裂解液加入其中，混合均匀；用细胞粉碎仪粉碎上述菌液，条件为25khz，10s工作/间隔，时间为20min；4℃离心后收集上清，即为酿酒酵母粗酶液样品。实施例16：重组蛋白s182y、h94p、n17q、t212i、a178y、h91s的纯化(重组大肠杆菌e.colibl21(de3)体系表达)利用his-traphpaffinitycolumn纯化重组蛋白s182y、h94p、n17q、t212i、a178y、h91s：挑取阳性克隆单菌落接种于5ml含50μg·ml-1硫酸卡那霉素的lb液体培养基中，于37℃，200rpm振荡培养过夜。取10ml培养液转接于1l含50μg·ml-1硫酸卡那霉素的lb液体培养基中，于37℃，200rpm振荡培养至od600约为0.6–0.8。向培养物中加入诱导物异丙基-β-d-硫代半乳糖苷0.5mm，在培养温度25℃下进行诱导培养12h。培养后的重组大肠杆菌细胞12,000rpm离心10min并用生理盐水洗涤三次后收集。称取2g湿菌体，加入适量20mmtris、150mmnacl(ph8.0)缓冲液悬浮细胞，于冰浴中进行超声破碎(工作2s，间隔3s，工作时间30min)。4℃条件下12,000rpm离心30min收集上清液作为粗酶液。利用ge公司生产的his-trap亲和层析对粗酶液进行纯化，纯酶液超滤脱盐后用于特异性转化2-/3-/4-位取代苯甲醛。实施例17：突变前后比酶活测定突变体酶活力的测定：苏氨酸醛缩酶在辅因子(5-磷酸吡哆醛，plp)存在下催化苏氨酸裂解成甘氨酸和乙醛，乙醛在nadh的存在下能够被乙醇脱氢酶还原，nad+的生成会引起340nm处吸光值的降低，因此可以通过测定反应过程中340nm处的吸光值的变化来衡量苯基丝氨酸醛缩酶的活力。酶活测定的标准条件：总反应体积250μl，分别加入100mmhepes-naoh缓冲液(ph8.0)，50umplp，50mml-苏氨酸，0.5mmnadh，30u乙醇脱氢酶，30℃保温2min，加入适量纯酶液后开始扫描340nm处吸光值的变化。蛋白质含量测定采用bradford法，以牛血清白蛋白bsa为标准品。酶活力定义为：在上述条件下，每分钟催化1μmol的nadh的酶量定义为一个单位u。酶活的计算公式为：酶活(u)＝ew×v×103/(6220×0.3)比活的计算公式：比活(u·mg-1)＝酶活(u)/蛋白量(mg)其中，ew：1min内340nm处吸光度的变化；v：反应液的体积(ml)；6220：摩尔消光系数(lmol-1cm-1)；0.3：光程距离(cm)。表1不同突变体的比酶活通过对野生酶进行定点突变，得到的突变体比酶活相较于野生酶比酶活有了明显提高。实施例18：催化4-甲砜基-苯甲醛与甘氨酸生成l-syn-β-4-甲砜基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，4-甲砜基苯甲醛(100mm)，plp(240μm)，2mg酶(s182y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。实施例19：hplc检测l-syn-β-4-甲砜基苯基丝氨酸将反应体系用opa/nac进行衍生，用安捷伦1260进行检测。液相条件：purospherstarrp18(250mm，5um)c18柱bufferph8.0(50mmkh2po4)/ch3cn＝81:19，流速0.8ml/min，运行时间20min，温度40℃。通过对实施例18的反应检测，得到hplc分析收率82％，ee值>99％，de值70％，如图1所示。实施例20：1h-nmr检测l-syn-β-4-甲砜基苯基丝氨酸将终止的反应液在5000rmp离心，取上清液通过0.2μm滤膜过滤，滤液在旋转蒸发仪上设定温度55度，压力40mbar旋干，收集的固体送核磁共振波谱仪检测。氘代溶剂为氘水，检测仪器为brukeravanceiii400mhz核磁共振波谱仪。核磁验证产品结构正确，其非对映异构体符合hplc的检测结果，如图2所示。实施例21：催化3-溴苯甲醛与甘氨酸生成l-syn-β-3-溴苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，3-溴苯甲醛(100mm)，plp(240μm)，2mg酶(s182y))。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率81％，ee值>99％，de值90％。实施例22：催化4-溴苯甲醛与甘氨酸生成l-syn-β-4-溴苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，4-溴苯甲醛(100mm)，plp(240μm)，2mg酶(s182y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率79％，ee值>99％，de值70％。实施例23：催化2-硝基苯甲醛与甘氨酸生成l-syn-β-2-硝基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，2-硝基苯甲醛(100mm)，plp(240μm)，2mg酶(a178y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率99％，ee值>99％，de值85％。实施例24：催化3-硝基苯甲醛与甘氨酸生成l-syn-β-3-硝基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，3-硝基苯甲醛(100mm)，plp(240μm)，2mg酶(a178y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率83％，ee值>99％，de值77％。实施例25：催化4-硝基苯甲醛与甘氨酸生成l-syn-β-4-硝基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，4-硝基苯甲醛(100mm)，plp(240μm)，2mg酶(a178y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率89％，ee值>99％，de值90％。实施例26：催化4-甲砜基苯甲醛与甘氨酸生成l-syn-β-4-甲砜基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，4-甲砜基苯甲醛(100mm)，plp(240μm)，2mg酶(a178y)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率91％，ee值>99％，de值92％。实施例27：催化3-羟基苯甲醛与甘氨酸生成l-syn-β-3-羟基苯基丝氨酸反应体系2mlph8.0：kh2po4(50mm)缓冲液，甘氨酸(1m)，3-羟基苯甲醛(100mm)，plp(240μm)，2mg酶(h91s)。30℃200rpm反应1h，加入1mhcl调节ph＝2终止反应。hplc分析收率76％，ee值>99％，de值71％。序列表<110>王喆明杭州法哲罗生物科技有限公司<120>苏氨酸醛缩酶、突变体及其在制备取代苯丝氨酸衍生物中的应用<130>1<160>88<170>siposequencelisting1.0<210>1<211>1074<212>dna<213>假单胞菌苏氨酸醛缩酶基因(人工序列)<400>1atgaatggtgaaaccagccgtccgccggcactgggttttagcagcgataatattgcaggt60gcctctccggaagtggcacaggcactggttaaacattcttcaggtcaggcaggtccgtat120ggtaccgatgaactgaccgcacaggttaaacgtaaattttgtgaaatttttgaacgtgat180gttgaagtgtttctggttccgaccggcaccgccgcaaatgcactgtgtctgagcgcaatg240accccgccgtggggtaatatttattgtcatccggcaagccatattaataatgatgaatgt300ggtgcaccggaattcttttctaatggtgcaaaactgatgaccgttgatggtccggccgca360aaactggatattgttcgtctgcgcgaacgcacccgtgaaaaagttggtgatgttcatacc420acacagccggcatgtgttagtattacccaggcgaccgaagttggtagcatttataccctg480gatgaaattgaagcgattggtgatgtttgtaaaagcagtagcctgggtctgcatatggat540ggtagccgttttgcgaatgcactggtgagcctgggttgcagtccggcagaaatgacctgg600aaagcgggcgttgatgcgctgtcttttggcgcaaccaaaaatggtgttctggcagcagaa660gcaattgttctgtttaataccagcctggcaaccgaaatgagctatcgtcgtaaacgcgca720ggtcatctgagcagtaaaatgcgttttctgagtgcacagattgatgcctatctgaccgat780gatctgtggctgcgtaatgcgcgtaaagccaatgccgcagcgcagcgtctggcgcagggt840ctggaaggtctgggtggtgttgaagtgctgggtggtaccgaagcaaatattctgttttgt900cgcctggatagcgccatgattgatgccctgctgaaagccggttttggtttttatcatgat960cgttggggtccgaatgtggttcgttttgttaccagctttgccaccaccgcagaagatgtg1020gatcatctgctgaatcaggttcgtctggcagcagatcgcacccaggaacgttaa1074<210>2<211>357<212>prt<213>假单胞菌苏氨酸醛缩酶氨基酸(人工序列)<400>2metasnglygluthrserargproproalaleuglypheserserasp151015asnilealaglyalaserprogluvalalaglnalaleuvallyshis202530serserglyglnalaglyprotyrglythraspgluleuthralagln354045vallysarglysphecysgluilephegluargaspvalgluvalphe505560leuvalprothrglythralaalaasnalaleucysleuseralamet65707580thrproprotrpglyasniletyrcyshisproalaserhisileasn859095asnaspglucysglyalaprogluphepheserasnglyalalysleu100105110metthrvalaspglyproalaalalysleuaspilevalargleuarg115120125gluargthrargglulysvalglyaspvalhisthrthrglnproala130135140cysvalserilethrglnalathrgluvalglyseriletyrthrleu145150155160aspgluileglualaileglyaspvalcyslysserserserleugly165170175leuhismetaspglyserargphealaasnalaleuvalserleugly180185190cysserproalaglumetthrtrplysalaglyvalaspalaleuser195200205pheglyalathrlysasnglyvalleualaalaglualailevalleu210215220pheasnthrserleualathrglumetsertyrargarglysargala225230235240glyhisleuserserlysmetargpheleuseralaglnileaspala245250255tyrleuthraspaspleutrpleuargasnalaarglysalaasnala260265270alaalaglnargleualaglnglyleugluglyleuglyglyvalglu275280285valleuglyglythrglualaasnileleuphecysargleuaspser290295300alametileaspalaleuleulysalaglypheglyphetyrhisasp305310315320argtrpglyproasnvalvalargphevalthrserphealathrthr325330335alagluaspvalasphisleuleuasnglnvalargleualaalaasp340345350argthrglngluarg355<210>3<211>1074<212>dna<213>假单胞菌苏氨酸醛缩酶基因突变(人工序列)<400>3atgaatggtgaaaccagccgtccgccggcactgggttttagcagcgataatattgcaggt60gcctctccggaagtggcacaggcactggttaaacattcttcaggtcaggcaggtccgtat120ggtaccgatgaactgaccgcacaggttaaacgtaaattttgtgaaatttttgaacgtgat180gttgaagtgtttctggttccgaccggcaccgccgcaaatgcactgtgtctgagcgcaatg240accccgccgtggggtaatatttattgtcatccggcaagccatattaataatgatgaatgt300ggtgcaccggaattcttttctaatggtgcaaaactgatgaccgttgatggtccggccgca360aaactggatattgttcgtctgcgcgaacgcacccgtgaaaaagttggtgatgttcatacc420acacagccggcatgtgttagtattacccaggcgaccgaagttggtagcatttataccctg480gatgaaattgaagcgattggtgatgtttgtaaaagcagtagcctgggtctgcatatggat540ggttaccgttttgcgaatgcactggtgagcctgggttgcagtccggcagaaatgacctgg600aaagcgggcgttgatgcgctgtcttttggcgcaaccaaaaatggtgttctggcagcagaa660gcaattgttctgtttaataccagcctggcaaccgaaatgagctatcgtcgtaaacgcgca720ggtcatctgagcagtaaaatgcgttttctgagtgcacagattgatgcctatctgaccgat780gatctgtggctgcgtaatgcgcgtaaagccaatgccgcagcgcagcgtctggcgcagggt840ctggaaggtctgggtggtgttgaagtgctgggtggtaccgaagcaaatattctgttttgt900cgcctggatagcgccatgattgatgccctgctgaaagccggttttggtttttatcatgat960cgttggggtccgaatgtggttcgttttgttaccagctttgccaccaccgcagaagatgtg1020gatcatctgctgaatcaggttcgtctggcagcagatcgcacccaggaacgttaa1074<210>4<211>357<212>prt<213>假单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>4metasnglygluthrserargproproalaleuglypheserserasp151015asnilealaglyalaserprogluvalalaglnalaleuvallyshis202530serserglyglnalaglyprotyrglythraspgluleuthralagln354045vallysarglysphecysgluilephegluargaspvalgluvalphe505560leuvalprothrglythralaalaasnalaleucysleuseralamet65707580thrproprotrpglyasniletyrcyshisproalaserhisileasn859095asnaspglucysglyalaprogluphepheserasnglyalalysleu100105110metthrvalaspglyproalaalalysleuaspilevalargleuarg115120125gluargthrargglulysvalglyaspvalhisthrthrglnproala130135140cysvalserilethrglnalathrgluvalglyseriletyrthrleu145150155160aspgluileglualaileglyaspvalcyslysserserserleugly165170175leuhismetaspglytyrargphealaasnalaleuvalserleugly180185190cysserproalaglumetthrtrplysalaglyvalaspalaleuser195200205pheglyalathrlysasnglyvalleualaalaglualailevalleu210215220pheasnthrserleualathrglumetsertyrargarglysargala225230235240glyhisleuserserlysmetargpheleuseralaglnileaspala245250255tyrleuthraspaspleutrpleuargasnalaarglysalaasnala260265270alaalaglnargleualaglnglyleugluglyleuglyglyvalglu275280285valleuglyglythrglualaasnileleuphecysargleuaspser290295300alametileaspalaleuleulysalaglypheglyphetyrhisasp305310315320argtrpglyproasnvalvalargphevalthrserphealathrthr325330335alagluaspvalasphisleuleuasnglnvalargleualaalaasp340345350argthrglngluarg355<210>5<211>1074<212>dna<213>假单胞菌苏氨酸醛缩酶基因突变(人工序列)<400>5atgaatggtgaaaccagccgtccgccggcactgggttttagcagcgataatattgcaggt60gcctctccggaagtggcacaggcactggttaaacattcttcaggtcaggcaggtccgtat120ggtaccgatgaactgaccgcacaggttaaacgtaaattttgtgaaatttttgaacgtgat180gttgaagtgtttctggttccgaccggcaccgccgcaaatgcactgtgtctgagcgcaatg240accccgccgtggggtaatatttattgtcatccggcaagccctattaataatgatgaatgt300ggtgcaccggaattcttttctaatggtgcaaaactgatgaccgttgatggtccggccgca360aaactggatattgttcgtctgcgcgaacgcacccgtgaaaaagttggtgatgttcatacc420acacagccggcatgtgttagtattacccaggcgaccgaagttggtagcatttataccctg480gatgaaattgaagcgattggtgatgtttgtaaaagcagtagcctgggtctgcatatggat540ggtagccgttttgcgaatgcactggtgagcctgggttgcagtccggcagaaatgacctgg600aaagcgggcgttgatgcgctgtcttttggcgcaaccaaaaatggtgttctggcagcagaa660gcaattgttctgtttaataccagcctggcaaccgaaatgagctatcgtcgtaaacgcgca720ggtcatctgagcagtaaaatgcgttttctgagtgcacagattgatgcctatctgaccgat780gatctgtggctgcgtaatgcgcgtaaagccaatgccgcagcgcagcgtctggcgcagggt840ctggaaggtctgggtggtgttgaagtgctgggtggtaccgaagcaaatattctgttttgt900cgcctggatagcgccatgattgatgccctgctgaaagccggttttggtttttatcatgat960cgttggggtccgaatgtggttcgttttgttaccagctttgccaccaccgcagaagatgtg1020gatcatctgctgaatcaggttcgtctggcagcagatcgcacccaggaacgttaa1074<210>6<211>357<212>prt<213>假单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>6metasnglygluthrserargproproalaleuglypheserserasp151015asnilealaglyalaserprogluvalalaglnalaleuvallyshis202530serserglyglnalaglyprotyrglythraspgluleuthralagln354045vallysarglysphecysgluilephegluargaspvalgluvalphe505560leuvalprothrglythralaalaasnalaleucysleuseralamet65707580thrproprotrpglyasniletyrcyshisproalaserproileasn859095asnaspglucysglyalaprogluphepheserasnglyalalysleu100105110metthrvalaspglyproalaalalysleuaspilevalargleuarg115120125gluargthrargglulysvalglyaspvalhisthrthrglnproala130135140cysvalserilethrglnalathrgluvalglyseriletyrthrleu145150155160aspgluileglualaileglyaspvalcyslysserserserleugly165170175leuhismetaspglyserargphealaasnalaleuvalserleugly180185190cysserproalaglumetthrtrplysalaglyvalaspalaleuser195200205pheglyalathrlysasnglyvalleualaalaglualailevalleu210215220pheasnthrserleualathrglumetsertyrargarglysargala225230235240glyhisleuserserlysmetargpheleuseralaglnileaspala245250255tyrleuthraspaspleutrpleuargasnalaarglysalaasnala260265270alaalaglnargleualaglnglyleugluglyleuglyglyvalglu275280285valleuglyglythrglualaasnileleuphecysargleuaspser290295300alametileaspalaleuleulysalaglypheglyphetyrhisasp305310315320argtrpglyproasnvalvalargphevalthrserphealathrthr325330335alagluaspvalasphisleuleuasnglnvalargleualaalaasp340345350argthrglngluarg355<210>7<211>1074<212>dna<213>假单胞菌苏氨酸醛缩酶基因突变(人工序列)<400>7atgaatggtgaaaccagccgtccgccggcactgggttttagcagcgatcagattgcaggt60gcctctccggaagtggcacaggcactggttaaacattcttcaggtcaggcaggtccgtat120ggtaccgatgaactgaccgcacaggttaaacgtaaattttgtgaaatttttgaacgtgat180gttgaagtgtttctggttccgaccggcaccgccgcaaatgcactgtgtctgagcgcaatg240accccgccgtggggtaatatttattgtcatccggcaagccatattaataatgatgaatgt300ggtgcaccggaattcttttctaatggtgcaaaactgatgaccgttgatggtccggccgca360aaactggatattgttcgtctgcgcgaacgcacccgtgaaaaagttggtgatgttcatacc420acacagccggcatgtgttagtattacccaggcgaccgaagttggtagcatttataccctg480gatgaaattgaagcgattggtgatgtttgtaaaagcagtagcctgggtctgcatatggat540ggtagccgttttgcgaatgcactggtgagcctgggttgcagtccggcagaaatgacctgg600aaagcgggcgttgatgcgctgtcttttggcgcaaccaaaaatggtgttctggcagcagaa660gcaattgttctgtttaataccagcctggcaaccgaaatgagctatcgtcgtaaacgcgca720ggtcatctgagcagtaaaatgcgttttctgagtgcacagattgatgcctatctgaccgat780gatctgtggctgcgtaatgcgcgtaaagccaatgccgcagcgcagcgtctggcgcagggt840ctggaaggtctgggtggtgttgaagtgctgggtggtaccgaagcaaatattctgttttgt900cgcctggatagcgccatgattgatgccctgctgaaagccggttttggtttttatcatgat960cgttggggtccgaatgtggttcgttttgttaccagctttgccaccaccgcagaagatgtg1020gatcatctgctgaatcaggttcgtctggcagcagatcgcacccaggaacgttaa1074<210>8<211>357<212>prt<213>假单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>8metasnglygluthrserargproproalaleuglypheserserasp151015glnilealaglyalaserprogluvalalaglnalaleuvallyshis202530serserglyglnalaglyprotyrglythraspgluleuthralagln354045vallysarglysphecysgluilephegluargaspvalgluvalphe505560leuvalprothrglythralaalaasnalaleucysleuseralamet65707580thrproprotrpglyasniletyrcyshisproalaserhisileasn859095asnaspglucysglyalaprogluphepheserasnglyalalysleu100105110metthrvalaspglyproalaalalysleuaspilevalargleuarg115120125gluargthrargglulysvalglyaspvalhisthrthrglnproala130135140cysvalserilethrglnalathrgluvalglyseriletyrthrleu145150155160aspgluileglualaileglyaspvalcyslysserserserleugly165170175leuhismetaspglyserargphealaasnalaleuvalserleugly180185190cysserproalaglumetthrtrplysalaglyvalaspalaleuser195200205pheglyalathrlysasnglyvalleualaalaglualailevalleu210215220pheasnthrserleualathrglumetsertyrargarglysargala225230235240glyhisleuserserlysmetargpheleuseralaglnileaspala245250255tyrleuthraspaspleutrpleuargasnalaarglysalaasnala260265270alaalaglnargleualaglnglyleugluglyleuglyglyvalglu275280285valleuglyglythrglualaasnileleuphecysargleuaspser290295300alametileaspalaleuleulysalaglypheglyphetyrhisasp305310315320argtrpglyproasnvalvalargphevalthrserphealathrthr325330335alagluaspvalasphisleuleuasnglnvalargleualaalaasp340345350argthrglngluarg355<210>9<211>1074<212>dna<213>假单胞菌苏氨酸醛缩酶基因突变(人工序列)<400>9atgaatggtgaaaccagccgtccgccggcactgggttttagcagcgataatattgcaggt60gcctctccggaagtggcacaggcactggttaaacattcttcaggtcaggcaggtccgtat120ggtaccgatgaactgaccgcacaggttaaacgtaaattttgtgaaatttttgaacgtgat180gttgaagtgtttctggttccgaccggcaccgccgcaaatgcactgtgtctgagcgcaatg240accccgccgtggggtaatatttattgtcatccggcaagccatattaataatgatgaatgt300ggtgcaccggaattcttttctaatggtgcaaaactgatgaccgttgatggtccggccgca360aaactggatattgttcgtctgcgcgaacgcacccgtgaaaaagttggtgatgttcatacc420acacagccggcatgtgttagtattacccaggcgaccgaagttggtagcatttataccctg480gatgaaattgaagcgattggtgatgtttgtaaaagcagtagcctgggtctgcatatggat540ggtagccgttttgcgaatgcactggtgagcctgggttgcagtccggcagaaatgacctgg600aaagcgggcgttgatgcgctgtcttttggcgcaatcaaaaatggtgttctggcagcagaa660gcaattgttctgtttaataccagcctggcaaccgaaatgagctatcgtcgtaaacgcgca720ggtcatctgagcagtaaaatgcgttttctgagtgcacagattgatgcctatctgaccgat780gatctgtggctgcgtaatgcgcgtaaagccaatgccgcagcgcagcgtctggcgcagggt840ctggaaggtctgggtggtgttgaagtgctgggtggtaccgaagcaaatattctgttttgt900cgcctggatagcgccatgattgatgccctgctgaaagccggttttggtttttatcatgat960cgttggggtccgaatgtggttcgttttgttaccagctttgccaccaccgcagaagatgtg1020gatcatctgctgaatcaggttcgtctggcagcagatcgcacccaggaacgttaa1074<210>10<211>357<212>prt<213>假单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>10metasnglygluthrserargproproalaleuglypheserserasp151015asnilealaglyalaserprogluvalalaglnalaleuvallyshis202530serserglyglnalaglyprotyrglythraspgluleuthralagln354045vallysarglysphecysgluilephegluargaspvalgluvalphe505560leuvalprothrglythralaalaasnalaleucysleuseralamet65707580thrproprotrpglyasniletyrcyshisproalaserhisileasn859095asnaspglucysglyalaprogluphepheserasnglyalalysleu100105110metthrvalaspglyproalaalalysleuaspilevalargleuarg115120125gluargthrargglulysvalglyaspvalhisthrthrglnproala130135140cysvalserilethrglnalathrgluvalglyseriletyrthrleu145150155160aspgluileglualaileglyaspvalcyslysserserserleugly165170175leuhismetaspglyserargphealaasnalaleuvalserleugly180185190cysserproalaglumetthrtrplysalaglyvalaspalaleuser195200205pheglyalailelysasnglyvalleualaalaglualailevalleu210215220pheasnthrserleualathrglumetsertyrargarglysargala225230235240glyhisleuserserlysmetargpheleuseralaglnileaspala245250255tyrleuthraspaspleutrpleuargasnalaarglysalaasnala260265270alaalaglnargleualaglnglyleugluglyleuglyglyvalglu275280285valleuglyglythrglualaasnileleuphecysargleuaspser290295300alametileaspalaleuleulysalaglypheglyphetyrhisasp305310315320argtrpglyproasnvalvalar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erhiscysaspargglyaspglutyrleucysglygln65707580glnalahisasntyrlysphegluglyglyglyalaalavalleugly859095serileglnproglnproleuthrasnglnproaspglyserileleu100105110leuseraspileglualaalailelysproaspaspphehispheala115120125argthrargleuleuserleugluasnthrileglyglylysvalleu130135140proglnsertyrleualaglualaglnalaleualapheasnlysarg145150155160leulysilehisleuaspglytyrargilealaasnalaalavalala165170175hisasnleuaspilealaaspilethrglntyrpheaspservalser180185190ilecysleuserlysglyleucysalaprovalglyserleuleuleu195200205glyaspgluargleuileasnlysalaargargtrparglysmetleu210215220glyglyglymetargglnalaglyileleualavalalaglygluile225230235240alaleuasngluglnvalserargleualavalasphisgluasnala245250255argtyrleualagluglnleusercysleuaspgluphegluvalasp260265270leuglygluvalglnthrasnmetleuphealaargvalvalglugly275280285valalaileasplysleualathrserleulysalaserglyileleu290295300ileserproglylysthrleuargmetvalthrhisalaaspilearg305310315320leugluaspileaspleupheileasplysleulysserleuleu325330335<210>73<211>335<212>prt<213>鲍希瓦氏菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>73metileasppheargseraspthrvalthrglnprothralaalamet151015argargalametalaaspalaglnvalglyaspaspvaltyrglyasp202530aspproservalasnargleuglualametalaalagluargphegly354045pheaspseralaleuphethrserserglythrglnalaasnleuleu505560alaleumetserhiscysaspargglyaspglutyrleucysglygln65707580glnalahisasntyrlysphegluglyglyglyalaalavalleugly859095serileglnproglnproleuthrasnglnproaspglyserileleu100105110leuseraspileglualaalailelysproaspaspphehispheala115120125argthrargleuleuserleugluasnthrileglyglylysvalleu130135140proglnsertyrleualaglualaglnalaleualapheasnlysarg145150155160leulysilehisleuaspglyalaargilealaasnalaalavalala165170175hisasnleuaspilealaaspilethrglntyrpheaspservalser180185190ilecysleuilelysglyleucysalaprovalglyserleuleuleu195200205glyaspgluargleuileasnlysalaargargtrparglysmetleu210215220glyglyglymetargglnalaglyileleualavalalaglygluile225230235240alaleuasngluglnvalserargleualavalasphisgluasnala245250255argtyrleualagluglnleusercysleuaspgluphegluvalasp260265270leuglygluvalglnthrasnmetleuphealaargvalvalglugly275280285valalaileasplysleualathrserleulysalaserglyileleu290295300ileserproglylysthrleuargmetvalthrhisalaaspilearg305310315320leugluaspileaspleupheileasplysleulysserleuleu325330335<210>74<211>339<212>prt<213>维龙气单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>74metargtyrileaspleuargseraspglnvalthrglnprothrasp151015alametargglnalametleuhisalagluvalglyaspaspvaltyr202530glygluaspproglyvalasnalaleuglualahisglyalaargleu354045leuglylysglnalaalaleuphevalproserglythrmetserasn505560leuleualavalmetserhiscysglnargglygluglyalaileleu65707580glyasnalaalahisiletyrargtyrglualaglnglyseralaval859095leuglyservalalaleuglnproleuprometglnargaspglythr100105110leualapheaspaspilelysalaalaleualaproaspaspalahis115120125phevalglnthrargleuilecysleugluasnthrhisasnglylys130135140valleuproleusertyrleuglnglumetglythrphevalalaglu145150155160argglyleulysleuhisleuaspglyalaargleupheasnalaala165170175valalasergluthrprovalalavalilealaalapropheaspser180185190ileserilecysleuserlysglyleuglyalaprovalglyserleu195200205leuvalglyserhisasppheilealaargalaargargleuarglys210215220metleuglyglyglymetargglnalaglyileleualaglnalagly225230235240leuphealaleugluglnhisvalthrargleualaaspasphisarg245250255argalalysargleualagluglyleualaalaleuproglyileglu260265270leuaspleuserleuvalglnserasnmetvalpheleuargleuarg275280285gluglygluseralaproleuleualaphemetlysgluargglyile290295300leupheserglytyrglygluleuargleuvalthrhisleuglnile305310315320asnaspaspaspileglugluvalileaspalaphethrglutyrleu325330335glyalaarg<210>75<211>339<212>prt<213>维龙气单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>75metargtyrileaspleuargseraspthrvalthrglnprothrasp151015alametargglnalametleuhisalagluvalglyaspaspvaltyr202530glygluaspproglyvalasnalaleuglualahisglyalaargleu354045leuglylysglnalaalaleuphevalproserglythrmetserasn505560leuleualavalmetserhiscysglnargglygluglyalaileleu65707580glyasnalaalatrpiletyrargtyrglualaglnglyseralaval859095leuglyservalalaleuglnproleuprometglnargaspglythr100105110leualapheaspaspilelysalaalaleualaproaspaspalahis115120125phevalglnthrargleuilecysleugluasnthrhisasnglylys130135140valleuproleusertyrleuglnglumetglythrphevalalaglu145150155160argglyleulysleuhisleuaspglyalaargleupheasnalaala165170175valalasergluthrprovalalavalilealaalapropheaspser180185190ileserilecysleuserlysglyleuglyalaprovalglyserleu195200205leuvalglyserhisasppheilealaargalaargargleuarglys210215220metleuglyglyglymetargglnalaglyileleualaglnalagly225230235240leuphealaleugluglnhisvalthrargleualaaspasphisarg245250255argalalysargleualagluglyleualaalaleuproglyileglu260265270leuaspleuserleuvalglnserasnmetvalpheleuargleuarg275280285gluglygluseralaproleuleualaphemetlysgluargglyile290295300leupheserglytyrglygluleuargleuvalthrhisleuglnile305310315320asnaspaspaspileglugluvalileaspalaphethrglutyrleu325330335glyalaarg<210>76<211>339<212>prt<213>维龙气单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>76metargtyrileaspleuargseraspthrvalthrglnprothrasp151015alametargglnalametleuhisalagluvalglyaspaspvaltyr202530glygluaspproglyvalasnalaleuglualahisglyalaargleu354045leuglylysglnalaalaleuphevalproserglythrmetserasn505560leuleualavalmetserhiscysglnargglygluglyalaileleu65707580glyasnalaalaproiletyrargtyrglualaglnglyseralaval859095leuglyservalalaleuglnproleuprometglnargaspglythr100105110leualapheaspaspilelysalaalaleualaproaspaspalahis115120125phevalglnthrargleuilecysleugluasnthrhisasnglylys130135140valleuproleusertyrleuglnglumetglythrphevalalaglu145150155160argglyleulysleuhisleuaspglyalaargleupheasnalaala165170175valalasergluthrprovalalavalilealaalapropheaspser180185190ileserilecysleuserlysglyleuglyalaprovalglyserleu195200205leuvalglyserhisasppheilealaargalaargargleuarglys210215220metleuglyglyglymetargglnalaglyileleualaglnalagly225230235240leuphealaleugluglnhisvalthrargleualaaspasphisarg245250255argalalysargleualagluglyleualaalaleuproglyileglu260265270leuaspleuserleuvalglnserasnmetvalpheleuargleuarg275280285gluglygluseralaproleuleualaphemetlysgluargglyile290295300leupheserglytyrglygluleuargleuvalthrhisleuglnile305310315320asnaspaspaspileglugluvalileaspalaphethrglutyrleu325330335glyalaarg<210>77<211>339<212>prt<213>维龙气单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>77metargtyrileaspleuargseraspthrvalthrglnprothrasp151015alametargglnalametleuhisalagluvalglyaspaspvaltyr202530glygluaspproglyvalasnalaleuglualahisglyalaargleu354045leuglylysglnalaalaleuphevalproserglythrmetserasn505560leuleualavalmetserhiscysglnargglygluglyalaileleu65707580glyasnalaalahisiletyrargtyrglualaglnglyseralaval859095leuglyservalalaleuglnproleuprometglnargaspglythr100105110leualapheaspaspilelysalaalaleualaproaspaspalahis115120125phevalglnthrargleuilecysleugluasnthrhisasnglylys130135140valleuproleusertyrleuglnglumetglythrphevalalaglu145150155160argglyleulysleuhisleuaspglytyrargleupheasnalaala165170175valalasergluthrprovalalavalilealaalapropheaspser180185190ileserilecysleuserlysglyleuglyalaprovalglyserleu195200205leuvalglyserhisasppheilealaargalaargargleuarglys210215220metleuglyglyglymetargglnalaglyileleualaglnalagly225230235240leuphealaleugluglnhisvalthrargleualaaspasphisarg245250255argalalysargleualagluglyleualaalaleuproglyileglu260265270leuaspleuserleuvalglnserasnmetvalpheleuargleuarg275280285gluglygluseralaproleuleualaphemetlysgluargglyile290295300leupheserglytyrglygluleuargleuvalthrhisleuglnile305310315320asnaspaspaspileglugluvalileaspalaphethrglutyrleu325330335glyalaarg<210>78<211>339<212>prt<213>维龙气单胞菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>78metargtyrileaspleuargseraspthrvalthrglnprothrasp151015alametargglnalametleuhisalagluvalglyaspaspvaltyr202530glygluaspproglyvalasnalaleuglualahisglyalaargleu354045leuglylysglnalaalaleuphevalproserglythrmetserasn505560leuleualavalmetserhiscysglnargglygluglyalaileleu65707580glyasnalaalahisiletyrargtyrglualaglnglyseralaval859095leuglyservalalaleuglnproleuprometglnargaspglythr100105110leualapheaspaspilelysalaalaleualaproaspaspalahis115120125phevalglnthrargleuilecysleugluasnthrhisasnglylys130135140valleuproleusertyrleuglnglumetglythrphevalalaglu145150155160argglyleulysleuhisleuaspglyalaargleupheasnalaala165170175valalasergluthrprovalalavalilealaalapropheaspser180185190ileserilecysleuilelysglyleuglyalaprovalglyserleu195200205leuvalglyserhisasppheilealaargalaargargleuarglys210215220metleuglyglyglymetargglnalaglyileleualaglnalagly225230235240leuphealaleugluglnhisvalthrargleualaaspasphisarg245250255argalalysargleualagluglyleualaalaleuproglyileglu260265270leuaspleuserleuvalglnserasnmetvalpheleuargleuarg275280285gluglygluseralaproleuleualaphemetlysgluargglyile290295300leupheserglytyrglygluleuargleuvalthrhisleuglnile305310315320asnaspaspaspileglugluvalileaspalaphethrglutyrleu325330335glyalaarg<210>79<211>348<212>prt<213>硫还原地杆菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>79metasnileileaspleuargseraspglnvalthrargproserpro151015alametargalaalametalaglyalagluvalglyaspaspvaltyr202530glygluaspprothrvalasnargleuglugluleuglyalaalathr354045leuglythrglualaalaleuphethralaserglythrglnalaasn505560leuleualaleuleualahiscysargargglyaspglutyrileala65707580glyglnthralahiscystyrargtyrgluglyglyglyalaalaala859095leuglyglyileglnproglnproleugluvalgluproaspglythr100105110leuaspleuseralavalalaalaalailelysproaspaspleuhis115120125phealaargthrargleuleucysleugluasnthrhisalaglyarg130135140valleuproleuasptyrleualaargalaargargleucysasnglu145150155160glnserleuglyleuhismetaspglyalaargilepheasnalaala165170175vallysleuglyvalprovalarggluilealaglyhisvalaspser180185190valservalcysleuserlysglyleuglyalaprovalglyserleu195200205leucysglyglyarggluphevalalathralaargargtrparglys210215220alavalglyglyglymetargglnalaglyileleualaalaalagly225230235240ileleualaleuthrgluasnvalaspargleualagluasphisala245250255asnalaargargleualagluglyleualaalaileproglyleugly260265270leuaspproserglnvalglnthrasnmetvalpheleucysleupro275280285leuargthralaaspargleualaserpheleuasngluglnglyile290295300leuileserglyarggluserileargleuvalthrhisleuaspval305310315320thrserseraspvalgluargvalilealaalapheglyalaphephe325330335alaglyhisglyglumetglnproglyalaserser340345<210>80<211>348<212>prt<213>硫还原地杆菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>80metasnileileaspleuargseraspthrvalthrargproserpro151015alametargalaalametalaglyalagluvalglyaspaspvaltyr202530glygluaspprothrvalasnargleuglugluleuglyalaalathr354045leuglythrglualaalaleuphethralaserglythrglnalaasn505560leuleualaleuleualahiscysargargglyaspglutyrileala65707580glyglnthralatrpcystyrargtyrgluglyglyglyalaalaala859095leuglyglyileglnproglnproleugluvalgluproaspglythr100105110leuaspleuseralavalalaalaalailelysproaspaspleuhis115120125phealaargthrargleuleucysleugluasnthrhisalaglyarg130135140valleuproleuasptyrleualaargalaargargleucysasnglu145150155160glnserleuglyleuhismetaspglyalaargilepheasnalaala165170175vallysleuglyvalprovalarggluilealaglyhisvalaspser180185190valservalcysleuserlysglyleuglyalaprovalglyserleu195200205leucysglyglyarggluphevalalathralaargargtrparglys210215220alavalglyglyglymetargglnalaglyileleualaalaalagly225230235240ileleualaleuthrgluasnvalaspargleualagluasphisala245250255asnalaargargleualagluglyleualaalaileproglyleugly260265270leuaspproserglnvalglnthrasnmetvalpheleucysleupro275280285leuargthralaaspargleualaserpheleuasngluglnglyile290295300leuileserglyarggluserileargleuvalthrhisleuaspval305310315320thrserseraspvalgluargvalilealaalapheglyalaphephe325330335alaglyhisglyglumetglnproglyalaserser340345<210>81<211>348<212>prt<213>硫还原地杆菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>81metasnileileaspleuargseraspthrvalthrargproserpro151015alametargalaalametalaglyalagluvalglyaspaspvaltyr202530glygluaspprothrvalasnargleuglugluleuglyalaalathr354045leuglythrglualaalaleuphethralaserglythrglnalaasn505560leuleualaleuleualahiscysargargglyaspglutyrileala65707580glyglnthralaprocystyrargtyrgluglyglyglyalaalaala859095leuglyglyileglnproglnproleugluvalgluproaspglythr100105110leuaspleuseralavalalaalaalailelysproaspaspleuhis115120125phealaargthrargleuleucysleugluasnthrhisalaglyarg130135140valleuproleuasptyrleualaargalaargargleucysasnglu145150155160glnserleuglyleuhismetaspglyalaargilepheasnalaala165170175vallysleuglyvalprovalarggluilealaglyhisvalaspser180185190valservalcysleuserlysglyleuglyalaprovalglyserleu195200205leucysglyglyarggluphevalalathralaargargtrparglys210215220alavalglyglyglymetargglnalaglyileleualaalaalagly225230235240ileleualaleuthrgluasnvalaspargleualagluasphisala245250255asnalaargargleualagluglyleualaalaileproglyleugly260265270leuaspproserglnvalglnthrasnmetvalpheleucysleupro275280285leuargthralaaspargleualaserpheleuasngluglnglyile290295300leuileserglyarggluserileargleuvalthrhisleuaspval305310315320thrserseraspvalgluargvalilealaalapheglyalaphephe325330335alaglyhisglyglumetglnproglyalaserser340345<210>82<211>348<212>prt<213>硫还原地杆菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>82metasnileileaspleuargseraspthrvalthrargproserpro151015alametargalaalametalaglyalagluvalglyaspaspvaltyr202530glygluaspprothrvalasnargleuglugluleuglyalaalathr354045leuglythrglualaalaleuphethralaserglythrglnalaasn505560leuleualaleuleualahiscysargargglyaspglutyrileala65707580glyglnthralahiscystyrargtyrgluglyglyglyalaalaala859095leuglyglyileglnproglnproleugluvalgluproaspglythr100105110leuaspleuseralavalalaalaalailelysproaspaspleuhis115120125phealaargthrargleuleucysleugluasnthrhisalaglyarg130135140valleuproleuasptyrleualaargalaargargleucysasnglu145150155160glnserleuglyleuhismetaspglytyrargilepheasnalaala165170175vallysleuglyvalprovalarggluilealaglyhisvalaspser180185190valservalcysleuserlysglyleuglyalaprovalglyserleu195200205leucysglyglyarggluphevalalathralaargargtrparglys210215220alavalglyglyglymetargglnalaglyileleualaalaalagly225230235240ileleualaleuthrgluasnvalaspargleualagluasphisala245250255asnalaargargleualagluglyleualaalaileproglyleugly260265270leuaspproserglnvalglnthrasnmetvalpheleucysleupro275280285leuargthralaaspargleualaserpheleuasngluglnglyile290295300leuileserglyarggluserileargleuvalthrhisleuaspval305310315320thrserseraspvalgluargvalilealaalapheglyalaphephe325330335alaglyhisglyglumetglnproglyalaserser340345<210>83<211>348<212>prt<213>硫还原地杆菌苏氨酸醛缩酶氨基酸突变(人工序列)<400>83metasnileileaspleuargseraspthrvalthrargproserpro151015alametargalaalametalaglyalagluvalglyaspaspvaltyr202530glygluaspprothrvalasnargleuglugluleuglyalaalathr354045leuglythrglualaalaleuphethralaserglythrglnalaasn505560leuleualaleuleualahiscysargargglyaspglutyrileala65707580glyglnthralahiscystyrargtyrgluglyglyglyalaalaala859095leuglyglyileglnproglnproleugluvalgluproaspglythr100105110leuaspleuseralavalalaalaalailelysproaspaspleuhis115120125phealaargthrargleuleucysleugluasnthrhisalaglyarg130135140valleuproleuasptyrleualaargalaargargleucysasnglu145150155160glnserleuglyleuhismetaspglyalaargilepheasnalaala165170175vallysleuglyvalprovalarggluilealaglyhisvalaspser180185190valservalcysleuilelysglyleuglyalaprovalglyserleu195200205leucysglyglyarggluphevalalathralaargargtrparglys210215220alavalglyglyglymetargglnalaglyileleualaalaalagly225230235240ileleualaleuthrgluasnvalaspargleualagluasphisala245250255asnalaargargleualagluglyleualaalaileproglyleugly260265270leuaspproserglnvalglnthrasnmetvalpheleucysleupro275280285leuargthralaaspargleualaserpheleuasngluglnglyile290295300leuileserglyarggluserileargleuvalthrhisleuaspval305310315320thrserseraspvalgluargvalilealaalapheglyalaphephe325330335alaglyhisglyglumetglnproglyalaserser340345<210>84<211>24<212>dna<213>引物(人工序列)<400>84ctggacggctaccggctggccaat24<210>85<211>24<212>dna<213>引物(人工序列)<400>85attggccagccggtagccgtccag24<210>86<211>22<212>dna<213>引物(人工序列)<400>86agcacgccagcatctgcacgga22<210>87<211>22<212>dna<213>引物(人工序列)<400>87tccgtgcagatgctggcgtgct22<210>88<211>1032<212>dna<213>新月柄杆菌苏氨酸醛缩酶基因(人工序列)<400>88atgacccagaccgcgccccgctacgatttcgcctccgacaacgtcgccggcgccatgccc60gaggtgatggaggcgttgatcgcggccaatgcggggacggcctcgggctacgggaccgac120catgtcagccgtgccgccgccgatcgcatccgcgcggcgctggacgccgacgcgcaggtg180cggttcacagcctcgggcacggcggccaacgccttcgccctgaccctgctggcccagccg240cacgaggcggtgctggcccacgagcacgcccacatctgcacggacgagaccggcgcgccc300ggcttctttggccagggcgtcggcctgatcggcttgccgggcgcatcgggcaagatggag360ttggcggccctggaggcggcgctggcccagccggacgtctcgtaccgccagccggcggcg420gccttgtcgctgaccaccgccaccgagtacggcacggtctattcggaagaccacctgcgg480gccctgatcgcgccggtgaaggccaagggctatggcgtccacctggacggcgcgcggctg540gccaatgcggtcgcggggggctttgacctgaaaagcatcgccaagatgggcgtcgacatc600ctggtgatgggcggcaccaaggccggctcgacgcccaccgaggccgtggtgttcctgaac660cccgaccacgccaaacgcctggacgcgcgcctcaagcacgccggccagctgatctcgaag720ggacgcttcctggccgcgccgtggctgggcctgctgggcgagaacggtcagaccgccccc780tgggccgcccgcgccgcccacgccaacgccatggcgcagaagctggccgcgctgatgccc840gtcccgatcaagcacccggtcgaggccaacggcatcttcgtcgagatggacgaacttgcc900ctggaacgcctgcgcggcgaaggctggttcgtctatcgcttcctggacggcacggtgcgc960ttcatgtgctcgtgggccacgacgcccgagatggtcgaggatctgggcgcggcgctgaag1020cgagtggcttag1032当前第1页12