CN115491361A - 扩环酶及其突变体在生产g-7-adca中的应用 - Google Patents
扩环酶及其突变体在生产g-7-adca中的应用 Download PDFInfo
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Abstract
本发明提供一种扩环酶及其突变体在生产G‑7‑ADCA中的应用。其氨基酸序列如SEQ ID No.5‑13之一所示。本发明通过NCBI序列比对、挖掘，以及定向进化等技术产生了一系列的具有扩环活性的酶，这些酶能够将底物青霉素G一步扩环为G‑7‑ADCA；G‑7‑ADCA可以再通过青霉素酰化酶的作用，脱去侧链，转化为7‑ADCA。因此，这些扩环酶在工业上具有重要的应用价值。
Description
技术领域
本发明涉及扩环酶及其突变体在生产G-7-ADCA中的应用，属于生物技术领域。
背景技术
头孢类抗生素是目前临床上广泛应用的一类抗感染药物，具有低毒性、广谱性等优点。7-氨基-3-脱乙酰氧基头孢烷酸(7-ADCA)是重要的头孢类抗生素合成的母核之一，可以用于合成头孢氨苄、头孢拉定及头孢羟氨苄等，市场用量巨大。
目前，工业上生产7-ADCA主要有两种方法：化学酶法和发酵法。化学酶法制备7-ADCA的工艺以青霉素G为原料，采用化学方法进行氧化、扩环、重排后生成苯乙酰-7-氨基去乙酰氧基头孢烯酸(G-7-ADCA)，然后在青霉素酰化酶的作用下脱去侧链生成7-ADCA。该工艺比较复杂，成本较高，且产生大量的废水和污染，对环境的破坏很大(Wei CL et al,.Applied and Environmental Mirobiology,2005)。在国家节能减排和环保压力下，该工艺正逐步被淘汰。
工业上生产7-ADCA还可以采用发酵法。1998年，Cho等研究发现了扩环酶(DAOCS)能够对青霉素G具有一定的活性，使从青霉素G生产G-7-ADCA成为可能(Cho et al.,PNAS,1998)。因此，目前一些企业中常用的方法是将扩环酶(DAOCS)基因，克隆至产黄青霉菌中，通过在发酵过程中添加己二酸，直接发酵生产G-7-ADCA，然后利用青霉素酰化酶脱去侧链制备7-ADCA。然而，发酵法存在工艺复杂、发酵周期长等问题，且核心技术主要掌握在国外公司手中，目前国内未见采用发酵法生产7-ADCA的工业方法。
全酶法制备7-ADCA具有绿色环保、成本低等优点。全酶法从廉价的青霉素G钾盐出发，经青霉素扩环酶的催化扩环反应生成G-7-ADCA，再经青霉素酰化酶的作用水解除去酰基侧链，生成7-ADCA(如图1所示)。青霉素酰化酶的研究已经很成熟，且已经在工业上应用了很多年。现在用的较多的是固定化的青霉素酰化酶；而扩环酶的活性较低，在从青霉素G合成7-ADCA的路径中是限速步骤。因此，迫切需要提高扩环酶的活性。
青霉素扩环酶(DAOCS)，也称为脱乙酰氧基头孢菌素C合成酶，是一种依赖于Fe2+、O2和α-酮戊二酸的酶，主要催化青霉素的五元噻唑环向六元噻嗪环的转化(扩环)，是催化棒状链霉菌和其他原核生物合成头孢类抗生素的关键步骤。因此，DAOCS具有重要的工业应用价值。
但在实际应用中，发现DAOCS对青霉素G的生物活性较低，无法满足工业应用，这成为了使用全酶法合成7-ADCA的限速步骤。因此，迫切需要对扩环酶进行改造，以期能够大幅提高其对青霉素G的转化效率，为工业应用奠定基础。
目前顶头孢霉菌(C.acremonium)，棒状链霉菌(S.clavuligerus)和诺卡氏菌(N.lactamdurans)来源的DAOCS已经表达纯化出来，并且来源于棒状链霉菌的scDAOCS已经获得了晶体结构，这使得通过蛋白质工程技术改造scDAOCS，提高其催化活性、拓宽其底物谱成为可能，并且已经取得了一些进展。
发明内容
本发明要解决的技术问题是如何提高扩环酶对青霉素G的扩环催化活性。
为了解决上述技术问题，本发明首先提供了一种蛋白质。
本发明提供的蛋白质如下(a)或(b)或(c)或(d)：
(a)其氨基酸序列如SEQ ID No.4所示(命名为H7)，或在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(a)衍生的蛋白质；
(b)其氨基酸序列如SEQ ID No.5所示(命名为E727)，或在(b)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(b)衍生的蛋白质；
(c)其氨基酸序列如SEQ ID No.6所示(命名为E735)，或在(c)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(c)衍生的蛋白质；
(d)其氨基酸序列如SEQ ID No.13(命名为scDAOCS-V303K)所示，或在(d)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(d)衍生的蛋白质。
进一步地，所述蛋白质为如下(1)—(6)中的任一种：
(1)第155位为n1，第184位为n2的SEQ ID NO:5所示的氨基酸序列表示的蛋白质；
(2)第275位为n3，第281位氨基酸残基为n4，第305位为n5的SEQ ID NO:5所示的氨基酸序列表示的蛋白质；
(3)第155位为n1，第184位为n2，第275位为n3，第281位为n4，第305位为n5的SEQID NO:5所示的氨基酸序列表示的蛋白质；
(4)第155位为m1，第184位为m2的SEQ ID NO:6所示的氨基酸序列表示的蛋白质；
(5)第275位为m3，第281位为m4，第305位为m5的SEQ ID NO:6所示的氨基酸序列表示的蛋白质；
(6)第155位为m1，第184位为m2，第275位为m3，第281位为m4，第305位为m5的SEQID NO:6所示的氨基酸序列表示的蛋白质；
具体地，SEQ ID NO：5所示的氨基酸序列中第155位的n1可为：半胱氨酸、酪氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第184位的n2可为：酪氨酸、组氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸或精氨酸，第275位的n3可为：缬氨酸、异亮氨酸、甘氨酸、丙氨酸、亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第281位的n4可为：半胱氨酸、酪氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第305位的n5可为：亮氨酸、甲硫氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸；
SEQ ID NO：6所示的氨基酸序列中第155位的m1可为苯丙氨酸、赖氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、精氨酸或组氨酸，第184位的m2可为天冬氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、赖氨酸、精氨酸或组氨酸，第275位的m3可谷氨酸、组氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、赖氨酸或精氨酸，第281位的m4可为：酪氨酸、脯氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、色氨酸、丝氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第305位的m5可为异亮氨酸、甲硫氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸。
进一步地，所述蛋白质为如下(1)—(6)中的任一种：
(1)将SEQ ID NO:5所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，且将第184位氨基酸残基由酪氨酸突变为组氨酸得到的蛋白质，命名为E727-M2(C155Y/Y184H)，其氨基酸序列如SEQ ID NO:7所示；
(2)将SEQ ID NO:5所示的氨基酸序列的第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由亮氨酸突变为甲硫氨酸得到的蛋白质，命名为E727-M3(V275I/C281Y/L305M)，其氨基酸序列如SEQID NO:8所示；
(3)将SEQ ID NO:5所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，将第184位氨基酸残基由酪氨酸突变为组氨酸，将第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由亮氨酸突变为甲硫氨酸得到的蛋白质，命名为E727-M5(C155Y/Y184H/V275I/C281Y/L305M)，其氨基酸序列如SEQ ID NO:9所示；
(4)将SEQ ID NO:6所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，且将第184位氨基酸残基由酪氨酸突变为组氨酸得到的蛋白质，命名为E735-M2(C155Y/Y184H)，其氨基酸序列如SEQ ID NO:10所示；
(5)将SEQ ID NO:6所示的氨基酸序列的第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由异亮氨酸突变为甲硫氨酸得到的蛋白质，命名为E735-M3(V275I/C281Y/I305M)，其氨基酸序列如SEQ ID NO:11所示；
(6)将SEQ ID NO:6所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，将第184位氨基酸残基由酪氨酸突变为组氨酸，将第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由异亮氨酸突变为甲硫氨酸得到的蛋白质，命名为E735-M5(C155Y/Y184H/V275I/C281Y/I305M)，其氨基酸序列如SEQ ID No.12所示；
编码上述蛋白质的核酸分子也属于本发明的保护范围。
所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA、hnRNA或tRNA等。
所述核酸分子具体为编码上述蛋白质的基因。
含上述核酸分子的重组载体、表达盒、转基因细胞系或重组菌也属于本发明的保护范围。
所述重组载体可为重组表达载体，也可为重组克隆载体。
所述重组表达载体可用现有的表达载体构建。所述表达载体还可包含外源基因的3’端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，它们可单独使用或与其它的启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。
所述表达盒由能够启动所述基因表达的启动子，所述基因，以及转录终止序列组成。
所述蛋白质在作为青霉素扩环酶中的应用也属于本发明的保护范围。
所述蛋白质在作为青霉素扩环酶催化青霉素G扩环生成G-7-ADCA提高青霉素G的转化率中的应用也属于本发明的保护范围。
所述蛋白质或所述核酸分子或所述重组载体、表达盒、转基因细胞系或重组菌在如下任一中的应用也属于本发明的保护范围：
(b1)制备具有青霉素扩环酶活性的产品；
(b2)制备G-7-ADCA。
本发明还保护一种制备G-7-ADCA的方法。
本发明所提供的制备G-7-ADCA的方法，具体可包括步骤：制备所述蛋白质；以所述蛋白质为青霉素扩环酶催化青霉素G扩环生成G-7-ADCA。
本发明还保护一种在青霉素扩环酶催化青霉素G扩环生成G-7-ADCA反应中提高青霉素G的转化率的方法的方法。
本发明所提供的青霉素扩环酶催化青霉素G扩环生成G-7-ADCA反应中提高青霉素G的转化率的方法，其中以所述蛋白质为青霉素扩环酶催化青霉素G扩环生成G-7-ADCA。
本发明通过NCBI序列比对、挖掘，以及定向进化等技术产生了一系列的具有扩环活性的酶，这些酶能够将底物青霉素G一步扩环为G-7-ADCA；G-7-ADCA可以再通过青霉素酰化酶的作用，脱去侧链，转化为7-ADCA。因此，这些扩环酶在工业上具有重要的应用价值。
附图说明
图1为扩环酶催化的7-ADCA反应流程图。
图2为SCHEMA技术用到的序列比对信息。
图3为依据Rossetta Design方法所获Loop环相关突变位点位置示意。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：基于SCHEMA技术产生的扩环酶突变体
1.基因获得以及载体构建
利用SCHEMA技术，分析模拟DNA shuffling进行重组。根据计算模拟的结果，筛选出20条序列。经过密码子优化后，利用全基因合成的方式获得全长基因，并将其构建到pET-24a表达载体上。SCHEMA技术用到的三个扩环酶序列(E632,SEQ ID No.1,8MUT SEQ IDNo.2,及E633 SEQ ID No.3)及比对如图2所示。
经SCHEMA技术比对，获得如表1序列，根据所获序列进行基因合成，表达后根据催化效率进行筛选。
2.蛋白的表达以及活性检测
将上述表达载体转化宿主菌BL21(DE3)中，同时设置阴性对照组(转pET-24a的空载体)和阳性对照组(pET-24a-scDAOCS)，37℃条件下过夜培养；挑取单菌落，转接到含有100μg/mL卡那霉素的LB培养基中，37℃、220rpm条件下培养8-10h；然后，按照1/100的接种量转接到含有100μg/mL的50mLTB培养基中，在37℃下培养至OD600达到0.6左右时，加入0.1mM IPTG诱导蛋白表达，诱导时间16h，获得表达扩环酶的重组菌体。
3.反应体系
收集菌体。将上述菌体倒入50mL离心管中，8000rpm离心10min，获取菌体；菌体用50mM pH 7.4的磷酸盐缓冲液重悬洗涤一次；再用8000rpm离心10min收集菌体；称重。
反应液配方：50mM pH 7.4的磷酸盐缓冲液，50μg/mL FeSO4，0.4mM抗坏血酸，5mM或10mMα-酮戊二酸，5mM或10mM青霉素G。
反应。按照1g湿菌体加入10mL液比例，在菌体中加入反应液，重悬。置于25℃摇床，220rpm，反应2h。
检测G-7-ADCA的生成量。反应2h后，12000rpm，离心1min，取上清进行HPLC检测。
液相色谱检测条件为：色谱柱：Agilent ZORBAX SB-C18 StableBondAnalytical4.6×250mm；流动相：20mM pH 3.0磷酸钠缓冲液：甲醇＝55:45。流速：1mL/min，检测波长215nm。
4.催化青霉素G转化的转化率数据结果
表1利用SCHEMA技术产生的扩环酶突变体转化率
反应条件：
50mM pH 7.4的磷酸盐缓冲液，50μg/mL FeSO4，0.4mM抗坏血酸，10mMα-酮戊二酸，10mM青霉素G,200μL粗酶液。置于25℃摇床，220rpm，反应30min。
5.酶的改造
将活性较高的E727及E735进行双突变、三突变及五突变体的改造，选取高活性突变体H7中的突变位点，移入E727以及E735，获得E727-M2(C155Y/Y184H)、E727-M3(V275I/C281Y/L305M)、E727-M5(C155Y/Y184H/V275I/C281Y/L305M)、E735-M2(C155Y/Y184H)、E735-M3(V275I/C281Y/I305M)、E727-M5(C155Y/Y184H/V275I/C281Y/I305M)突变体。其活性如下：
表2利用SCHEMA程序产生的扩环酶突变体转化率
由表2可知，E727-M2催化青霉素G转化的转化率最高并优于H7。
6.纯酶反应及其HPLC检测
1mL体系进行纯酶活性验证。pH 7.4的PBS缓冲溶液，50μg/mL FeSO4，0.4mM抗坏血酸，5mMα-酮戊二酸，5mM青霉素G,100μL酶液。置于25℃摇床，220rpm，反应30min。HPLC检测检测结果见表3。
表3利用扩环酶突变体催化的酶活
酶活(U)定义为25℃下，在1分钟内能转化1微摩尔底物的酶量；比酶活(U/mg)为每mg蛋白的酶活。
经实验验证，突变体E727-M2的酶活比scH7提高了25％，具有较好的应用前景。
实施例2、基于Rosetta Design方法针对扩环酶H7进行改造所获突变体
1.基因的获得。
所有计算设计都使用美国华盛顿大学David Baker团队开发的Rosetta design软件完成。选择满足所有必需条件的前20个构象进行分析及实验验证。以扩环酶7突变体scDAOCS H7为模板，经过动力学模拟loop环相关突变位点，构建了4个组合突变库，突变库如下表4。突变位点在扩环酶上的位置如图3所示。
表4依据RosettaDesign方法所建立的突变体库
2.突变体库的建立。
用所选残基的相应引物进行位点组合突变，相应的引物如表5。
表5引物序列表
以质粒scDAOCS H7为模板，将表5中引物T72-F1、T72-F2、T72-F3、T72-F4、T73-F1、T73-F2、T73-F3、T73-F4按3：1：1：1：3：1：1：1比例混合，取2μL上述的混合液作为上游引物，取表5中T72-T73-R74-R 2μL作为下游引物，扩增基因片段L1。以质粒scDAOCS H7为模板，将表5中Y94-D95-Y97-F1,Y94-D95-Y97-F2,Y94-D95-Y97-F3按2:1:1比例混合，取2μL作为上游引物，取表4中Y94-D95-Y97-R2μL作为下游引物，扩增基因片段L2。以质粒scDAOCS H7为模板，将表5中L179-F1,L179-F2,L179-F3按2:1:1比例混合，取2μL作为上游引物，取表5中R242-H244-R1，R242-H244-R2按2:1混合，取混合液2μL作为下游引物，扩增基因片段L3。以质粒scDAOCS H7为模板，将表4中N301-F1,N301-F2,Y302-F1，Y302-F2，Y302-F3，V303-F1，V303-F2按2:2:6:6:3:6:2比例混合，取2μL作为上游引物，取表5中N301-Y302-V303-R 2μL作为下游引物，扩增基因片段L4。第一次轮PCR程序：94℃保温2min，(98℃保温15s，55℃保温30s，72℃保温30s)×28个循环，72℃保温5min。
分别取2μL第一轮PCR产物L1,L2,L3,L4为引物，以质粒scDAOCS H7为模板，进行第二PCR,构建构建ML1，ML2，ML3，ML4突变库。第二轮PCR程序：94℃保温2min，(98℃保温15s，60℃保温30s，72℃保温5min)×28个循环，72℃保温10min。
将获得的第二轮PCR产物进行如下操作：
向第二轮PCR的产物中加入1μL的Dpn I酶用于消化质粒模板，37℃恒温条件下处理3h。取2μL酶消化后的第二轮PCR产物电转化进大肠杆菌BL21(DE3)中，将电转后的大肠杆菌BL21(DE3)菌液均匀的涂布在卡那霉素抗性(浓度为50μg/mL)的LB平板上，37℃恒温条件下培养14h后长出单菌落，即突变体库。将一个平板上的所有单菌落刮下送测序验证突变体库的多样性。
3.突变体筛选及反应体系
将突变体库中的单菌落分别接种于96个深孔板中含终浓度50μg/mL卡那霉素的300μL LB培养基中，37℃培养8-12h，同时设置阴性对照组(pET-24a的空载体)和阳性对照组(pET-24a-scDAOCSH7)，然后将200μL种子液接种于含终浓度0.1-0.2mM IPTG和50μg/mL卡那霉素的800μLTB培养基中。在25℃下表达16h后，离心收集细胞菌体，加入400μL的50mM，含50μg/mL FeSO4，0.4mM抗坏血酸，5mM或10mMα-酮戊二酸，5mM或10mM青霉素G的pH7.4磷酸盐缓冲液，25℃反应2h。反应2h后，12000rpm，离心1min，取上清进行HPLC检测。液相色谱检测条件见实施例一。获得酶催化反应转化率数据如下：
表6.突变体转化率
以pET24a为阴性对照，scDAOCS-H7、为阳性对照，scDAOCS-V303K突变体进行摇瓶复筛，同时，对该突变体进行测序。
结果见表7，突变体scDAOCS-H7-V303K全细胞和粗酶相较于scDAOCS-H7均有活力提升。
表7.复筛高活性突变体库的转化率
综上所述，本发明构建了扩环酶突变体，相比scDAOCS-H7扩环酶，酶活力得到了较大的提升，转化率提升了25％，具有广阔的应用前景。
序列表
野生型WT-scDAOCS
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA
SEQ IDNo.1(E632)
MDTTVPTFSLDELQEGLHQDEFRRCLTEKGVFYLTDSGLSEADHKSAKDVAVDFFEHGTEEEKRAVTSPIPTIRRGFSGLESESTAQITNTGTYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVAAPGRDQRAGSSRTSSVFFLRPKSDFSFSVPLARECGFDVSLDGETATFGDWIGGNYVNIRRTSKA
SEQ IDNo.2(8MUT)
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLLMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA
SEQ IDNo.3(E633)
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVVDFFEHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.4(ScDAOCSH7)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA
SEQ ID No.5(E727)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.6(E735)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.7(E727-M2)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.8(E727-M3)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.9(E727-M5)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEH
RSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.10(E735-M2)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.11(E735-M3)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.12(E735-M5)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.13(scDAOCS-V303K)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYKNMRRTSKA。
SEQUENCE LISTING
<110> 中国科学院天津工业生物技术研究所
<120> 扩环酶及其突变体在生产G-7-ADCA中的应用
<130> GNCAQ211831
<160> 1
<170> PatentIn version 3.5
<210> WT-scDAOCS
<211> 311
<212> PRT
<213> C. acremonium
<400> WT-scDAOCS
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
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Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
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Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
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Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
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Ala Val Thr Ser Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu
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Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
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Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
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Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
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Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
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Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
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Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
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Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
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Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
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Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
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Ile Arg Arg Thr Ser Lys Ala
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<210> 1
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<212> PRT
<213> Artificial sequence
<400> 1
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Asp Glu Leu Gln Glu Gly
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Leu His Gln Asp Glu Phe Arg Arg Cys Leu Thr Glu Lys Gly Val Phe
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Tyr Leu Thr Asp Ser Gly Leu Ser Glu Ala Asp His Lys Ser Ala Lys
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Asp Val Ala Val Asp Phe Phe Glu His Gly Thr Glu Glu Glu Lys Arg
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Ala Val Thr Ser Pro Ile Pro Thr Ile Arg Arg Gly Phe Ser Gly Leu
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Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Thr Tyr Thr Asp
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Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr
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Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
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Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
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Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
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Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
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Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile
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Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
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Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
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Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
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Pro Arg His His Val Ala Ala Pro Gly Arg Asp Gln Arg Ala Gly Ser
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Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Lys Ser Asp Phe Ser
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Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp
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Gly Glu Thr Ala Thr Phe Gly Asp Trp Ile Gly Gly Asn Tyr Val Asn
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Ile Arg Arg Thr Ser Lys Ala
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<210> 2
<211> 311
<212> PRT
<213> Artificial sequence
<400> 2
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
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Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
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Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
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Pro Leu Leu Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
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Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
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Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
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Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
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Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp
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Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
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Met Arg Arg Thr Ser Lys Ala
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<210> 3
<211> 311
<212> PRT
<213> Artificial sequence
<400> 3
Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe
20 25 30
Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys
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Asp Val Val Val Asp Phe Phe Glu His Gly Thr Glu Glu Glu Lys Arg
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Ala Val Thr Ser Pro Val Pro Thr Ile Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
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Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
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Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp
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Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Leu Arg Thr Lys Thr Lys Ala
305 310
<210> 4
<211> 311
<212> PRT
<213> Artificial sequence
<400> 4
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
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Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
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Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
290 295 300
Met Arg Arg Thr Ser Lys Ala
305 310
<210> 5
<211> 311
<212> PRT
<213> Artificial sequence
<400> 5
Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe
20 25 30
Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys
35 40 45
Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Leu Arg Thr Lys Thr Lys Ala
305 310
<210> 6
<211> 311
<212> PRT
<213> Artificial sequence
<400> 6
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr
100 105 110
Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Leu Arg Thr Lys Thr Lys Ala
305 310
<210> 7
<211> 311
<212> PRT
<213> Artificial sequence
<400> 7
Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe
20 25 30
Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys
35 40 45
Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His His Asp Leu Ser Ile Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Leu Arg Thr Lys Thr Lys Ala
305 310
<210> 8
<211> 311
<212> PRT
<213> Artificial sequence
<400> 8
Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe
20 25 30
Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys
35 40 45
Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Met Arg Thr Lys Thr Lys Ala
305 310
<210> 9
<211> 311
<212> PRT
<213> Artificial sequence
<400> 9
Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe
20 25 30
Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys
35 40 45
Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His His Asp Leu Ser Ile Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Met Arg Thr Lys Thr Lys Ala
305 310
<210> 10
<211> 311
<212> PRT
<213> Artificial sequence
<400> 10
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr
100 105 110
Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Leu Arg Thr Lys Thr Lys Ala
305 310
<210> 11
<211> 311
<212> PRT
<213> Artificial sequence
<400> 11
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr
100 105 110
Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Met Arg Thr Lys Thr Lys Ala
305 310
<210> 12
<211> 311
<212> PRT
<213> Artificial sequence
<400> 12
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr
100 105 110
Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp
115 120 125
Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu
165 170 175
Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala
225 230 235 240
Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp
275 280 285
Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn
290 295 300
Met Arg Thr Lys Thr Lys Ala
305 310
<210> 13
<211> 311
<212> PRT
<213> Artificial sequence
<400> 13
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
165 170 175
Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Lys Asn
290 295 300
Met Arg Arg Thr Ser Lys Ala
305 310
Claims (10)
1.蛋白质，为如下(b)或(c)或(d)：
(b)其氨基酸序列如SEQ ID No.5所示，或在(b)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(b)衍生的蛋白质；
(c)其氨基酸序列如SEQ ID No.6所示，或在(c)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(c)衍生的蛋白质；
(d)其氨基酸序列如SEQ ID No.13所示，或在(d)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有扩环酶活性的由(d)衍生的蛋白质。
2.根据权利要求1所述的蛋白质，其特征在于：所述蛋白质为如下(1)—(6)中的任一种：
(1)第155位为n1，第184位为n2的SEQ ID NO:5所示的氨基酸序列表示的蛋白质；
(2)第275位为n3，第281位氨基酸残基为n4，第305位为n5的SEQ ID NO:5所示的氨基酸序列表示的蛋白质；
(3)第155位为n1，第184位为n2，第275位为n3，第281位为n4，第305位为n5的SEQ IDNO:5所示的氨基酸序列表示的蛋白质；
(4)第155位为m1，第184位为m2的SEQ ID NO:6所示的氨基酸序列表示的蛋白质；
(5)第275位为m3，第281位为m4，第305位为m5的SEQ ID NO:6所示的氨基酸序列表示的蛋白质；
(6)第155位为m1，第184位为m2，第275位为m3，第281位为m4，第305位为m5的SEQ IDNO:6所示的氨基酸序列表示的蛋白质。
3.根据权利要求2所述的蛋白质，其特征在于：SEQ ID NO：5所示的氨基酸序列中第155位的n1为：半胱氨酸、酪氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第184位的n2为：酪氨酸、组氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸或精氨酸，第275位的n3为：缬氨酸、异亮氨酸、甘氨酸、丙氨酸、亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第281位的n4为：半胱氨酸、酪氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第305位的n5为：亮氨酸、甲硫氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸；
SEQ ID NO：6所示的氨基酸序列中第155位的m1为苯丙氨酸、赖氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、精氨酸或组氨酸，第184位的m2为天冬氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、赖氨酸、精氨酸或组氨酸，第275位的m3谷氨酸、组氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、赖氨酸或精氨酸，第281位的m4为：酪氨酸、脯氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、色氨酸、丝氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸，第305位的m5为异亮氨酸、甲硫氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸。
4.根据权利要求1-3中任一项所述的蛋白质，其特征在于：所述蛋白质为如下(1)—(6)中的任一种：
(1)将SEQ ID NO:5所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，且将第184位氨基酸残基由酪氨酸突变为组氨酸得到的蛋白质，其氨基酸序列如SEQ IDNO:7所示；
(2)将SEQ ID NO:5所示的氨基酸序列的第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由亮氨酸突变为甲硫氨酸得到的蛋白质，其氨基酸序列如SEQ ID NO:8所示；
(3)将SEQ ID NO:5所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，将第184位氨基酸残基由酪氨酸突变为组氨酸，将第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由亮氨酸突变为甲硫氨酸得到的蛋白质，其氨基酸序列如SEQ ID NO:9所示；
(4)将SEQ ID NO:6所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，且将第184位氨基酸残基由酪氨酸突变为组氨酸得到的蛋白质，其氨基酸序列如SEQ IDNO:10所示；
(5)将SEQ ID NO:6所示的氨基酸序列的第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由异亮氨酸突变为甲硫氨酸得到的蛋白质，其氨基酸序列如SEQ ID NO:11所示；
(6)将SEQ ID NO:6所示的氨基酸序列的第155位氨基酸残基由半胱氨酸突变为酪氨酸，将第184位氨基酸残基由酪氨酸突变为组氨酸，将第275位氨基酸残基由缬氨酸突变为异亮氨酸，将第281位氨基酸残基由半胱氨酸突变为酪氨酸，且将第305位氨基酸残基由异亮氨酸突变为甲硫氨酸得到的蛋白质，其氨基酸序列如SEQ ID No.12所示。
5.编码权利要求1-4中任一项所述蛋白质的核酸分子，所述核酸分子为编码权利要求1-4中任一项所述蛋白质的基因。
6.含有权利要求5所述核酸分子的重组载体、表达盒、转基因细胞系或重组菌。
7.权利要求1-4中任一项所述的蛋白质在作为青霉素扩环酶中的应用。
8.权利要求1-4中任一项所述的蛋白质在作为青霉素扩环酶催化青霉素G扩环生成G-7-ADCA提高青霉素G的转化率中的应用。
9.权利要求1-4中任一项所述的蛋白质或权利要求5所述的核酸分子或权利要求6所述的重组载体、表达盒、转基因细胞系或重组菌在如下任一中的应用：
(b1)制备具有青霉素扩环酶活性的产品；
(b2)制备G-7-ADCA。
10.一种制备G-7-ADCA的方法或一种在青霉素扩环酶催化青霉素G扩环生成G-7-ADCA反应中提高青霉素G的转化率的方法，为下述方法中的一种：
1)制备权利要求1-4中任一项所述的蛋白质；以所述蛋白质为青霉素扩环酶催化青霉素G扩环生成G-7-ADCA；
2)发酵含有编码权利要求权利要求1-4中任一项所述蛋白质基因的微生物，以获得的发酵物催化青霉素G扩环生成G-7-ADCA；
3)将编码权利要求1-4中任一项所述蛋白质的基因克隆到产青霉素G的菌体中得到重组菌，发酵该重组菌催化青霉素G扩环得到G-7-ADCA。
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