CN1246147A - 由大肠杆菌产生的修饰的三角酵母D-氨基酸氧化酶制备头孢菌素烷基7β-(4-羧丁基酰胺)酸的方法 - Google Patents
由大肠杆菌产生的修饰的三角酵母D-氨基酸氧化酶制备头孢菌素烷基7β-(4-羧丁基酰胺)酸的方法 Download PDFInfo
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Abstract
酶法制备头孢菌素烷基7β-(4-羧丁基酰胺)酸,是通过在大肠杆菌中产生的修饰过了的三角酵母(TrigonopsisVariabilis)D-氨基酸氧化酶进行的,该方法包含:(1)分离相应于编码D-氨基酸氧化酶基因的DNA;(2)去除所述的基因中的内含子;(3)将得到的DNA片段插入在大肠杆菌中能够复制的质粒;(4)合成含有编码6个组氨酸的一段核苷酸序列,融合到基因结构区的5’端;(5)用所得的重组质粒转化大肠杆菌;(6)培养转化了的大肠杆菌细胞;和(7)通过亲和层析回收前面培养细胞的D-氨基酸氧化酶。
Description
本发明涉及酶法制备7β-(4-羧丁基酰胺)头孢菌素烷酸(7β-(4-carboxybutanamide)cephalosporanic acid)的一种方法。具体地说,描述了一种利用重组技术分离一个基因的方法,该基因编码一个具有D-氨基酸氧化酶活性的酶,所述的基因被克隆到大肠杆菌(E.coli)属微生物,利用蛋白质工程修饰该酶,通过所述的微物生发酵的方法可以得到高产的所述的修饰酶,提取修饰酶用于制备7β-(4-羧丁基酰胺)头孢菌素烷酸。该酸是制备7-氨基头孢菌素烷酸的一个中间体,该中间体是制备头孢菌素家族中抗细菌制剂的一个已知中间体。
7β-(4-羧丁基酰胺)头孢菌素烷酸也称为戊二酰-7-氨基头孢菌素烷酸(GL-7ACA)是从头孢菌素C用D-氨基酸氧化酶(以下简称DAO)作用产生的。该酶可从各种微生物中得到,例如,三角酵母(Trigonopsisvariabilis)(Biochem.Biophys.Res.Commun.(1993)31:709),Rhodotorula gracilis(J.Biol.Chem.(1994)269:17809)and Fusariumsolani(J.Biochem.1990)108:1063。用这些微生物产生的DAO存在许多缺点。其一,所产生的DAO活性水平很低;其二,一些不希望有的酶活性,例如:酯酶类和过氧化氢酶类与DAO酶同时存在,前者,酯酶会裂解GL7ACA,降低其产率而增加纯化过程的成本。后者,过氧化氢酶会分解催化反应中所需的过氧化氢。必需添加该化合物,也增加了利用该方法的成本,同时造成酶活丧失,以至降低它重复使用的可能性。要想避免所述的对该酶的污染必需要纯化DAO活性,不然会大大增加从头孢菌素C得到GL-7ACA酶法制备的成本和难度。
最近,一个方法已经被描述,即从三角酵母(T.variabilis)分离编码DAO基因,并在大肠杆菌和三角酵母中得以表达,如日本专利,(JapanesePatent Application Laid-Open No.71180/1988;European PatentApplication No.93202219.7,Publication No.0583817A2)。
此外,由腐皮镰孢菌(F.solani)编码DAO活性的基因也已被克隆,并在大肠杆菌和产黄半知菌(Achremonium Chrysogenum)中表达(日本专利,Japanese Patent Application Laid-Open No.2000181/1990;J.Biochem.(1990)108:1063;Bio/Technology(1991)9:188)。最近,来自细长红酵母(R.gracilis)编码DAO的基因也已被克隆,并在大肠杆菌中表达(西班牙专利,Spanish Patent P9600906)。
鉴于利用纯的DAO活性生产GL-7ACA已产业化,本发明致力于生产一种具有DAO活性的酶,并且能非常容易地被纯化。众所周知纯化蛋白质最有效的方法是用亲和层析(Sassenfeld,H.M.(1990)Trends Biotechnol.8:88)。为此,需要寻找一个层析的载体,该载体有利于蛋白被选择性地连结和/或洗脱。该层析载体必需含有一个为该蛋白质可识别的分子或配基,并使蛋白质和配体间相互作用是特异的同时其强度足以使该蛋白质选择性的洗脱。开发一种亲和层析的载体是不容易的,至今尚未见采用简单的一步亲和层析法纯化DAO酶活性的报导。
此外,众所周知,某些遗传工程方法可修饰蛋白质,改变其某些特性使其更易被纯化。因此,修饰一个蛋白质的结构使其适合于亲和性纯化的各种体系已被建立(Sii,D.And Sadana,A.(1991)J.Biotechnol.19:83;Narayanan,S.R.and Crane,L.J.(1990)Trends Biotechnol.8:12;Scouten,W.H.(1991)Curr.Opinion Biotechnol.2:37;Sassenfeld,H.M.(1990)Trends Biotechnol.8:88)。这些修饰的要素是将一个多肽融合到蛋白质上,该多肽具有与一个特别的层析载体相互作用的特异性。因此它能使融合蛋白质通过亲和层析被纯化,这些修饰之一是在于融合一个聚组氨酸到这蛋白质上。该聚组氨酸序列使融合蛋白质能在含有二价金属离子载体的亲合层析柱上纯化。(Arnols,F.H.(1991)Bio/Technology9:151;Hochuli et al.(1988)Bio/Technology 6:1321)。
虽然,利用得到的融合蛋白质改进纯化方法原则上是简单而有效的技术,但它的缺点是蛋白质的一级结构被改变也可能对其二级结构有很大的影响。这些结构上的改变可能影响到蛋白质的功能至完全或部分被丧失的程度,针对蛋白质的功能,它已转化为没有价值的蛋白质。因此,得到一种融合蛋白质总是要担一种不确定性的风险,特别是当进行第一次融合试验时,即在以前的融合结果未知时。正是因为最终结果的这种不确定性是获得融合蛋白的方法具有新颖性,因为事先不能确切预见进行蛋白质融合试验将发生些什么情况。
在科技文献中,尚未见用任何方法描述遗传修饰三角酵母(T.variabilis)生产DAO酶,并使其能一步被纯化,同时在大肠杆菌或在另一个微生物中以活性的形式高产DAO酶的报道。本发明的详细描述
描述本发明的起点是酵母(三角酵母,T.variabilis)ATCC 20931作为脱氧核糖核酸(以下称为之为DNA)的供体。一旦得到这种酵母的基因组DNA(该基因组DNA含有与生成DAO有关的遗传信息的基因,以下称之为dao基因),用噬菌体载体λ-GEM12,在大肠杆菌中构建一个DNA文库。根据不同DAO之间保守的碱基序列,设计并合成寡核苷酸,并将其作为探针通过标准的杂交技术分析该DNA文库。这样,一系列含有能编码dao基因的三角酵母DNA片段的大肠杆菌重组克隆被发离出来。将得到的DNA片段再克隆到一个大肠杆菌质粒载体。用这个重组载体来得到含有三角酵母(T.variabilis)dao基因的DNA片段的序列(SEQ ID NO:1)。该序列分析表明这dao基因具有2个外显子和一个内含子的结构特征。
如前所述所得到的含有三角酵母(T.variabilis)dao基因基因组序列的DNA片段有一个内含子,这不能直接用于在大肠杆菌中的表达。因此要得到缺少该内含子的dao基因。基于这目的,用两个合成的寡粒苷酸通过PCR扩增这dao基因。第一个寡核苷酸,要这样设计需包含以下成分:一个核糖体联结位点、一个转译起始位点,第一个外显子的全序列和第二个外显子的5’端前面一些核苷酸。第二寡核苷酸相应于dao基因第二个外显子3’端的互补序列并包括一个转译终止密码。为克隆DNA片段,不同的限制酶位点也要包括在这些合成的寡核酸中。通过这样的步骤得到了一个新的DNA片段,分离后被克隆到大肠杆菌质粒载体(图1)。用限制酶切过的含有缺失内含子的完整dao基因DNA片段又被克隆到不同的大肠杆菌质粒载体,这些载体具有一些启动子,这些启动子能使外源基因在这宿主细菌中过量表达。各克隆产生的DAO活性用显色技术和HPLC层析来测定。这样,得到了能产生大量具活性的DAO酶重组大肠杆菌克隆。
将该dao基因通过加一段可编码聚组氨酸的核苷酸序列进行修饰。为此目的,将含dao基因质粒用一能切开转译起始密码子的限制酶酶解。酶解后将产生的DNA粘性末端用大肠杆菌DNA聚合酶I片段补平。然后与一个编码聚组氨酸的合成的寡核苷酸连接子(linker)连接起来,以此可产生一个在编码区5’端含有被修饰的dao基因(SEQ ID NO:2)。产生的重组质粒被转化到一株大肠杆菌,用聚组氨酸的连接子作为探针,通过杂交技术来筛选阳生克隆。修饰的dao基因再次被测序以证明按设计正确地产生融合。
用不同株系大肠杆菌作为先前构建的上述质粒的宿主来研究聚组氨酸修饰的DAO产生(以下称hisDAO)。经测试该hisDAO酶是有活性的。
将上述选择出来的重组大肠杆菌克隆培养在含碳源、氮源和无机盐的培养基上,使其产生hisDAO。培养温度是在18℃-37℃之间,pH必需维持在5-9之间。小规模发酵可用各种体积50ml-1000ml的三角瓶,培养基的量可占三角瓶体积的10%至50%之间,发酵周期可控制在12-90小时的范围。
如果选用适于保持重组载体稳定性的培养条件,可以改善该重组微生物对DAO的产生,这是通过在培养基中加入抗菌素来实现的,该含有dao基因的重组质粒具有对该抗菌素的抗性标记(抗氨苄青霉素、氯霉素、卡那霉素、四环素等等)。除了使DAO能稳定产生以外,还能防止培养基被其它一些不必要的微生物所污染,同时也排除了因重组载体丢失而停止产生DAO的菌株。
重组的产hisDAO细胞通过离心方法使其从培养液中分离出来,再用化学的、酶学的或机械的方法进行细胞破碎或透析。为了得到高纯度的hisDAO酶提取物,用Co2+-IDA柱通过亲和层析法一步纯化hisDAO。为此,将粗酶液样品注进Co2+-IDA树脂柱上,用20mM磷酸-缓冲液,pH7.0,含0.2MNaCl的洗脱液洗脱除去杂蛋白。然后用10mM咪唑洗脱,得到hisDAO酶。
如果用Cu2+-IDA或Zn2+-IDA替代Co2+-IDA作为层析载体,his-DAO酶与层析的载体保持很强的结合,不可能用高浓度咪唑将his-DAO酶洗脱下来。这样载体通过适当的洗涤除去杂蛋白可用于固定化酶系统。
利用从表达该镶嵌基因的重组大肠杆菌克隆中纯化的hisDAO酶,由头孢菌素C制备了GL-7ACA。
从Co2+-IDA载体柱层析上得到的,经过透析和浓缩的酶提取物也能与另外一些适当的隋性固相载体反应成为固定化酶,使循环使用固定酶成为可能。
本发明的新颖性在于首次将三角酵母(T.vaniabilis)的DAO酶修饰成hisDAO使其在一个原核微生物如大肠杆菌中以活性形式表达并通过亲和层析一步就被纯化。此外,其产量相对于三角酵母(T.vaniabilis)中得到的量备提高,从而使其适合于工业规模生产。再不含有不需要的酶,如果氧化氢酶,酯酶,的不同的大肠杆菌菌株中产生hisDAO的可能性,以及用蛋白质工程技术进一步修饰,改进酶的稳定性和催化特性诸多方面的可能性,均增加了本专利的新颖性。
本发明在下述实施例中将被详细阐述,而不对其进行任何限定。实施例1
1.DAO活性的测定
DAO活性测定是按照早先描述的方法进行的(J.Biol.Chem.(1967)242:3957),用D-苯基甘氨酸(25mM)为底物。在50mM磷酸盐缓冲液pH8.0保温15至30分钟,用1/10体积的纯醋酸终止反应。根据在252nm OD值的大小测定酶活,同时考虑在反应中所形成的89.77nmol苯酰甲酸在252mM的吸收值为1.0。
2.DNA载体和用于转化的大肠杆菌感受态细胞的制备
用碱法溶菌的方法制备质粒载体pBluescript I KS(+)(Apr)(Stratagene)和pkk233.2(Apr)(pharmacia)(Sambrook et al.(1989)Molecular cloning:A Laboratory manual,Cold Spring Harbor Laboratory,Cold spring Harbor,New York,USA)。为此目的,携带前面谈及的质粒的大肠杆菌在振荡摇床培养16hr,转速为250rpm,培养温度37℃,500毫升LB培养基含有100μg/ml浓度的氨苄青霉素,通过这种方法得到的质粒DNA用氯化铯CsCl梯度离心进行纯化。
大肠杆菌TG1和DH5α的感受态细胞通过Rbcl方法获得。(Sambrooket al.(1989)Molecular cloning:A Laboratory Manual,cold SpringHarbor Laboratory,Cold Spring Harbor,New York,USA)。
3.含有D关于氨基酸氧化酶DAO产生的遗传信息的DNA供体的制备
三角酵母T.variabilis ATCC 20931菌株在YMPG培养基(麦芽浸提物0.3%,酵母抽提物0.3%,蛋白胨0.5%和葡萄糖1%)中培养,连续振荡培养36小时(OD660=5.0),转速250rpm,培养温度30℃。
沉淀和洗涤细胞后,用溶菌酶溶解细胞,用前述的方法提取DNA(Sherman et al.(1986)Laboratory Course for Methods in yeastGenetics,Cold Spring Harbor Laboratory,Cold Spring Harbor,Newyork)。为了达到更高的纯度,将DNA用RNase消化,用苯酚和氯仿-异戊醇24/1(CIA)抽提几次,并用异丙醇沉淀。将DNA沉淀物用100%乙醇和70%乙醇洗涤,并溶解于含有1mM EDTA的10mM Tris-HCl缓冲液,pH7.5(TE缓冲液)。实施例2
1.三角酵母DNA文库的构建
按照实施例1-3部分所述的方法,得到了三解酵母ATCC20931菌株的总DNA。所述的总DNA取300μg,用20单位的Sau 3AI部分裂解,反应体积600μl,37℃保温,按45秒,1分和2分钟不同反应时间,分别取出200μl等体积的反应液,用20mM冷的EDTA液终止裂解反应。用0.7%的琼脂糖凝胶电泳检测裂解物后,将他们合并到一起并加热至68℃10分钟,慢慢冷却至室温,放在一个38毫升含10-40%的蔗糖梯度离心管上面,进行离心,转速为2600rpm,时间24小时,温度15℃,按0.5毫升一个分部采集样品,取10微升样品在0.4%的琼脂糖凝胶电泳上分析。
合并含有18至22kb之间的DNA的分部,用蒸馏水稀释至大约10%的蔗糖浓度。DNA用乙醇沉淀和重新悬浮在50μl的TE缓冲液中取后者3μl进行0.4%琼脂糖凝胶电脉分析,电泳检测表明DNA片段的大小是对的,他们的浓度大约50ng/μl。
前面描述的方法稍做修改同样可制备细菌噬菌体λ-GEM12(Promega)DNA(Sambrook et al.(1989)Molecular Cloning;A Laboratory Manual,Cold Spring Harbor Laboratory,Cold Spring Harbor,New York,USA)。
为此目的,大肠杆菌菌株NM 538(promega)在NZCYM-0.2%麦芽糖培养基上培养10小时,在600nm检测菌体生长的OD。培养细胞的浓度相当于3×109细胞时,用台式离心机离心收集菌体细胞,转速4000rpm;时间10分钟;温度4℃。细胞用1.2ml的SM缓冲液再悬浮;λ-GEM12噬菌体(3×107噬图斑形成单位)加入悬浮的细菌细胞溶液中,37℃静止保温培养30分钟,每500毫升摇瓶装100毫升NZCYM-0.2%麦芽糖培养基,瓶子予热至37℃,接种200μl感染的细胞。37℃摇瓶培养直到培养出现溶菌(5-6小时)。裂解物在室温条件下用DNase(1μg/ml)和RNase(2μg/ml)处理45分钟。接着,每100ml裂解物,加5.8克的NaCl,混合物在冰浴放置60分钟,随后过滤除去细胞残存物,每100ml溶解物加入20ml 50%PEG-6000,其混合物于冰浴放置60分钟,然后在4℃条件下,10,000g离心20分钟。该沉淀物用1ml的TE缓冲液再悬浮,用CIA提取二次除去多余的FEG-6000。然后再用中性酚提取二次,酚-CIA提取一次,CIA提取一次。水相用4M NaCl调至0.5M NaCl,DNA用二倍体积的乙醇在-20℃沉淀,用小台式离心机,4℃,12,000rpm离心20分钟,沉淀用70%的乙醇洗,干燥后重新悬浮在50μl的TE缓冲液中。
50μg细菌噬菌体DNA用限制酶BamHI和XbaI于37℃酶解2小时。双酶切后经酚-CIA和CIA抽提,再用乙醇沉淀DNA,加TE缓冲液50μl悬浮后,取出2μl后为了促进载体粘着端臂的重新环化剩余液体加MgCl2至10mM;42℃保温1小时,一份2μl的样品和前面的样品一同在0.5%琼脂胶分析并收集。经过确认粘着末端确实重新环化了。混合液放到38ml 10-40%蔗糖密度梯度上。在混合液铺放梯度上之前DNA是没有受到68℃的热处理,不然会导致噬菌体的粘着末端的分离。梯度离心转速26000rpm,离心24小时,温度15℃,随后以0.5ml一个分部进行收集。每个分部取15μl用0.5%琼脂糖凝胶电泳分析后,合并那些缺少非必需中心片段或“stuffer”的部分,用蒸馏水稀释至大约10%蔗糖浓度。DNA用乙醇沉淀,沉淀部分再用50μl的TE悬浮,取2μl用0.5%琼脂糖凝胶电泳检测用以确认缺少中心片段,估计它的浓度大约是100ng/μl。
以插入DNA片段/载体=0.25μg/0.25-0.75μg的比率进行一系列的连接反应。连接反应在12-14℃进行16小时。在0.4%琼脂糖凝胶上检测连接结果。连接产生的DNA片段要比载体或插入片段大,全部连接反应的混合物用乙醇沉淀,并重悬浮在4μl的连接缓冲液中。
用Packagene(Promega)的“体外”包装抽提物来包装连接后的重组噬菌体。包装后的产生重悬浮在500μl SM并使其感染大肠杆菌NM538,为了滴定存在的噬菌体数目和大肠杆菌NM539(Promega)的数目(测定重组噬菌体的百分比)。大肠杆菌NM539是噬菌体P2的一个溶菌株。当感染该大肠杆菌噬菌体缺少非必需的中心区时才产生噬菌斑。构建的DNA文库含有200,000噬菌斑,大约85%噬菌体携带一个外源的DNA片段。
经计算后,大肠杆菌NM539是被感染,完整的基因文库涂布在150毫米直径的培养皿上。
2.含dao基因克隆的鉴定
完整的DNA文库被转移到硝酸纤维素膜上按照前面所述的杂交方法(Sambrook et al.(1989)Molecular Cloning:A Laboratory Manual,ColdSpring Harbor Laboratory,Cold Spring Harbor,New York,USA)筛选阳性噬菌体。硝酸纤维素膜在杂交缓冲液中保温于42℃预杂交3小时,除去预杂交时用的缓冲液后,加入新的杂交缓冲液和10pmol的32P标记寡核苷酸OA(5’-TCTTGTCCTCGACACC-3’)和OB(5’-GACGTGGATTGTCAACTG-3’)进行杂交。按(Sambrook et al.(1989)Molecular Cloning:A LaboratoryManual,Cold Spring Harbor Laboratory,Cold Spring Harbor,New York,USA)标准方法,用噬菌体T4聚核酸激酶和[γ-32P]ATP(ICN Biochemicals)标记上述寡核苷酸的5’端。膜在42℃保温16小时后于室温在2×SSC-0.1%SDS中洗2次,在同样长的时间,同样温室下再用1×SSC-0.1%SDS缓冲液洗2次。最后,硝酸纤维素膜加增感屏后用Hyperfilm-MP(Amershan)压片,在-70℃放48小时。
一旦预杂交、杂交、洗涤和放射身显影完成后,经两个探针OA和OB筛选出63个阳性克隆。其中的9个阳性噬菌斑从培养皿中分别被收集,每一个均被悬浮在1ml SM(加50μl氯仿)中。滴定该溶液中的噬菌体。为此,该噬菌体必需被稀释5000倍,以保证用20μl进行感染时,每个培养皿中的噬菌斑数目在500-1000之间。一旦每个培养皿上物质被转移到相应的硝酸纤维素膜上,硝酸纤维素膜再用OA、OB探针进行杂交。放射自显影表明每个培养皿中有20%-50%噬菌体产生阳性信号。因此,需要用第三次杂交来纯化每一个阳性噬菌体。为此用一吸管去收集那些阳性的噬菌斑使它们与其它的物质进一步分开,或者被也是阳性的噬菌斑包围,然后被悬浮在1ml SM(加50μl氯仿)。稀释100倍后,取15μl进行感染,得到了大约每培养皿大约有300噬菌斑的滴度。在经过与前面2个循环相同的条件下进行处理后,可达到每个培养中100%噬菌体为阳性。这样,纯化了9个独立的噬菌斑。将每个噬菌斑重悬浮在100μl SM(加10μl氯仿),取出2μl汇合溶菌斑溶液涂布在固相培养基上,用于扩增阳性噬菌体。收集上层琼脂并悬浮在5ml SM,溶液中噬菌体浓度约为107噬菌斑形成单位/μl。
按(Sambrook et al.(1989)Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,Cold Spring Harbor,New York,USA),采用Southern方法,以上述OA、OB为探针,测定基因组DNA片段是否包含三角酵母(T.variabilis)dao基因。为此目的,用限制酶(pharmacia)BamH、EcoRI、HindIII、KnpI、PstI、PvuII、XbaI和XhoI酶解的DNA在琼脂糖凝胶上分离并在下述条件下进行杂交。将DNA片段按分子大小被分离开的琼脂糖胶,于室温在0.25M HCl中轻轻振荡15分钟,在变性溶液中振荡1小时,在中和溶液中振荡1小时。然后把凝胶放在一小块浸过10×SSCWhatman 3MM滤纸上,一张与凝胶大小相同的在2×SSC中浸过的BA85硝酸纤维素膜(0.45μM)(Schleicher and Schuell)放在凝胶的上面(小心,不要有气泡)。两张在2×SSC浸过的Whatman 3MM滤纸再放在硝酸纤维素膜上,最后在上面复盖8-10厘米高的同样大小的一叠干滤纸,并压上约500g重的东西,经16小时可完成印迹转移。一旦DNA转印到硝酸纤维膜,将膜小心地放在6×SSC中浸5分钟,然后在室温中放1小时使其变干,再放在两张Whatman 3MM滤纸之间在80℃(抽真空)保温3小时,使DNA固定在滤纸上。如DNA文库筛选部分所描述的相同条件下进行预杂交和杂交。放射自显影结果表明每钟酶解均显现特异的杂交带。详述这些带的大小如下:EcoRI酶解显现10.2kb的带,BamHI酶解显现3.7kb的带,HindIII酶解显现3.5kb的带,KpnI酶解显现11.6kb的带,PstI酶解显现11.3kb的带PvuII酶解显现4.8kb带,XbaI酶解显现4.8kb的带,XhoI酶解显现4.7kb的带。
2.克隆编码dao基因DNA片段
噬菌体DNA纯化后(实施例2-1)用SalI酶解,用噬菌体T4(Amersham)DNA连接酶在ATP存在条件下和所推荐的缓冲液下与经SalI酶解的pBluescriptI KS(+)(Stratagene)在12℃保温5小时,并用于转化感受态大肠杆菌TG1细胞(Amersham)。在含氨苄青霉素(100μg/ml)、X-gal(40μg/ml)和0.2mM IPTG的LB固体培养基上,挑选出转化子。在一些白色的选择性表型克隆中质粒pALT1被分离出来。用Southern技术和探针OB,dao基因被定位于pALT1质粒3.7kb BamHI片段。这BamHI片段亚克隆到经BamHI酶解的质粒pBluescript I KS(+)(Stratagene)。质粒pALT2和pALT3含有3.7kb BamHI片段(图1)。
4.含Dao基因的片段的测序
按前面描述的方法(Sanger et al.(1977)Proc.Natl.Acad.Sci.USA.74,5663-5464)用T7 DNA聚合酶试剂盒(Pharmacia)和[35S]dATP测定了包含在质粒pALT2中片段的核苷酸序列。这序列请见SEQ ID NO:1。与在基因库(Gene Bank/EMBL)中其它已知序列进行比较后指示克隆的片段可编码三角酵母(T.variabilis)dao基因。实施例3
1.用PCR消除dao基因内含子
从质粒pALT2得到0.15μg DNA与10μl(25μM)下列各寡核苷酸混合。
寡核苷酸:DAO1(5’-CATGCCATGGCTAAAATCGTTGTTATTGGGGCCGGTGTTGCCGGTTTAAC-3’),它编码第一个外显子的全序列和第二个外显子的5’端前几个核苷酸,同时还包含一个NcoI限制酶位点。DAO2(5’-CCCAAGCTTCTAAAGGTTTGGACGAG-3’),它含有一个HindIII限制酶位点和带有一个转译终止密码子的相应于dao基因第二个外显子的3’端序列。在这混合物中加入2.5单位Tag聚合酶(Perkin-Elmer)与相应的缓冲液在PCR仪(Gene-ATAQ,Pharmacia)上进行30个循环的扩增。每个循环是:95℃(1分钟),50℃(2分钟)和72℃(2.5分钟)。少量反应混合物在1%琼脂糖凝胶电泳后经EB染色观察扩增结果。
由PCR得到的DNA片段其大小约0.9kb,按说明书推荐方法用β-agarose(BioLabs)从琼脂糖凝胶中提取纯化该DNA片段。2μg纯化的片段与1μg经HincII(Pharmacia)酶切的载体M13tg130(Amersham)用噬菌体T4(Amersham)DNA连接酶在存在ATP和所推荐的缓冲液条件下进行连接。获得了噬菌体M13DAO(图1)测序后证明内含子已被排除。
在噬菌体M13DAO中所含该片段的核苷酸序列按Sanger et al.(1977)Proc.Natl.Aca.Sci.USA 74:5463-5464所描述的方法用T7 DNA聚合酶试剂盒(Pharmacia)和[35S]dATP进行序列测定。这核苷酸序列揭示所克隆的0.9kb片段为没有内含子的三角酵母(T.variabilis)dao基因序列。
2.pKK233.2中无任何内含子dao基因的亚克隆
来自质粒pkk232.2的0.1μg DNA样品用限制酶NcoI和HindIII(pharmacia)于37℃在所推荐的缓冲液中酶解1小时,然后在65℃加热10分钟终止该反应。酶切过的质粒与用上述相同限制酶酶解的0.2μg噬菌体M13DAO混合,在存在ATP和所推荐的缓冲液条件下通过噬菌体T4 DNA连接酶(AmerSham)将2个DNA连接起来。
上述连接产物用于转化感受态大肠杆菌TG1细胞。在含100μg/ml氨苄青霉素的LB培养基上选择转化子。通过这方法获得了含重组质粒pKDA003(图1)的克隆。该重组质粒含有通过PCR扩增得到的0.9-kb DNA片段,并已插入到质粒pkk233.2(即在trc启动子操纵下可表达的质粒)的NcoI和HindIII酶切位点之间。
实施例4
1.设计一个含有6个组氨酸尾巴的嵌合DNA酶
为了在三角酵母(T.variabilis)DAO酶的氨基末端插入聚组氨酸尾巴,用限制酶NcoI(Pharmacia)于37℃在推荐的缓冲液中酶解,0.1μg质粒pKDAO3 1个小时,然后65℃加热终止其反应。酶解后的质粒用大肠杆菌DNA聚合酶I Klenow片段(Pharmacia)按说明书推荐的方法进行处理。样品在65℃加热10分钟终止反应。得到的线性质粒在存在ATP和所推荐的缓冲液条件下通过噬菌体T4 DNA连接酶(Amersham)连接到一个含有编码6个组氨酸残基的核苷酸序列的DNA片段。编码6个组氨酸残基的DNA片段是通过两个被称为HIS1(5’-CATCATCACCACCATCACTT-3’)和HIS2(5’-AAGTGATGGTGGTGATGATG-3’)的2个互补寡苷酸的1.5μg混合物,经100℃加热5分钟,冷却到室温,由杂交形成一个双链DNA片段而得到的。
得到的连接混合物用于转化感受大肠杆菌TG1细胞,在含100μg/ml氨苄青霉素LB培养基上挑选出转化子。通过这种方法,得了一个含有重组质粒pKDAOHIS的克隆,该质粒的dao基因连接在一个5’端编码6个组氨酸残基的一段18个核苷酸序列上。)
为了证明质粒pKDAOHIS(图1)含有预期的相嵌结构,其序列按(Sangeret al.(1977).Proc.Natl.Aca.Sci.USA 74:5463-5464)方法,用T7 DNA聚合酶试剂盒(Pharmacia)和[35S]dATP在同一重组质粒上进行测定。这序列如Seq ID NO:1所示。
经质粒pKDAOHIS转化的大肠杆菌TG1在LB培养液在25℃和250rpm条件下发酵20小时。细胞通过离心收集(5000Xg;10分钟)并用超声波破碎细胞,其DAO活性如同实施例1-1部分描述的方法测定,其DAO活性是每毫克蛋白质350单位。表明用这样的方法得到的具有组氨酸链(hisDAO)的嵌合DAO酶是有活性的。SDS-PAGE实验也表明hisDAO酶分子量略大于天然DAO酶。被质粒pKDAOHIS转化的大肠杆菌TG1菌株已于1997年10月6日保存在西班牙菌种保藏中心(CECP),保藏号:CECT4888。保藏中心位于Valencia大学生物学院微生物系,46100 Burjasot(Valencia)。
实施例5
1.琼脂糖-金属螯合载体的制备
5.7ml 3-氯-1,2-环氧丙烷与11.4ml乙二醇二甲醚的混合物是被缓慢地加入到含7g琼脂糖CL6B的57ml 0.1N NOH(含340mg NaBH4)悬浮液中于25℃轻轻地连接搅拌4小时。所得到的琼脂糖-环氧化物用足量的蒸馏水洗后,再补加2.5ml 2M亚胺双醋酸钠和19ml 0.1M碳酸盐缓冲液pH11,于25℃轻轻地连续搅拌该悬浮液12小时。最后,载体用蒸馏水洗,并重悬浮在5mg/ml所选的金属盐溶液中,形成的琼脂糖-金属螯合载体用足量蒸馏水洗以备下步使用。当CoCl2作为金属盐被加入时,得到的是一种琼脂糖-钴螯合载体。
2.嵌合hisDAO酶在琼脂糖-钴螯合载体上的纯化
含琼脂糖-钴螯合载体的柱用含0.2M NaCl的20mM磷酸盐缓冲液(pH7.0)平衡,可溶性细胞提取液注入到该柱,该可溶性细胞提取液是从上述实施例所描述的转化了质粒pKDAOHIS的大肠杆菌TG1细胞中得到的。当提取液完全进入柱以后,用足够量的相同的平衡缓冲液洗柱,除hisDAO留在柱上外,其它所有蛋白质都被洗脱下来。然后用含10mM咪唑的20mM磷酸钠缓冲液(pH7.0)将hisDAO洗脱下来。收集含有该酶的分部,经20mM磷酸钠缓冲液(pH7.0)透析后,酶(9000单位/毫克)的纯度达90%,可用于将头孢菌素C转化成GL-7ACA。
图表的详细描述
图1.质粒pKDAOHIS的构建方法
缺少内含子,在编码蛋白质的第一个甲硫氨酸ATG有一NcoI位点的dao基因通过PCR从质粒pALT2得到,PCR扩增的片段然后被亚克隆在M13DAO的M13tg130载体的HincII位点。从M13DAO得到的NcoI-HindIII片段被亚克隆到pKDAO3的质粒pKK233.2的NcoI-HindIII位点。这质粒有dao基因,在大肠杆菌trc启动子操纵下该基因得以表达。最后,将编码聚组氨酸尾巴DNA片段引入dao基因的5’端,产生质粒pKDAOHIS。
序列表<110>抗生素有限公司<120>由大肠杆菌产生的修饰的三角酵母D-氨基酸氧化酶制备头孢菌素烷基
7β-(4-羧丁基酰胺)酸的方法<130>PCT-47<150>ES 9702008<151>25.09.97<160>2<210>SEQ ID NO:1<211>3668碱基对<212>ADN<213>三角酵母(Trigonopsis variabilis)<220><221>CDS<222>(1481...1503,1543...2506)<223>dao基因<220><221>内含子<222>1504...1442<220><221>CDS<222>2952...3668<223>ORF1<400>GGATCCTTAC GAGGAAGATC TGATAATGGA GAATTCTCTC GCTTTATGCC ATGACTTGCA 60GGTTCCTCGG GTAATGGAAC CACTGACAAA TCGGCTGCTT GAGGTTTTGT TCCCTTCAAC 120ACTTCATCTT CCAAGGTTCT AATTCGTAGT TTCTTCTCAT TCAATAAAGC TGCAAACCGC 180TCCAACATTT GTAGATCATT ATTCTTTGTC TTCACGGCAA CTGTATCTAG TTCCTTCTGA 240AGGTCCTTCG TTTCTTTGGT TAGTAGATCT ACCATGCCAG TTAAGCGATC AATCTCCTCT 300TGGACCTCCT TCTTGAGCGA TAGCTCTGAC CAGAACCAAT CAAAAAGAAC ACCAGGATCG 360GTACATGTTC CTTGTCGGGA TAGGGACAGT GATCCCAAAG TAATTGATAG ATCCTGAACT 420TTCTGGGTAA TCGAGATAAT AACCGAACTC AAATCTTGTG ATGGCTTGGC GTTCAATTGA 480ATGTTTTCGC TAGAAAGCCT CGGACTCTTA CGTTGGTCTT CATCTGTAAG CGACCGCGTG 540AACAGTTGTT GGAATAAGTC ATTCCATTCA CTTTCACTTT GAGGACTTCT ATTTGACCGT 600AGATCTTTAA TTGACTCATT TCCAGTTACT GAAAGTTAGC TGGAAGCTTT CAACATGTCC 660TCACCAGAGG TTTGATAGAG GTCTAACGAT GGCTTCATAC AGGCTGTGAA TGTGATTGTT 720TCGCCAGTCT CAACTCTGAC GATCAAACGC TTAGATCCAC CAATCTCAAT ACCGAACGAG 780TTAATTCGAC TCATTGCTCA ATATGATTGA TCGCGCGGGA TGACATCGAA GGTGACAACC 840GTACGCGAAA GATGCGTGAC GATAAGGACA ACGACTAAGG GAGTAGATGG ACTGGGGAGT 900GAAGGAAATG TGAGACTAGA GAAAAGCCAC TGACTGAGAG TAAAACAGCC ATGATTAGAC 960AATCAGCCAT GACAGCACTA TAACGTGATA TGATAAGTAA GGCTCTGTTG CCCGCTGACG 1020GCCAACGGCT GACGGCCAAC TTGATGATTC TACCACAAAA AATCATACGA GAAGTCAACG 1080AAAAGTCCTT AGTTTGGAAT TCCAGACATG GCAGAATTTA ACGGCCACTA CAGTTGGCCG 1140TTCGTAAACG AGACAAGTGA CTCATGGCAG CACCGTCTCA GTCCACCGGT CTAAAGCACT 1200TGGTGCCAGA TGAATTTGGA AACTGTCACC TTATAGAATT ACTTTTGGAT AGTTTTTGTA 1260AGGCTGGAGA CTTGTAAGCC TGACTCAGTT GACTCATCGG CGAAAGCTTC CTATCTTGGA 1320GCTAAGATCG CCTGATCGTT TTGCCCTACT TATCTTGGTT GCATGAGTTG GCCGGTCAGA 1380GCCGCATTCT AGCCAAAGGG TTATAGCGTT ACACTCTTGA TAGGCAAATC CGTGCTCGGA 1440TTATATATAA GGCAAAAGTC GATTCAACGG ATCAATAAAA ATG GCT AAA ATC GTT 1495
Met Ala Lys Ile Val
1 5GTT ATT GG GTAAGTGCCT TGATACCAGA CGGCTGACAT TTGTTTAG T GCC GGT 1548Val Ile Gly Ala Gly
10GTT GCC GGT TTA ACT ACA GCT CTT CAA CTT CTT CGT AAA GGA CAT GAG 1596Val Ala Gly Leu Thr Thr Ala Leu Gln Leu Leu Arg Lys Gly His Glu
15 20 25GTT ACA ATT GTG TCC GAG TTT ACG CCC GGT GAT CTT AGT ATC GGA TAT 1644Val Thr Ile Val Ser Glu Phe Thr Pro Gly Asp Leu Ser Ile Gly Tyr
30 35 40ACC TCG CCT TGG GCA GGT GCC AAC TGG CTC ACA TTT TAC GAT GGA GGC 1692Thr Ser Pro Trp Ala Gly Ala Asn Trp Leu Thr Phe Tyr Asp Gly Gly
45 50 55AAG TTA GCC GAC TAC GAT GCC GTC TCT TAT CCT ATC TTG CGA GAG CTG 1740Lys Leu Ala Asp Tyr Asp Ala Val Ser Tyr Pro Ile Leu Arg Glu Leu
60 65 70GCT CGA AGC AGC CCC GAG GCT GGA ATT CGA CTC ATC AAC CAA CGC TCC 1788Ala Arg Ser Ser Pro Glu Ala Gly Ile Arg Leu Ile Asn Gln Arg Ser75 80 85 90CAT GTT CTC AAG CGT GAT CTT CCT AAA CTG GAA GGT GCC ATG TCG GCC 1836His Val Leu Lys Arg Asp Leu Pro Lys Leu Glu Gly Ala Met Ser Ala
95 100 105ATC TGT CAA CGC AAC CCC TGG TTC AAA AAC ACA GTC GAT TCT TTC GAG 1884Ile Cys Gln Arg Asn Pro Trp Phe Lys Asn Thr Val Asp Ser Phe Glu
110 115 120ATT ATC GAG GAC AGG TCC AGG ATT GTC CAC GAT GAT GTG GCT TAT CTA 1932Ile Ile Glu Asp Arg Ser Arg Ile Val His Asp Asp Val Ala Tyr Leu
125 130 135GTC GAA TTT GCT TCC GTT TGT ATC CAC ACC GGA GTC TAC TTG AAC TGG 1980Val Glu Phe Ala Ser Val Cys Ile His Thr Gly Val Tyr Leu Asn Trp
140 145 150CTG ATG TCC CAA TGC TTA TCG CTC GGC GCC ACG GTG GTT AAA CGT CGA 2028Leu Met Ser Gln Cys Leu Ser Leu Gly Ala Thr Val Val Lys Arg Arg155 160 165 170GTG AAC CAT ATC AAG GAT GCC AAT TTA CTA CAC TCC TCA GGA TCA CGC 2076Val Asn His Ile Lys Asp Ala Asn Leu Leu His Ser Ser Gly Ser Arg
175 180 185CCC GAC GTG ATT GTC AAC TGT AGT GGT CTC TTT GCC CGG TTC TTG GGA 2124Pro Asp Val Ile Val Asn Cys Ser Gly Leu Phe Ala Arg Phe Leu Gly
190 195 200GGC GTC GAG GAC AAG AAG ATG TAC CCT ATT CGA GGA CAA GTC GTC CTT 2172Gly Val Glu Asp Lys Lys Met Tyr Pro Ile Arg Gly Gln Val Val Leu
205 210 215GTT CGA AAC TCT CTT CCT TTT ATG GCC TCC TTT TCC AGC ACT CCT GAA 2220Val Arg Asn Ser Leu Pro Phe Met Ala Ser Phe Ser Ser Thr Pro Glu
220 225 230AAA GAA AAT GAA GAC GAA GCT CTA TAT ATC ATG ACC CGA TTC GAT GGT 2268Lys Glu Asn Glu Asp Glu Ala Leu Tyr Ile Met Thr Arg Phe Asp Gly235 240 245 250ACT TCT ATC ATT GGC GGT TGT TTC CAA CCC AAC AAC TGG TCA TCC GAA 2316Thr Ser Ile Ile Gly Gly Cys Phe Gln Pro Asn Asn Trp Ser Ser Glu
255 260 265CCC GAT CCT TCT CTC ACC CAT CGA ATC CTG TCT AGA GCC CTC GAC CGA 2364Pro Asp Pro Ser Leu Thr His Arg Ile Leu Ser Arg Ala Leu Asp Arg
270 275 280TTC CCG GAA CTG ACC AAA GAT GGC CCT CTT GAC ATT GTG CGC GAA TGC 2412Phe Pro Glu Leu Thr Lys Asp Gly Pro Leu Asp Ile Val Arg Glu Cys
285 290 295GTT GGC CAC CGT CCT GGT AGA GAG GGC GGT CCC CGA GTA GAA TTA GAG 2460Val Gly His Arg Pro Gly Arg Glu Gly Gly Pro Arg Val Glu Leu Glu
300 305 310AAG ATC CCC GGC GTT GGC TTT GTT GTC CAT AAC TAT GGT GCC GCC GGT 2508Lys Ile Pro Gly Val Gly Phe Val Val His Asn Tyr Gly Ala Ala Gly315 320 325 330GCT GGT TAC CAG TCC TCT TAC GGC ATG GCT GAT GAA GCT GTT TCT TAC 2556Ala Gly Tyr Gln Ser Ser Tyr Gly Met Ala Asp Glu Ala Val Ser Tyr
335 340 345GTC GAA AGA GCT CTT ACT CGT CCA AAC CTT TAGAAATCAT GTATACAATT 2606Val Glu Arg Ala Leu Thr Arg Pro Asn Leu
350 355ATTCTCTCTC TATAAATCTA ATTTTTTTGT GTGGTCTAAT ATTCGTAAAC ACGTCGCAGT 2666CGTCTATGTC GCCCTCGTCA CCGTGTCCAA AGTCGTAAAG TGACTGATTG CAATTGCGAC 2726AACACGTGAC TCGACCTGCC TCCTTACCTC CCATCAACAA CAAAAGAAGC TGGCTAAGAT 2786AGAGGTCTGT TGACGAGCAC TCGTAAGAAC GGCAAACATA GAAAGGAGGC TCTATAATTA 2846CCTGGAAACT GTGTTATATA CTATCACTAG TGAGGGTGAG TGATTAGAAG CAAGGGGACT 2906AGAATACTGA CATGGATAGA GATCCAGGAG CCTTATAAAT AATCA ATG AAT ACA ATT 2963
Met Asn Thr IleGAT TTG GAA TCT TGG GAT GAT GAT CCA GAT TTC GCA GAT GAC TTT GAG 3011Asp Leu Glu Ser Trp Asp Asp Asp Pro Asp Phe Ala Asp Asp Phe Glu360 365 370 375AAT TTA AAG ACT CCA GCT CCT ACG TTT TAT GAA GCC AAC GAT GAG ATT 3059Asn Leu Lys Thr Pro Ala Pro Thr Phe Tyr Glu Ala Asn Asp Glu Ile
380 385 390AGG GAT GGA GAA GAA GAG GAT GAT TTT TTC TCT CAA GAT TTC GAG TTG 3107Arg Asp Gly Glu Glu Glu Asp Asp Phe Phe Ser Gln Asp Phe Glu Leu
395 400 405GAT GAT AAG AAT ACA CTC GGA CGA CAG AAC AAG ATT TCG ACT AGT CAC 3155Asp Asp Lys Asn Thr Leu Gly Arg Gln Asn Lys Ile Ser Thr Ser His
410 415 420CTA AAG TCC GCG AGT CAG GAA CAA GCA GAG ACC TCC TTT CGT GAC TCG 3203Leu Lys Ser Ala Ser Gln Glu Gln Ala Glu Thr Ser Phe Arg Asp Ser
425 430 435AAC GCT GGC GTC AAT GCT TTC AGC TGC GGG ACT ATA AAA GCC TTA GGA 3251Asn Ala Gly Val Asn Ala Phe Ser Cys Gly Thr Ile Lys Ala Leu Gly440 445 450 455AAG AAT AGG ATG ACG ACG GTG GAA GAG AAG TGG GAA AAG GAA GTT AGA 3299Lys Asn Arg Met Thr Thr Val Glu Glu Lys Trp Glu Lys Glu Val Arg
460 465 470CGC GAT CAA ATT GGG TTC AAT GAA GCT ACT CTT AGA GCT CAT GAG ACT 3347Arg Asp Gln Ile Gly Phe Asn Glu Ala Thr Leu Arg Ala His Glu Thr
475 480 485ACC AGA GAA TGG TTA AAA TCC CAG ACT GGC GAA GCT GGG ACT AAA AGC 3395Thr Arg Glu Trp Leu Lys Ser Gln Thr Gly Glu Ala Gly Thr Lys Ser
490 495 500AAG GTC TTT AGC CCA ATT CTC GAC GGA TCA TTC TTC GAA CCG CCT TTG 3443Lys Val Phe Ser Pro Ile Leu Asp Gly Ser Phe Phe Glu Pro Pro Leu
505 510 515GAA TCT AAA GTC AGG CGT TAT CAT TCA CCC CGG AAA CAG GCA CCT CCT 3491Glu Ser Lys Val Arg Arg Tyr His Ser Pro Arg Lys Gln Ala Pro Pro
520 525 530CCT CCT CCT GAC GAT TTT TCA GAT GCA TTT GAA CTA TCT ACA GAA GAG 3539Pro Pro Pro Asp Asp Phe Ser Asp Ala Phe Glu Leu Ser Thr Glu Glu535 540 545 550CCA CTG AAA TTA AAA GTC CAA CCA GTT CAA CCT CAT ATG ACG CCT GCT 3587Pro Leu Lys Leu Lys Val Gln Pro Val Gln Pro His Met Thr Pro Ala
555 560 565CTG AGT GAT AAT GAT CTA TGG GGT GAG GAA TCT ATT GGT GTG CGA CGA 3635Leu Ser Asp Asn Asp Leu Trp Gly Glu Glu Ser Ile Gly Val Arg Arg
570 575 580GGA GGC AGG GAC TCG TCA AGT ATG GGA GGA TCC 3668Gly Gly Arg Asp Ser Ser Ser Met Gly Gly Ser
585 590<210>SEQ ID NO:2<211>1128碱基对<212>ADN<213>三角酵母(Trigonopsis variabilis)<220><221>CDS<222>16...1107<223>5′端具有编码6聚体组氨酸的核苷酸的dao基因<400>CACAGGAAAC AGACC ATG CAT CAT CAC CAC CAT CAC TTC ATG GCT AAA ATC 51
Met His His His His His His Phe Met Ala Lys Ile
1 5 10GTT GTT ATT GGG GCC GGT GTT GCC GGT TTA ACT ACA GCT CTT CAA CTT 99Val Val Ile Gly Ala Gly Val Ala Gly Leu Thr Thr Ala Leu Gln Leu
15 20 25CTT CGT AAA GGA CAT GAG GTT ACA ATT GTG TCC GAG TTT ACG CCC GGT 147Leu Arg Lys Gly His Glu Val Thr Ile Val Ser Glu Phe Thr Pro Gly
30 35 40GAT CTT AGT ATC GGA TAT ACC TCG CCT TGG GCA GGT GCC AAC TGG CTC 195Asp Leu Ser Ile Gly Tyr Thr Ser Pro Trp Ala Gly Ala Asn Trp Leu45 50 55 60AGA TTT TAC GAT GGA GGC AAG TTA GCC GAC TAC GAT GCC GTC TCT TAT 243Thr Phe Tyr Asp Gly Gly Lys Leu Ala Asp Tyr Asp Ala Val Ser Tyr
65 70 75CCT ATC TTG CGA GAG CTG GCT CGA AGC AGC CCC GAG GCT GGA ATT CGA 291Pro Ile Leu Arg Glu Leu Ala Arg Ser Ser Pro Glu Ala Gly Ile Arg
80 85 90CTC ATC AAC CAA CGC TCC CAT GTT CTC AAG CGT GAT CTT CCT AAA CTG 339Leu Ile Asn Gln Arg Ser His Val Leu Lys Arg Asp Leu Pro Lys Leu
95 100 105GAA GGT GCC ATG TCG GCC ATC TGT CAA CGC AAC CCC TGG TTC AAA AAC 387Glu Gly Ala Met Ser Ala Ile Cys Gln Arg Asn Pro Trp Phe Lys Asn
110 115 120ACA GTC GAT TCT TTC GAG ATT ATC GAG GAC AGG TCC AGG ATT GTC CAC 435Thr Val Asp Ser Phe Glu Ile Ile Glu Asp Arg Ser Arg Ile Val His125 130 135 140GAT GAT GTG GCT TAT CTA GTC GAA TTT GCT TCC GTT TGT ATC CAC ACC 483Asp Asp Val Ala Tyr Leu Val Glu Phe Ala Ser Val Cys Ile His Thr
145 150 155GGA GTC TAC TTG AAC TGG CTG ATG TCC CAA TGC TTA TCG CTC GGC GCC 531Gly Val Tyr Leu Asn Trp Leu Met Ser Gln Cys Leu Ser Leu Gly Ala
160 165 170ACG GTG GTT AAA CGT CGA GTG AAC CAT ATC AAG GAT GCC AAT TTA CTA 579Thr Val Val Lys Arg Arg Val Asn His Ile Lys Asp Ala Asn Leu Leu
175 180 185CAC TCC TCA GGA TCA CGC CCC GAC GTG ATT GTC AAC TGT AGT GGT CTC 627His Ser Ser Gly Ser Arg Pro Asp Val Ile Val Asn Cys Ser Gly Leu
190 195 200TTT GCC CGG TTC TTG GGA GGC GTC GAG GAC AAG AAG ATG TAC CCT ATT 675Phe Ala Arg Phe Leu Gly Gly Val Glu Asp Lys Lys Met Tyr Pro Ile205 210 215 220CGA GGA CAA GTC GTC CTT GTT CGA AAC TCT CTT CCT TTT ATG GCC TCC 723Arg Gly Gln Val Val Leu Val Arg Asn Ser Leu Pro Phe Met Ala Ser
225 230 235TTT TCC AGC ACT CCT GAA AAA GAA AAT GAA GAC GAA GCT CTA TAT ATC 771Phe Ser Ser Thr Pro Glu Lys Glu Asn Glu Asp Glu Ala Leu Tyr Ile
240 245 250ATG ACC CGA TTC GAT GGT ACT TCT ATC ATT GGC GGT TGT TTC CAA CCC 819Met Thr Arg Phe Asp Gly Thr Ser Ile I1e Gly Gly Cys Phe Gln Pro
255 260 265AAC AAC TGG TCA TCC GAA CCC GAT CCT TCT CTC ACC CAT CGA ATC CTG 867Asn Asn Trp Ser Ser Glu Pro Asp Pro Ser Leu Thr His Arg Ile Leu
270 275 280TCT AGA GCC CTC GAC CGA TTC CCG GAA CTG ACC AAA GAT GGC CCT CTT 915Ser Arg Ala Leu Asp Arg Phe Pro Glu Leu Thr Lys Asp Gly Pro Leu285 290 295 300GAC ATT GTG CGC GAA TGC GTT GGC CAC CGT CCT GGT AGA GAG GGC GGT 963Asp Ile Val Arg Glu Cys Val Gly His Arg Pro Gly Arg Glu Gly Gly
305 310 315CCC CGA GTA GAA TTA GAG AAG ATC CCC GGC GTT GGC TTT GTT GTC CAT 1011Pro Arg Val Glu Leu Glu Lys Ile Pro Gly Val Gly Phe Val Val His
320 325 330AAC TAT GGT GCC GCC GGT GCT GGT TAC CAG TCC TCT TAC GGC ATG GCT 1059Asn Tyr Gly Ala Ala Gly Ala Gly Tyr Gln Ser Ser Tyr Gly Met Ala
335 340 345GAT GAA GCT GTT TCT TAC GTC GAA AGA GCT CTT ACT CGT CCA AAC CTT 1107Asp Glu Ala Val Ser Tyr Val Glu Arg Ala Leu Thr Arg Pro Asn Leu
350 355 360 364TAGAAGCTTG GCTGTTTTGG C 1128
Claims (8)
1.一种在非人寄主的生物体中生产修饰的三角酵母(TrigonopsisVariabilis)D-氨基酸氧化酶的方法,其特征包含如下步骤:
(a)分离产生所述的D-氨基酸氧化酶的任何一种微生物的编码所述酶的基因的DNA;
(b)通过用合成的寡核苷酸和聚合酶链反应(PCR)的方法,去除所述的基因所含的内含子;
(c)将(b)所得到的寡核苷酸DNA片段插入能够在宿主中复制的质粒中;
(d)将一种合成的含有编码六个组氨酸的核苷酸序列融合于编码D-氨基酸氧化酶活性的结构基因区的5’端,该基因不含任何内含子;
(e)用由(d)产生的重组质粒转化宿主;
(f)用适当的培养基,在得以D-氨基酸氧化酶产生的条件下,培养由(e)得到的转化了的宿主细胞;
(g)由(f)的培养物中通过亲和层析回收D-氨基酸氧化酶。
2.按照权利要求1所述的方法,其特征在于:编码D-氨基酸氧化酶的基因是三角酵母(Trigonopsis Variabilis)的编码D-氨基酸氧化酶的基因。
3.按照权利要求1或2所述的方法,其特征在于,该非人宿主是大肠杆菌。
4.按照前述任何一项权利要求所述的一种方法,其用于回收修饰酶的亲和层析法使用了基于二价阳离子的载体。
5.由前述任何一项权利要求所述的方法生产和纯化的经修饰的D-氨基酸氧化酶。
6.使用权利要求5所述的D-氨基酸氧化酶酶促合成7β-(4-羧丁基酰胺)头孢菌素烷酸的方法。
7.按照权利要求6所述的方法,其特征在于,将头孢菌素C与D-氨基酸氧化酶在一种水溶性介质中反应。
8.按照权利要求6或7所述的方法,其特征在于,酶促反应是在D-氨基酸氧化酶被固相化的条件下进行的。
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Application Number | Priority Date | Filing Date | Title |
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ES009702008A ES2131018B1 (es) | 1997-09-25 | 1997-09-25 | Un procedimiento enzimatico para preparar acido 7beta-(4-carboxibutanamido)cefalosporanico utilizando la enzima d-aminoacido oxidasa de trigonopsis variabilis modificada producida en escherichia coli. |
ESP9702008 | 1997-09-25 |
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CN1246147A true CN1246147A (zh) | 2000-03-01 |
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CN98801336A Pending CN1246147A (zh) | 1997-09-25 | 1998-09-24 | 由大肠杆菌产生的修饰的三角酵母D-氨基酸氧化酶制备头孢菌素烷基7β-(4-羧丁基酰胺)酸的方法 |
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US (1) | US6635458B2 (zh) |
EP (1) | EP0969088A1 (zh) |
JP (1) | JP2001506868A (zh) |
KR (1) | KR20000069110A (zh) |
CN (1) | CN1246147A (zh) |
AU (1) | AU9162898A (zh) |
CA (1) | CA2272354A1 (zh) |
CZ (1) | CZ182399A3 (zh) |
EE (1) | EE9900205A (zh) |
ES (1) | ES2131018B1 (zh) |
HU (1) | HUP0000797A2 (zh) |
IL (1) | IL129533A0 (zh) |
LT (1) | LT4628B (zh) |
LV (1) | LV12359B (zh) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005098000A1 (en) * | 2004-04-08 | 2005-10-20 | Bioright Worldwide Company Limited | Recombinant d-amino acid oxidase |
WO2007016861A1 (fr) * | 2005-08-08 | 2007-02-15 | Bioright Worldwide Company Limited | Procédé enzymatique en deux étapes pour préparer un acide 7-aminocéphalosporanique |
WO2007090317A1 (fr) * | 2006-02-10 | 2007-08-16 | Taiwan Advance Bio-Pharm Inc. | Procédé de production d'une protéine hoxb4 recombinée dont la terminaison c présente une étiquette his et son utilisation |
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FR2268022B1 (zh) * | 1974-04-22 | 1977-06-24 | Rhone Poulenc Ind | |
JPS62262994A (ja) * | 1986-05-12 | 1987-11-16 | Asahi Chem Ind Co Ltd | D−アミノ酸オキシダ−ゼ遺伝子 |
CA1340522C (en) * | 1987-03-10 | 1999-05-04 | Heinz Dobeli | Fusion proteins containing neighbouring histidines for improved purification |
CA2100987C (en) | 1992-07-27 | 1999-06-15 | Kaoru Furuya | A transformant capable of producing d-amino acid oxidase |
US5397653A (en) | 1993-04-30 | 1995-03-14 | Westinghouse Electric Corporation | Filler wire composition and method of welding turbine component with filter wire composition and its product thereof |
ES2109194B1 (es) | 1996-04-22 | 1998-07-16 | Antibioticos Sa | Un procedimiento para producir la enzima d-aminoacido oxidasa de rhodotorula gracilis en celulas hospedantes. |
-
1997
- 1997-09-25 ES ES009702008A patent/ES2131018B1/es not_active Expired - Fee Related
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- 1998-09-24 CZ CZ991823A patent/CZ182399A3/cs unknown
- 1998-09-24 CN CN98801336A patent/CN1246147A/zh active Pending
- 1998-09-24 US US09/308,683 patent/US6635458B2/en not_active Expired - Fee Related
- 1998-09-24 WO PCT/ES1998/000262 patent/WO1999015632A1/es not_active Application Discontinuation
- 1998-09-24 KR KR1019997004579A patent/KR20000069110A/ko not_active Application Discontinuation
- 1998-09-24 AU AU91628/98A patent/AU9162898A/en not_active Abandoned
- 1998-09-24 EP EP98943898A patent/EP0969088A1/en not_active Withdrawn
- 1998-09-24 CA CA002272354A patent/CA2272354A1/en not_active Abandoned
- 1998-09-24 SK SK648-99A patent/SK64899A3/sk unknown
- 1998-09-24 EE EEP199900205A patent/EE9900205A/xx unknown
- 1998-09-24 IL IL12953398A patent/IL129533A0/xx unknown
- 1998-09-24 JP JP51859999A patent/JP2001506868A/ja not_active Ceased
- 1998-09-24 HU HU0000797A patent/HUP0000797A2/hu unknown
-
1999
- 1999-05-11 LT LT99-051A patent/LT4628B/lt not_active IP Right Cessation
- 1999-05-25 LV LVP-99-87A patent/LV12359B/en unknown
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005098000A1 (en) * | 2004-04-08 | 2005-10-20 | Bioright Worldwide Company Limited | Recombinant d-amino acid oxidase |
US7517677B2 (en) | 2004-04-08 | 2009-04-14 | Jun Wang | Recombinant D-amino acid oxidases |
WO2007016861A1 (fr) * | 2005-08-08 | 2007-02-15 | Bioright Worldwide Company Limited | Procédé enzymatique en deux étapes pour préparer un acide 7-aminocéphalosporanique |
WO2007090317A1 (fr) * | 2006-02-10 | 2007-08-16 | Taiwan Advance Bio-Pharm Inc. | Procédé de production d'une protéine hoxb4 recombinée dont la terminaison c présente une étiquette his et son utilisation |
Also Published As
Publication number | Publication date |
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EP0969088A1 (en) | 2000-01-05 |
US6635458B2 (en) | 2003-10-21 |
JP2001506868A (ja) | 2001-05-29 |
KR20000069110A (ko) | 2000-11-25 |
WO1999015632A1 (es) | 1999-04-01 |
AU9162898A (en) | 1999-04-12 |
LV12359A (lv) | 1999-10-20 |
LT99051A (en) | 1999-10-25 |
CZ182399A3 (cs) | 1999-09-15 |
ES2131018B1 (es) | 2000-04-01 |
LV12359B (en) | 2000-03-20 |
IL129533A0 (en) | 2000-02-29 |
EE9900205A (et) | 1999-12-15 |
ZA988754B (en) | 1999-06-28 |
CA2272354A1 (en) | 1999-04-01 |
ES2131018A1 (es) | 1999-07-01 |
SK64899A3 (en) | 2000-05-16 |
US20030119087A1 (en) | 2003-06-26 |
LT4628B (lt) | 2000-02-25 |
HUP0000797A2 (en) | 2000-07-28 |
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