CN107099523B - 头孢拉定合成酶突变体及其编码基因 - Google Patents
头孢拉定合成酶突变体及其编码基因 Download PDFInfo
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Abstract
本发明公开了一种头孢拉定合成酶突变体及其编码基因。本发明提供了如下A)或B)或C)所示蛋白质:A)将大肠杆菌天然青霉素G酰化酶的β链的第24位苯丙氨酸替换为丙氨酸,β链的第67位丝氨酸替换为丙氨酸后得到的;B)将大肠杆菌天然青霉素G酰化酶的α链的第142位甲硫氨酸替换为亮氨酸,β链的第24位苯丙氨酸替换为丙氨酸,β链的第67位丝氨酸替换为丙氨酸后得到的;C)将A)或B)所限定的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有合成头孢拉定能力的由A)或B)衍生的蛋白质。本发明所提供的蛋白质均具较高的合成头孢拉定的活性和Vs/Vh以及较低的α,为酶法合成头孢拉定的工业化奠定了基础。
Description
技术领域
本发明属于生物化学领域,涉及一种头孢拉定合成酶突变体及其编码基因,特别涉及大肠杆菌青霉素G酰化酶组合突变体、编码基因及其在合成头孢拉定中的应用。
背景技术
半合成β-内酰胺类抗生素是医药行业中使用最广泛的抗生素,每年产量达3万吨,年销售额超过150亿美元,占整个抗生素市场的65%,其中头孢菌素类占比约2/3。与此同时,用于合成β-内酰胺类抗生素及β-内酰胺母核的青霉素G酰化酶的用量也高达1000~3000万吨(H,M,Grulich M,et al.Current state and perspectivesof penicillin G acylase-based biocatalyses.Applied Microbiology andBiotechnology,2014,98(7):2867-2879)。头孢拉定是美国施贵宝制药公司于1972年研究成功的第一代半合成头孢类抗生素,1977年首次在日本上市,也称先锋霉素VI,因其临床使用中的具有很多优点在我国占据很大市场。
头孢拉定的合成方法包括化学法和酶法。目前工业生产中多采用化学法,尤其是混合酸酐法来制备头孢拉定。化学法生产头孢拉定的过程中存在着活化、缩合、基团保护与去保护等多个步骤,合成过程繁琐,反应条件苛刻,产生大量三废。酶法合成头孢拉定则工艺简单,反应条件温和,生产周期短,并且绿色环保(芮菊,张体磊.酶法合成7-ACA及头孢菌素类抗生素的研究进展.中国药学会学术年会暨中国药师周.2008:348-348)。酶法合成头孢拉定如图1所示。目前报道的青霉素G酰化酶及其突变体合成头孢拉定的收率不够高,酶法未能工业化。但随着环保要求越来越高,开发高质量的头孢拉定合成酶显得尤为重要和迫切。
目前酶法合成头孢拉定的工艺主要是,利用固定的青霉素G酰化酶及其突变体催化二氢苯甘氨酸甲酯(2,5-dihydrophenylglycine methyl ester,DHME)与7-氨基去乙酰氧基头孢烷酸(7-aminodesacetoxycephalosporanic acid,7-ADCA)缩合生成头孢拉定(Cephradine)和甲醇(YE Shu-xiang,XU Cheng-miao,WANG Jia-bing.Synthesis ofCephradine with the Immobilized Penicillin Acylase.Chinese Journal ofPharmaceuticals,2007,38:619-620)。上述反应是动力学控制的过程,除合成反应外,还有头孢拉定水解和DHME水解两个反应,如图2所示。在动力学控制的合成反应中,有两个参数非常重要。第一个参数是Vs/Vh,即初始合成水解比,反映的是在一定反应条件下,酶对合成与水解反应的倾向性。Vs/Vh越大越好,越大说明越有利于合成头孢拉定,反之则倾向于水解生成二氢苯甘氨酸(2,5-dihydrophenylglycine,DHPG)。另外一个参数是α,为酶催化水解头孢拉定的催化效率(kcat/Km)cephradine与水解DHME的催化效率(kcat/Km)DHME之比。α越小越好,α越小表明酶越不容易水解产物头孢拉定,从而有利于头孢拉定的积累,否则合成得到的头孢拉定又很快水解掉,导致头孢拉定的总收率低。工业化要求Vs/Vh大于10,α小于0.1,这样有利于底物7-ADCA和DHME尽可能地转化成头孢拉定,减少水解产物的生成(WynandB.L.Alkema,Anne-Jan Dijkhuis,Erik de Vries and Dick B.Janssen.The role ofhydrophobic active-site residues in substrate specificity and acyl transferactivity of penicillin acylase.European Journal of Biochemistry,2002,269:2093–2100)。。
天然青霉素G酰化酶水解与合成头孢拉定的活性较高,但合成头孢拉定的Vs/Vh很低,而α很高。虽然目前有关酶法合成β-内酰胺类抗生素的报道较多,但研究主要集中在氨苄西林、阿莫西林、头孢氨苄、头孢羟氨苄以及头孢克洛等几种抗生素上(H,M,Grulich M,Kyslík P.Current state and perspectives of penicillin Gacylase-based biocatalyses.Applied Microbiology and Biotechnology,2014,98:2867–2879)。利用青霉素G酰化酶及其突变体合成头孢拉定的相关报道较少。目前只有专利WO2005/003367、WO2008/110527和WO2011/073166报道了利用青霉素G酰化酶单点突变体βF24A合成头孢拉定时,在相同反应条件下其Vs/Vh比野生型酶高,但并没有基于βF24A实现酶法合成头孢拉定的工业化报道。
发明内容
本发明的目的是在大肠杆菌天然青霉素G酰化酶及单点突变βF24A基础上,提高合成头孢拉定Vs/Vh与降低α的组合突变。
本发明首先要求保护如下(A)或(B)或(C)所示的蛋白质:
(A)将大肠杆菌天然青霉素G酰化酶的β链的第24位苯丙氨酸替换为丙氨酸,β链的第67位丝氨酸替换为丙氨酸后得到的;
(B)将大肠杆菌天然青霉素G酰化酶的α链的第142位甲硫氨酸替换为亮氨酸,β链的第24位苯丙氨酸替换为丙氨酸,β链的第67位丝氨酸替换为丙氨酸后得到的;
(C)将(A)或(B)所限定的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有合成头孢拉定能力的由(A)或(B)衍生的蛋白质。
进一步,所述蛋白质为如下(a)或(b)或(c):
(a)由序列表中序列2所示的氨基酸序列组成的蛋白质(PGA_M1);
(b)由序列表中序列4所示的氨基酸序列组成的蛋白质(PGA_M2);
(c)将序列表中序列2或序列4的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有合成头孢拉定能力的由(a)或(b)衍生的蛋白质。
其中,序列表中序列2所示的蛋白PGA_M1有846个氨基酸残基,第1~26位为信号肽,第27~235位为PGA_M1的α链、第236-289位为连接肽、第290-846位为PGA_M1的β链。序列表中序列4所示的蛋白PGA_M2有846个氨基酸残基,第1~26位为信号肽,第27-235位为PGA_M2的α链、第236-289位为连接肽、第290-846位为PGA_M2的β链。蛋白质PGA_M1的具体示意图如图3所示,PGA_M2的具体示意图与之相同。
为了便于所述蛋白质的纯化,可在所述蛋白质的氨基末端或羧基末端连接上如下表所示的标签。
表:标签的序列
标签 | 残基 | 序列 |
Poly-Arg | 5-6(通常为5个) | RRRRR |
Poly-His | 2-10(通常为6个) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
编码所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。
在本发明的一个实施例中,所述核酸分子具体为编码所述蛋白质的基因,所述基因具体可为如下任一所示的DNA分子:
1)序列表中序列1所示的DNA分子;
2)序列表中序列3所示的DNA分子;
3)在严格条件下与1)或2)限定的DNA分子杂交且编码所述蛋白质的DNA分子;
4)与1)或2)或3)限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性,且编码所述蛋白质的DNA分子。
上述严格条件可为用6×SSC,0.5%SDS的溶液,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
其中,序列表中的序列1由2538个碱基组成,其开放阅读框架(ORF)为第1-2538位碱基,编码具有序列表中序列2的氨基酸序列的蛋白,其中第79-705位碱基编码PGA_M1的α链;第868-2538位碱基编码PGA_M1的β链。序列表中的序列3由2538个碱基组成,其开放阅读框架(ORF)为第1-2538位碱基,编码具有序列表中序列4的氨基酸序列的蛋白,其中第79-705位碱基编码PGA_M2的α链;第868-2538位碱基编码PGA_M2的β链。
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。
在本发明的一个实施例中,所述重组载体为在pET28a(+)载体的多克隆位点间插入所述基因得到的重组质粒。
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。
在本发明的一个实施例中,所述重组菌为含有所述重组载体的大肠杆菌;所述大肠杆菌具体如BL21(DE3)。
所述蛋白质在作为青霉素G酰化酶中的应用也属于本发明的保护范围。
所述蛋白质在如下任一中的应用也属于本发明的保护范围:
(a1)作为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中提高Vs/Vh;
(a2)作为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中降低α。
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G酰化酶活性的产品;
(b2)制备头孢拉定或其他β-内酰胺类抗生素。
本发明还保护一种制备头孢拉定的方法。
本发明所提供的制备头孢拉定的方法,具体可包括步骤:制备所述蛋白质;以所述蛋白质为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定。
其中,制备所述蛋白质的方法包括如下步骤:将编码所述蛋白质的编码基因导入大肠杆菌后,培养重组菌,并加入终浓度为0.5mM的IPTG,在20℃下诱导培养14h。
本发明还保护一种在青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中提高Vs/Vh和/或降低α的方法。
本发明所提供的在青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中提高Vs/Vh和/或降低α的方法,其中以所述蛋白质为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定。
上述出现的所述Vs/Vh为初始合成水解比。所述Vs/Vh反映的是在一定反应条件下,酶对合成与水解反应的倾向性。Vs/Vh越大越好,越大说明越有利于合成头孢拉定,反之则倾向于水解生成二氢苯甘氨酸(2,5-dihydrophenylglycine,DHPG)。
上述出现的所述α为酶催化水解头孢拉定的催化效率(kcat/Km)cephradine与水解DHME的催化效率(kcat/Km)DHME之比。α越小越好,α越小表明酶越不容易水解产物头孢拉定,从而有利于头孢拉定的积累,否则合成得到的头孢拉定又很快水解掉,导致头孢拉定的总收率低。
与现有技术相比,本发明具有如下优点:
(1)本发明中青霉素G酰化酶的两个突变体PGA_M1、PGA_M2表达水平高,可在大肠杆菌细胞内高水平表达;(2)本发明中青霉素G酰化酶的两个突变体之一PGA_M1与其野生型酶相比,Vs/Vh从1.23提高到7.19,同时α从6.14降低到0.31,合成头孢拉定的酶活力(0~30分钟内)从0.59U/mg提高到0.77U/mg(1U为1个酶活力单位,是指在本实验测定条件下在1分钟内合成出1微摩尔头孢拉定的酶量,下同);(3)本发明中青霉素G酰化酶的两个突变体之一PGA_M1与突变体βF24A相比Vs/Vh从1.75提高到7.19,同时α从1.72降低到0.31,合成头孢拉定的酶活力(0~30分钟内)从0.54U/mg提高到0.77U/mg;(4)本发明中青霉素G酰化酶的两个突变体之一PGA_M2与野生型酶相比,Vs/Vh从1.23提高到14.42,同时α从6.14降低到0.51,合成头孢拉定的酶活力(0~30分钟内)从0.59U/mg提高到0.87U/mg;(5)本发明中青霉素G酰化酶的两个突变体之一PGA_M2与突变体βF24A相比,Vs/Vh从1.75提高到14.42,同时α从1.72降低到0.51,合成头孢拉定的酶活力(0~30分钟内)从0.54U/mg提高到0.87U/mg;(6)本发明PGA_M1与PGA_M2均具较高的合成头孢拉定的活性和Vs/Vh以及较低的α,为酶法合成头孢拉定的工业化奠定了基础。
附图说明
图1为酶催化DHME和7-ADCA生成头孢拉定和甲醇的反应式。
图2为动力学控制的头孢拉定合成过程。当DHME与酶作用形成酰化态酶后,可以有两种去向,7-ADCA亲核进攻合成得到头孢拉定,水分子亲核进攻水解生成DHPG。头孢拉定也能水解生成DHPG与7-ADCA。
图3为本发明提供的青霉素G酰化酶突变体pET28a-PGA_M1、pET28a-PGA_M2构成示意图,均由四部分组成。分别为:(1)信号肽,包含PGA_M1、PGA_M2上第1-26位的26个氨基酸残基;(2)酶突变体α链的肽段,包含PGA_M1、PGA_M2上27-235位的209个氨基酸残基;(3)连接肽段,包含PGA_M1、PGA_M2上236-289位的54个氨基酸残基;(4)酶突变体β链的肽段,包含PGA_M1、PGA_M2上290-846位的557个氨基酸残基。
图4为HPLC检测青霉素G酰化酶突变体PGA_M1水解头孢拉定和DHME反应后的样品图谱。其中图(A)为头孢拉定的水解,(B)为DHME的水解。图中Cephradine即为头孢拉定。
图5为HPLC检测青霉素G酰化酶突变体PGA_M1合成头孢拉定反应后的样品色谱图;其中,(A)为含有失活目的蛋白的头孢拉定合成空白实验;(B)为含有目的蛋白的样品反应20小时后的图谱。可以看到图谱(A)中在15.0min附近基本没有合成产物头孢拉定的生成,而在4.0min左右出现巨大的7-ADCA的峰;图谱(B)中在15.0min附近有明显的合成产物头孢拉定的生成,同时在4.0min左右7-ADCA的峰面积和峰高有明显降低,说明产物头孢拉定确实由底物7-ADCA转化生成。图中Cephradine即为头孢拉定。
图6为HPLC检测青霉素G酰化酶突变体PGA_M2合成头孢拉定反应后的样品色谱图;其中,(A)为含有失活目的蛋白的头孢拉定合成空白实验;(B)为含有目的蛋白的样品反应20小时后的图谱。可以看到图谱(A)中在15.0min附近基本没有合成产物头孢拉定的生成,而在4.0min左右出现巨大的7-ADCA的峰;图谱(B)中在15.0min附近有明显的合成产物头孢拉定的生成,同时在4.0min左右7-ADCA的峰面积和峰高有明显降低,说明产物头孢拉定确实由底物7-ADCA转化生成。并且与图5对比可以看出,在相同的条件下,图6(B)中头孢拉定的峰面积更大。图中Cephradine即为头孢拉定。
图7为青霉素G酰化酶野生型酶(WT)、突变体βF24A、突变PGA_M1和突变PGA_M2在5小时反应时间内合成得到的头孢拉定与水解得到的DHPG的浓度变化。其中,WT组的酶浓度为0.18μM;突变体βF24A组的酶浓度为0.13μM;突变体PGA_M1组的酶浓度为0.42μM;突变体PGA_M2组的酶浓度为0.35μM。可以看出:1)WT反应中,5小时后头孢拉定浓度曲线已经走平,但DHPG浓度仍在升高,且超过头孢拉定;2)突变体βF24A反应中,头孢拉定和DHPG的浓度都在上升,且DHPG的浓度在不断接近头孢拉定;3)和4)突变体PGA_M1和PGA_M2的反应中,头孢拉定的浓度远高于DHPG的浓度,说明突变体PGA_M1和PGA_M2合成头孢拉定的Vs/Vh高,比单点突变βF24A更好。
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、青霉素G酰化酶突变体的制备纯化
一、青霉素G酰化酶突变体编码基因与重组表达载体的构建
根据文献获得了野生的编码大肠杆菌青霉素G酰化酶的基因。序列通过Overlapping PCR扩增出本发明中的pET28a-PGA_M1基因(序列1)和pET28a-PGA_M2基因(序列3)。组氨酸标签His6-tag添加于基因序列碳端末尾以利于后续的纯化步骤。利用NcoI和XhoI双酶切并纯化后,与用同样内切酶进行双切的表达载体pET28a(+)(含卡那霉素抗性基因)过夜连接,随后转入化转感受态细胞BL21(DE3)中,即获得重组表达质粒pET28a-PGA_M1和pET28a-PGA_M2。
经测序,pET28a-PGA_M1和pET28a-PGA_M2序列正确。
重组质粒pET28a-PGA_M1结构描述:将序列表中序列1所示DNA片段插入pET28a(+)的NcoI和XhoI位点间后得到的重组质粒。
重组质粒pET28a-PGA_M2结构描述:将序列表中序列3所示DNA片段插入pET28a(+)的NcoI和XhoI位点间后得到的重组质粒。
二、重组菌的获得
将步骤一获得的重组质粒pET28a-PGA_M1和pET28a-PGA_M2分别转化大肠杆菌BL21(DE3)(Takara公司,大连),得到重组菌Escherichia coli BL21(DE3)/pET28a-PGA_M1以及Escherichia coli BL21(DE3)/pET28a-PGA_M2。
将得到的重组菌分别在含卡那霉素(50μg/mL)的LB平板上划线,37℃下过夜培养,再挑取生长良好的的单菌落接种于100mL LB液体培养基(含50μg/mL卡那霉素)中,在37℃、200rpm培养7h以上,制备成种子液,以1mL:100mL的接种量进行步骤三的实验。
三、青霉素G酰化酶突变体的获得
1、酶突变体的表达
以上述1mL:100mL的接种量,取5mL种子液转接于500mL的新鲜LB液体培养基(含50μg/mL卡那霉素)中,在37℃、200rpm的条件下,继续培养至OD600为0.6~0.8,制备成发酵液。然后加入终浓度为0.5mM的IPTG,在20℃,120rpm条件下培养14h,诱导青霉素G酰化酶突变体基因的表达。
诱导表达结束后,将所有500mL菌液在4℃,10,000rpm(相当于11,000g)的条件下离心10min,离心得到的菌体沉淀用磷酸盐缓冲液重悬(100mM磷酸钾盐,pH7.0)。重悬液用超声破碎方法提取可溶蛋白(超声时间4s、间隔时间6s,60%功率,20min)。将裂解液在4℃,10,000rpm(相当于11,000g)条件下离心10min,取上清即为含目的蛋白粗酶液;再将含目的蛋白粗酶液在4℃,12,000rpm(相当于13,500g)条件下离心10min,进一步去除超声破碎带来的细胞杂质。
2、酶突变体的纯化
将步骤1处理过的含目的蛋白的粗酶液上样至Ni-NTA柱,通过镍离子作为亲和离子,利用不同咪唑浓度梯度洗脱目的蛋白。由于青霉素G酰化酶及其突变体在280nm有特定吸收峰,故在纯化过程中,用280nm检测蛋白峰可以有效防止杂蛋白干扰。
首先,使用结合缓冲液A(100mM磷酸钾盐,pH 8.0,含500mM NaCl和20mM咪唑)预平衡柱床,随后,使用结合缓冲液B(100mM磷酸钾盐,pH 8.0,含500mM NaCl和50mM咪唑)洗脱杂蛋白,至洗脱液在280nm下的吸光度与缓冲液B基本相同。使用洗脱缓冲液(100mM磷酸钾盐,pH 8.0,含500mM NaCl和200mM咪唑)收集目的蛋白,并用10kDaUltra-0.5超滤离心管(Millipore)浓缩目的蛋白,并进行两次脱盐,移除目的蛋白中的咪唑和NaCl组分。纯化后的目的蛋白保存于磷酸盐缓冲液中(100mM磷酸钾盐,pH 7.0),并置于4℃冷藏保存。目的蛋白纯度由SDS-PAGE(5%积层胶,12%分离胶)检验,根据SDS-PAGE结果,收集的蛋白纯度大于90%。纯化后得到的目的蛋白浓度通过Bradford法并使用牛血清蛋白作为标准试剂测得,测定所使用的吸光度为595nm。
采用类似的方法制备并纯化获得野生型青霉素G酰化酶,以及青霉素G酰化酶单点突变体βF24A。其中,野生型青霉素G酰化酶的编码基因如序列表中序列5所示;青霉素G酰化酶单点突变体βF24A的编码基因如序列表中序列6所示。
实施例2、酶突变体的水解DHME催化活性测定
通过HPLC(LC-20AT,岛津公司)分析DHME转化产物,从而测定实施例1制备的两种酶突变体PGA_M1和PGA_M2水解DHME的催化活性和动力学参数。所选用色谱柱为InertsilC18反相柱(GL Sciences,5μm,150×4.6mm)。反应过程中酶突变体与底物配比为:0.5mL纯化后的目的蛋白(足量,保存于100mM磷酸钾盐缓冲液,pH 7.0)与0.5mL DHME溶液(pH 7.0,浓度呈梯度变化,最高浓度为1g/100mL),在22℃下反应8min。反应结束后添加1mL甲醇终止反应。HPLC的流动相配比为:75%磷酸钾盐缓冲液(30mM,pH 4.5):25%甲醇(v/v),流速0.8mL/min,检测波长为230nm,柱温箱恒定为25℃。
实验同时设置以等量野生型青霉素G酰化酶(或者青霉素G酰化酶单点突变体βF24A)替换酶突变体PGA_M1(或PGA_M2)的对照。
结果显示:PGA_M1和PGA_M2均在保留时间tR=3.1min附近出现产物DHPG色谱峰,在保留时间tR=6.0min附近出现底物DHME色谱峰。HPLC检测青霉素G酰化酶突变体PGA_M1水解DHME的色谱图如图4中(B)所示。
酶催化动力学参数(kcat、Km、kcat/Km)通过Lineweaver-Burk法拟合实验数据得到。
本发明中头孢菌素酰化酶的突变体PGA_M1水解DHME的kcat/Km为0.21mM-1s-1,与野生型酶相比(kcat/Km=0.15mM-1s-1),水解DHME的活性提高了40%,与突变体βF24A相比(kcat/Km=0.13mM-1s-1),水解DHME的活性提高了62%;突变体PGA_M2水解DHME的催化效率kcat/Km为0.78mM-1s-1,与野生型酶相比提高了4.2倍,与突变体βF24A相比提高了5倍。
实施例3、酶突变体的水解头孢拉定催化活性测定
通过HPLC(LC-20AT,岛津公司)分析头孢拉定转化产物,从而测定实施例1制备的两种酶突变体PGA_M1和PGA_M2水解头孢拉定的催化活性和动力学参数。所选用色谱柱为Inertsil C18反相柱(GL Sciences,5μm,150×4.6mm)。反应过程中酶突变体与底物配比为:0.5mL纯化后的目的蛋白(足量,保存于100mM磷酸钾盐缓冲液,pH 7.0)与0.5mL头孢拉定溶液(pH 7.0,浓度呈梯度变化,最高浓度为2g/100mL),在22℃下反应8min。反应结束后添加1mL甲醇终止反应。HPLC的流动相配比为:75%磷酸钾盐缓冲液(30mM,pH 4.5):25%甲醇(v/v),流速0.8mL/min,检测波长为254nm,柱温箱恒定为25℃。
实验同时设置以等量野生型青霉素G酰化酶(或者青霉素G酰化酶单点突变体βF24A)替换酶突变体PGA_M1(或PGA_M2)的对照。
结果显示:PGA_M1和PGA_M2均在保留时间tR=2.8min附近出现水解产物7-ADCA的色谱峰,在保留时间tR=7.2min附近出现底物头孢拉定的色谱峰。酶催化动力学参数(kcat、Km、kcat/Km)通过Lineweaver-Burk法拟合实验数据得到。HPLC检测青霉素G酰化酶突变体PGA_M1水解头孢拉定的色谱图如图4中(A)所示。
本发明中头孢菌素酰化酶的突变体PGA_M1水解头孢拉定的kcat/Km为0.06mM-1s-1,降低为野生型酶(kcat/Km=0.90mM-1s-1)的6.7%,降低为βF24A(kcat/Km=0.23mM-1s-1)的26%;突变体PGA_M2水解头孢拉定的kcat/Km为0.27mM-1s-1,降低为野生型酶的30%,与βF24A相比提高了4%。
实施例4、酶突变体合成头孢拉定Vs/Vh测定
通过HPLC(LC-20AT,岛津公司)分析头孢拉定转化产物,从而测定实施例1制备的两种酶突变体PGA_M1和PGA_M2水解头孢拉定的催化活性和动力学参数。所选用色谱柱为hypersil C18反相柱(伊利特,5μm,250×4.6mm)。反应过程中酶突变体与底物配比为:0.5mL纯化后的目的蛋白(足量,保存于100mM磷酸钾盐缓冲液,pH 7.0)与0.5mL底物混合溶液(pH 7.0,DHME为36mM,7-ADCA为30mM),在22℃下分别反应0.5、1、2、3、4、5小时。反应结束后添加1mL甲醇终止反应。HPLC的流动相配比为:75%磷酸钾盐缓冲液(30mM,pH 4.5):25%甲醇(v/v),流速0.8mL/min,检测波长为230nm,柱温箱恒定为25℃
实验同时设置将实施例1制备的两种酶突变体PGA_M1和PGA_M2进行失活处理的对照,失活处理的方法为:取10mL圆底离心管,加入0.5mL纯化后的目的蛋白(足量,保存于100mM磷酸钾盐缓冲液,pH 7.0),然后加入1mL色谱醇甲醇,使酶失活,然后再加入相应量的底物溶液。
实验同时设置以等量野生型青霉素G酰化酶(或者青霉素G酰化酶单点突变体βF24A)替换酶突变体PGA_M1(或PGA_M2)的对照。
结果如图5和图6所示:PGA_M1和PGA_M2均在保留时间tR=15.0min附近出现合成产物头孢拉定的色谱峰,在保留时间tR=4.0min附近出现底物7-ADCA的色谱峰。而在tR=4.7min附近出现DHPG的色谱峰。
通过实施例2和实施例3中测定的催化效率,可以计算出野生型酶、βF24A、突变体PGA_M1和PGA_M2的α分别为6.14、0.31和0.51。此外本发明还测定了专利WO2005/003367、WO2008/110527和WO2011/073166报道的突变βF24A的α为1.72。
青霉素G酰化酶野生型酶(WT)、突变体βF24A、突变PGA_M1和突变PGA_M2在5小时反应时间内合成得到的头孢拉定与水解得到的DHPG的浓度变化如图7所示。本发明中以反应0.5h时反应液中头孢拉定的浓度与DHPG的浓度比作为初始合成水解比Vs/Vh。本发明中测得野生型酶、βF24A、突变体PGA_M1和PGA_M2合成头孢拉定的Vs/Vh分别为1.23、1.75、7.19和14.42。
<110> 清华大学
<120> 头孢拉定合成酶突变体及其编码基因
<130> CGGNQALN176076
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 2538
<212> DNA
<213> 人工序列
<220>
<223>
<400> 1
atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60
agcttacctg cactggctga acagtctagc tctgagatta agattgtgcg tgacgaatac 120
ggcatgcctc atatctacgc caacgacacc tggcacctgt tctacggcta tggctacgtg 180
gtagcacagg accgtctgtt tcagatggaa atggctcgtc gtagcaccca gggcaccgta 240
gcagaagtgc tgggcaaaga cttcgtgaag ttcgacaaag acattcgtcg caactactgg 300
ccggacgcga tccgtgcgca gattgcggcg ctgagcccgg aagacatgag catcctgcaa 360
ggttacgctg atggtatgaa cgcatggatc gataaagtga acacgaaccc tgaaaccctg 420
ctgccgaaac agttcaacac ctttggcttc accccgaaac gctgggaacc gttcgatgtg 480
gcgatgatct tcgtgggcac tatggccaat cgcttctctg attctacctc cgagatcgac 540
aatctggccc tgctgaccgc actgaaagac aagtatggtg tcagccaggg catggcggtg 600
ttcaaccagc tgaaatggct ggtcaacccg tccgcgccga ctacgatcgc ggtgcaggag 660
tctaactacc cgctgaaatt caaccaacag aacagccaga cggctgcact gctgccgcgt 720
tatgatctgc cagcgccaat gctggatcgc ccggctaaag gtgcagacgg tgctctgctg 780
gcgctgactg ctggcaaaaa tcgcgaaacc atcgttgctc aattcgcaca gggcggtgcg 840
aatggtctgg ctggctatcc gaccacctct aacatgtggg tgatcggtaa atctaaagcg 900
caggacgcga aagcgatcat ggttaacggt ccgcaggcgg gctggtacgc tccggcctat 960
acctacggta tcggcctgca tggtgcaggc tatgacgtca ctggtaacac tccgttcgcg 1020
tatcctggtc tggttttcgg tcacaacggt gttatcagct ggggtgcgac cgcaggcttt 1080
ggtgatgatg ttgacatttt tgctgaacgt ctgagcgcag aaaaaccggg ctactacctg 1140
cacaacggta aatgggtaaa aatgctgtct cgcgaagaga ccatcacggt taaaaacggt 1200
caggcggaaa ctttcactgt gtggcgcacc gtacacggca acatcctgca gaccgaccag 1260
actactcaga ctgcttacgc taaatcccgt gcctgggacg gtaaggaagt agcatccctg 1320
ctggcgtgga cgcaccagat gaaagccaaa aactggcagg agtggaccca gcaagcggcc 1380
aaacaggcac tgacgattaa ctggtattac gcagacgtga acggtaacat cggttatgtt 1440
cacaccggcg catacccgga ccgtcagtct ggccatgatc cgcgtctgcc ggtgccaggc 1500
actggcaaat gggattggaa aggtctgctg ccgttcgaaa tgaatccaaa agtatacaac 1560
ccgcagtccg gttacattgc caactggaac aactccccgc agaaagacta cccggcatct 1620
gatctgtttg cgttcctgtg gggtggtgcc gatcgtgtta ccgagattga ccgcctgctg 1680
gaacagaaac cgcgcctgac ggccgatcag gcatgggacg ttatccgtca aacttcccgt 1740
caggacctga acctgcgtct gttcctgccg actctgcaag cagcaacgtc cggtctgact 1800
cagagcgatc ctcgtcgtca actggttgag acgctgactc gttgggatgg catcaacctg 1860
ctgaacgacg acggtaaaac ctggcaacaa ccaggttctg ctatcctgaa cgtttggctg 1920
acctccatgc tgaaacgtac cgtcgttgcg gctgtaccga tgccgtttga taagtggtac 1980
tctgctagcg gctatgaaac cacccaggat ggcccaaccg gctccctgaa catttctgtt 2040
ggcgcgaaaa tcctgtatga agcggtacag ggtgataaat cccctatccc acaggctgtt 2100
gatctgttcg ccggcaaacc gcagcaggaa gtagttctgg ctgcgctgga agacacctgg 2160
gaaactctgt ctaagcgtta cggtaacaac gttagcaact ggaaaacccc ggccatggct 2220
ctgaccttcc gtgcgaataa tttcttcggt gttccgcagg ctgcggcgga agaaacccgc 2280
catcaggctg aataccaaaa ccgcggcacc gaaaacgaca tgatcgtttt ttccccgact 2340
acctctgatc gtccggtcct ggcttgggac gtcgtagctc cgggtcagag cggttttatt 2400
gcaccggatg gtaccgtcga taagcactat gaagatcagc tgaagatgta cgagaacttt 2460
ggccgcaagt ctctgtggct gaccaaacag gacgtggagg cccacaaaga atctcaggaa 2520
gttctgcacg ttcagcgt 2538
<210> 2
<211> 846
<212> PRT
<213> 人工序列
<220>
<223>
<400> 2
Met Lys Asn Arg Asn Arg Met Ile Val Asn Cys Val Thr Ala Ser Leu
1 5 10 15
Met Tyr Tyr Trp Ser Leu Pro Ala Leu Ala Glu Gln Ser Ser Ser Glu
20 25 30
Ile Lys Ile Val Arg Asp Glu Tyr Gly Met Pro His Ile Tyr Ala Asn
35 40 45
Asp Thr Trp His Leu Phe Tyr Gly Tyr Gly Tyr Val Val Ala Gln Asp
50 55 60
Arg Leu Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Thr Val
65 70 75 80
Ala Glu Val Leu Gly Lys Asp Phe Val Lys Phe Asp Lys Asp Ile Arg
85 90 95
Arg Asn Tyr Trp Pro Asp Ala Ile Arg Ala Gln Ile Ala Ala Leu Ser
100 105 110
Pro Glu Asp Met Ser Ile Leu Gln Gly Tyr Ala Asp Gly Met Asn Ala
115 120 125
Trp Ile Asp Lys Val Asn Thr Asn Pro Glu Thr Leu Leu Pro Lys Gln
130 135 140
Phe Asn Thr Phe Gly Phe Thr Pro Lys Arg Trp Glu Pro Phe Asp Val
145 150 155 160
Ala Met Ile Phe Val Gly Thr Met Ala Asn Arg Phe Ser Asp Ser Thr
165 170 175
Ser Glu Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Lys Tyr
180 185 190
Gly Val Ser Gln Gly Met Ala Val Phe Asn Gln Leu Lys Trp Leu Val
195 200 205
Asn Pro Ser Ala Pro Thr Thr Ile Ala Val Gln Glu Ser Asn Tyr Pro
210 215 220
Leu Lys Phe Asn Gln Gln Asn Ser Gln Thr Ala Ala Leu Leu Pro Arg
225 230 235 240
Tyr Asp Leu Pro Ala Pro Met Leu Asp Arg Pro Ala Lys Gly Ala Asp
245 250 255
Gly Ala Leu Leu Ala Leu Thr Ala Gly Lys Asn Arg Glu Thr Ile Val
260 265 270
Ala Gln Phe Ala Gln Gly Gly Ala Asn Gly Leu Ala Gly Tyr Pro Thr
275 280 285
Thr Ser Asn Met Trp Val Ile Gly Lys Ser Lys Ala Gln Asp Ala Lys
290 295 300
Ala Ile Met Val Asn Gly Pro Gln Ala Gly Trp Tyr Ala Pro Ala Tyr
305 310 315 320
Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr Asp Val Thr Gly Asn
325 330 335
Thr Pro Phe Ala Tyr Pro Gly Leu Val Phe Gly His Asn Gly Val Ile
340 345 350
Ser Trp Gly Ala Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala
355 360 365
Glu Arg Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Leu His Asn Gly Lys
370 375 380
Trp Val Lys Met Leu Ser Arg Glu Glu Thr Ile Thr Val Lys Asn Gly
385 390 395 400
Gln Ala Glu Thr Phe Thr Val Trp Arg Thr Val His Gly Asn Ile Leu
405 410 415
Gln Thr Asp Gln Thr Thr Gln Thr Ala Tyr Ala Lys Ser Arg Ala Trp
420 425 430
Asp Gly Lys Glu Val Ala Ser Leu Leu Ala Trp Thr His Gln Met Lys
435 440 445
Ala Lys Asn Trp Gln Glu Trp Thr Gln Gln Ala Ala Lys Gln Ala Leu
450 455 460
Thr Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly Asn Ile Gly Tyr Val
465 470 475 480
His Thr Gly Ala Tyr Pro Asp Arg Gln Ser Gly His Asp Pro Arg Leu
485 490 495
Pro Val Pro Gly Thr Gly Lys Trp Asp Trp Lys Gly Leu Leu Pro Phe
500 505 510
Glu Met Asn Pro Lys Val Tyr Asn Pro Gln Ser Gly Tyr Ile Ala Asn
515 520 525
Trp Asn Asn Ser Pro Gln Lys Asp Tyr Pro Ala Ser Asp Leu Phe Ala
530 535 540
Phe Leu Trp Gly Gly Ala Asp Arg Val Thr Glu Ile Asp Arg Leu Leu
545 550 555 560
Glu Gln Lys Pro Arg Leu Thr Ala Asp Gln Ala Trp Asp Val Ile Arg
565 570 575
Gln Thr Ser Arg Gln Asp Leu Asn Leu Arg Leu Phe Leu Pro Thr Leu
580 585 590
Gln Ala Ala Thr Ser Gly Leu Thr Gln Ser Asp Pro Arg Arg Gln Leu
595 600 605
Val Glu Thr Leu Thr Arg Trp Asp Gly Ile Asn Leu Leu Asn Asp Asp
610 615 620
Gly Lys Thr Trp Gln Gln Pro Gly Ser Ala Ile Leu Asn Val Trp Leu
625 630 635 640
Thr Ser Met Leu Lys Arg Thr Val Val Ala Ala Val Pro Met Pro Phe
645 650 655
Asp Lys Trp Tyr Ser Ala Ser Gly Tyr Glu Thr Thr Gln Asp Gly Pro
660 665 670
Thr Gly Ser Leu Asn Ile Ser Val Gly Ala Lys Ile Leu Tyr Glu Ala
675 680 685
Val Gln Gly Asp Lys Ser Pro Ile Pro Gln Ala Val Asp Leu Phe Ala
690 695 700
Gly Lys Pro Gln Gln Glu Val Val Leu Ala Ala Leu Glu Asp Thr Trp
705 710 715 720
Glu Thr Leu Ser Lys Arg Tyr Gly Asn Asn Val Ser Asn Trp Lys Thr
725 730 735
Pro Ala Met Ala Leu Thr Phe Arg Ala Asn Asn Phe Phe Gly Val Pro
740 745 750
Gln Ala Ala Ala Glu Glu Thr Arg His Gln Ala Glu Tyr Gln Asn Arg
755 760 765
Gly Thr Glu Asn Asp Met Ile Val Phe Ser Pro Thr Thr Ser Asp Arg
770 775 780
Pro Val Leu Ala Trp Asp Val Val Ala Pro Gly Gln Ser Gly Phe Ile
785 790 795 800
Ala Pro Asp Gly Thr Val Asp Lys His Tyr Glu Asp Gln Leu Lys Met
805 810 815
Tyr Glu Asn Phe Gly Arg Lys Ser Leu Trp Leu Thr Lys Gln Asp Val
820 825 830
Glu Ala His Lys Glu Ser Gln Glu Val Leu His Val Gln Arg
835 840 845
<210> 3
<211> 2538
<212> DNA
<213> 人工序列
<220>
<223>
<400> 3
atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60
agcttacctg cactggctga acagtctagc tctgagatta agattgtgcg tgacgaatac 120
ggcatgcctc atatctacgc caacgacacc tggcacctgt tctacggcta tggctacgtg 180
gtagcacagg accgtctgtt tcagatggaa atggctcgtc gtagcaccca gggcaccgta 240
gcagaagtgc tgggcaaaga cttcgtgaag ttcgacaaag acattcgtcg caactactgg 300
ccggacgcga tccgtgcgca gattgcggcg ctgagcccgg aagacatgag catcctgcaa 360
ggttacgctg atggtatgaa cgcatggatc gataaagtga acacgaaccc tgaaaccctg 420
ctgccgaaac agttcaacac ctttggcttc accccgaaac gctgggaacc gttcgatgtg 480
gcgatgatct tcgtgggcac tctggccaat cgcttctctg attctacctc cgagatcgac 540
aatctggccc tgctgaccgc actgaaagac aagtatggtg tcagccaggg catggcggtg 600
ttcaaccagc tgaaatggct ggtcaacccg tccgcgccga ctacgatcgc ggtgcaggag 660
tctaactacc cgctgaaatt caaccaacag aacagccaga cggctgcact gctgccgcgt 720
tatgatctgc cagcgccaat gctggatcgc ccggctaaag gtgcagacgg tgctctgctg 780
gcgctgactg ctggcaaaaa tcgcgaaacc atcgttgctc aattcgcaca gggcggtgcg 840
aatggtctgg ctggctatcc gaccacctct aacatgtggg tgatcggtaa atctaaagcg 900
caggacgcga aagcgatcat ggttaacggt ccgcaggcgg gctggtacgc tccggcctat 960
acctacggta tcggcctgca tggtgcaggc tatgacgtca ctggtaacac tccgttcgcg 1020
tatcctggtc tggttttcgg tcacaacggt gttatcagct ggggtgcgac cgcaggcttt 1080
ggtgatgatg ttgacatttt tgctgaacgt ctgagcgcag aaaaaccggg ctactacctg 1140
cacaacggta aatgggtaaa aatgctgtct cgcgaagaga ccatcacggt taaaaacggt 1200
caggcggaaa ctttcactgt gtggcgcacc gtacacggca acatcctgca gaccgaccag 1260
actactcaga ctgcttacgc taaatcccgt gcctgggacg gtaaggaagt agcatccctg 1320
ctggcgtgga cgcaccagat gaaagccaaa aactggcagg agtggaccca gcaagcggcc 1380
aaacaggcac tgacgattaa ctggtattac gcagacgtga acggtaacat cggttatgtt 1440
cacaccggcg catacccgga ccgtcagtct ggccatgatc cgcgtctgcc ggtgccaggc 1500
actggcaaat gggattggaa aggtctgctg ccgttcgaaa tgaatccaaa agtatacaac 1560
ccgcagtccg gttacattgc caactggaac aactccccgc agaaagacta cccggcatct 1620
gatctgtttg cgttcctgtg gggtggtgcc gatcgtgtta ccgagattga ccgcctgctg 1680
gaacagaaac cgcgcctgac ggccgatcag gcatgggacg ttatccgtca aacttcccgt 1740
caggacctga acctgcgtct gttcctgccg actctgcaag cagcaacgtc cggtctgact 1800
cagagcgatc ctcgtcgtca actggttgag acgctgactc gttgggatgg catcaacctg 1860
ctgaacgacg acggtaaaac ctggcaacaa ccaggttctg ctatcctgaa cgtttggctg 1920
acctccatgc tgaaacgtac cgtcgttgcg gctgtaccga tgccgtttga taagtggtac 1980
tctgctagcg gctatgaaac cacccaggat ggcccaaccg gctccctgaa catttctgtt 2040
ggcgcgaaaa tcctgtatga agcggtacag ggtgataaat cccctatccc acaggctgtt 2100
gatctgttcg ccggcaaacc gcagcaggaa gtagttctgg ctgcgctgga agacacctgg 2160
gaaactctgt ctaagcgtta cggtaacaac gttagcaact ggaaaacccc ggccatggct 2220
ctgaccttcc gtgcgaataa tttcttcggt gttccgcagg ctgcggcgga agaaacccgc 2280
catcaggctg aataccaaaa ccgcggcacc gaaaacgaca tgatcgtttt ttccccgact 2340
acctctgatc gtccggtcct ggcttgggac gtcgtagctc cgggtcagag cggttttatt 2400
gcaccggatg gtaccgtcga taagcactat gaagatcagc tgaagatgta cgagaacttt 2460
ggccgcaagt ctctgtggct gaccaaacag gacgtggagg cccacaaaga atctcaggaa 2520
gttctgcacg ttcagcgt 2538
<210> 4
<211> 846
<212> PRT
<213> 人工序列
<220>
<223>
<400> 4
Met Lys Asn Arg Asn Arg Met Ile Val Asn Cys Val Thr Ala Ser Leu
1 5 10 15
Met Tyr Tyr Trp Ser Leu Pro Ala Leu Ala Glu Gln Ser Ser Ser Glu
20 25 30
Ile Lys Ile Val Arg Asp Glu Tyr Gly Met Pro His Ile Tyr Ala Asn
35 40 45
Asp Thr Trp His Leu Phe Tyr Gly Tyr Gly Tyr Val Val Ala Gln Asp
50 55 60
Arg Leu Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Thr Val
65 70 75 80
Ala Glu Val Leu Gly Lys Asp Phe Val Lys Phe Asp Lys Asp Ile Arg
85 90 95
Arg Asn Tyr Trp Pro Asp Ala Ile Arg Ala Gln Ile Ala Ala Leu Ser
100 105 110
Pro Glu Asp Met Ser Ile Leu Gln Gly Tyr Ala Asp Gly Met Asn Ala
115 120 125
Trp Ile Asp Lys Val Asn Thr Asn Pro Glu Thr Leu Leu Pro Lys Gln
130 135 140
Phe Asn Thr Phe Gly Phe Thr Pro Lys Arg Trp Glu Pro Phe Asp Val
145 150 155 160
Ala Met Ile Phe Val Gly Thr Leu Ala Asn Arg Phe Ser Asp Ser Thr
165 170 175
Ser Glu Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Lys Tyr
180 185 190
Gly Val Ser Gln Gly Met Ala Val Phe Asn Gln Leu Lys Trp Leu Val
195 200 205
Asn Pro Ser Ala Pro Thr Thr Ile Ala Val Gln Glu Ser Asn Tyr Pro
210 215 220
Leu Lys Phe Asn Gln Gln Asn Ser Gln Thr Ala Ala Leu Leu Pro Arg
225 230 235 240
Tyr Asp Leu Pro Ala Pro Met Leu Asp Arg Pro Ala Lys Gly Ala Asp
245 250 255
Gly Ala Leu Leu Ala Leu Thr Ala Gly Lys Asn Arg Glu Thr Ile Val
260 265 270
Ala Gln Phe Ala Gln Gly Gly Ala Asn Gly Leu Ala Gly Tyr Pro Thr
275 280 285
Thr Ser Asn Met Trp Val Ile Gly Lys Ser Lys Ala Gln Asp Ala Lys
290 295 300
Ala Ile Met Val Asn Gly Pro Gln Ala Gly Trp Tyr Ala Pro Ala Tyr
305 310 315 320
Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr Asp Val Thr Gly Asn
325 330 335
Thr Pro Phe Ala Tyr Pro Gly Leu Val Phe Gly His Asn Gly Val Ile
340 345 350
Ser Trp Gly Ala Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala
355 360 365
Glu Arg Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Leu His Asn Gly Lys
370 375 380
Trp Val Lys Met Leu Ser Arg Glu Glu Thr Ile Thr Val Lys Asn Gly
385 390 395 400
Gln Ala Glu Thr Phe Thr Val Trp Arg Thr Val His Gly Asn Ile Leu
405 410 415
Gln Thr Asp Gln Thr Thr Gln Thr Ala Tyr Ala Lys Ser Arg Ala Trp
420 425 430
Asp Gly Lys Glu Val Ala Ser Leu Leu Ala Trp Thr His Gln Met Lys
435 440 445
Ala Lys Asn Trp Gln Glu Trp Thr Gln Gln Ala Ala Lys Gln Ala Leu
450 455 460
Thr Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly Asn Ile Gly Tyr Val
465 470 475 480
His Thr Gly Ala Tyr Pro Asp Arg Gln Ser Gly His Asp Pro Arg Leu
485 490 495
Pro Val Pro Gly Thr Gly Lys Trp Asp Trp Lys Gly Leu Leu Pro Phe
500 505 510
Glu Met Asn Pro Lys Val Tyr Asn Pro Gln Ser Gly Tyr Ile Ala Asn
515 520 525
Trp Asn Asn Ser Pro Gln Lys Asp Tyr Pro Ala Ser Asp Leu Phe Ala
530 535 540
Phe Leu Trp Gly Gly Ala Asp Arg Val Thr Glu Ile Asp Arg Leu Leu
545 550 555 560
Glu Gln Lys Pro Arg Leu Thr Ala Asp Gln Ala Trp Asp Val Ile Arg
565 570 575
Gln Thr Ser Arg Gln Asp Leu Asn Leu Arg Leu Phe Leu Pro Thr Leu
580 585 590
Gln Ala Ala Thr Ser Gly Leu Thr Gln Ser Asp Pro Arg Arg Gln Leu
595 600 605
Val Glu Thr Leu Thr Arg Trp Asp Gly Ile Asn Leu Leu Asn Asp Asp
610 615 620
Gly Lys Thr Trp Gln Gln Pro Gly Ser Ala Ile Leu Asn Val Trp Leu
625 630 635 640
Thr Ser Met Leu Lys Arg Thr Val Val Ala Ala Val Pro Met Pro Phe
645 650 655
Asp Lys Trp Tyr Ser Ala Ser Gly Tyr Glu Thr Thr Gln Asp Gly Pro
660 665 670
Thr Gly Ser Leu Asn Ile Ser Val Gly Ala Lys Ile Leu Tyr Glu Ala
675 680 685
Val Gln Gly Asp Lys Ser Pro Ile Pro Gln Ala Val Asp Leu Phe Ala
690 695 700
Gly Lys Pro Gln Gln Glu Val Val Leu Ala Ala Leu Glu Asp Thr Trp
705 710 715 720
Glu Thr Leu Ser Lys Arg Tyr Gly Asn Asn Val Ser Asn Trp Lys Thr
725 730 735
Pro Ala Met Ala Leu Thr Phe Arg Ala Asn Asn Phe Phe Gly Val Pro
740 745 750
Gln Ala Ala Ala Glu Glu Thr Arg His Gln Ala Glu Tyr Gln Asn Arg
755 760 765
Gly Thr Glu Asn Asp Met Ile Val Phe Ser Pro Thr Thr Ser Asp Arg
770 775 780
Pro Val Leu Ala Trp Asp Val Val Ala Pro Gly Gln Ser Gly Phe Ile
785 790 795 800
Ala Pro Asp Gly Thr Val Asp Lys His Tyr Glu Asp Gln Leu Lys Met
805 810 815
Tyr Glu Asn Phe Gly Arg Lys Ser Leu Trp Leu Thr Lys Gln Asp Val
820 825 830
Glu Ala His Lys Glu Ser Gln Glu Val Leu His Val Gln Arg
835 840 845
<210> 5
<211> 2538
<212> DNA
<213> 野生型青霉素G酰化酶
<400> 5
atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60
agcttacctg cactggctga acagtctagc tctgagatta agattgtgcg tgacgaatac 120
ggcatgcctc atatctacgc caacgacacc tggcacctgt tctacggcta tggctacgtg 180
gtagcacagg accgtctgtt tcagatggaa atggctcgtc gtagcaccca gggcaccgta 240
gcagaagtgc tgggcaaaga cttcgtgaag ttcgacaaag acattcgtcg caactactgg 300
ccggacgcga tccgtgcgca gattgcggcg ctgagcccgg aagacatgag catcctgcaa 360
ggttacgctg atggtatgaa cgcatggatc gataaagtga acacgaaccc tgaaaccctg 420
ctgccgaaac agttcaacac ctttggcttc accccgaaac gctgggaacc gttcgatgtg 480
gcgatgatct tcgtgggcac tatggccaat cgcttctctg attctacctc cgagatcgac 540
aatctggccc tgctgaccgc actgaaagac aagtatggtg tcagccaggg catggcggtg 600
ttcaaccagc tgaaatggct ggtcaacccg tccgcgccga ctacgatcgc ggtgcaggag 660
tctaactacc cgctgaaatt caaccaacag aacagccaga cggctgcact gctgccgcgt 720
tatgatctgc cagcgccaat gctggatcgc ccggctaaag gtgcagacgg tgctctgctg 780
gcgctgactg ctggcaaaaa tcgcgaaacc atcgttgctc aattcgcaca gggcggtgcg 840
aatggtctgg ctggctatcc gaccacctct aacatgtggg tgatcggtaa atctaaagcg 900
caggacgcga aagcgatcat ggttaacggt ccgcagttcg gctggtacgc tccggcctat 960
acctacggta tcggcctgca tggtgcaggc tatgacgtca ctggtaacac tccgttcgcg 1020
tatcctggtc tggttttcgg tcacaacggt gttatcagct ggggttccac cgcaggcttt 1080
ggtgatgatg ttgacatttt tgctgaacgt ctgagcgcag aaaaaccggg ctactacctg 1140
cacaacggta aatgggtaaa aatgctgtct cgcgaagaga ccatcacggt taaaaacggt 1200
caggcggaaa ctttcactgt gtggcgcacc gtacacggca acatcctgca gaccgaccag 1260
actactcaga ctgcttacgc taaatcccgt gcctgggacg gtaaggaagt agcatccctg 1320
ctggcgtgga cgcaccagat gaaagccaaa aactggcagg agtggaccca gcaagcggcc 1380
aaacaggcac tgacgattaa ctggtattac gcagacgtga acggtaacat cggttatgtt 1440
cacaccggcg catacccgga ccgtcagtct ggccatgatc cgcgtctgcc ggtgccaggc 1500
actggcaaat gggattggaa aggtctgctg ccgttcgaaa tgaatccaaa agtatacaac 1560
ccgcagtccg gttacattgc caactggaac aactccccgc agaaagacta cccggcatct 1620
gatctgtttg cgttcctgtg gggtggtgcc gatcgtgtta ccgagattga ccgcctgctg 1680
gaacagaaac cgcgcctgac ggccgatcag gcatgggacg ttatccgtca aacttcccgt 1740
caggacctga acctgcgtct gttcctgccg actctgcaag cagcaacgtc cggtctgact 1800
cagagcgatc ctcgtcgtca actggttgag acgctgactc gttgggatgg catcaacctg 1860
ctgaacgacg acggtaaaac ctggcaacaa ccaggttctg ctatcctgaa cgtttggctg 1920
acctccatgc tgaaacgtac cgtcgttgcg gctgtaccga tgccgtttga taagtggtac 1980
tctgctagcg gctatgaaac cacccaggat ggcccaaccg gctccctgaa catttctgtt 2040
ggcgcgaaaa tcctgtatga agcggtacag ggtgataaat cccctatccc acaggctgtt 2100
gatctgttcg ccggcaaacc gcagcaggaa gtagttctgg ctgcgctgga agacacctgg 2160
gaaactctgt ctaagcgtta cggtaacaac gttagcaact ggaaaacccc ggccatggct 2220
ctgaccttcc gtgcgaataa tttcttcggt gttccgcagg ctgcggcgga agaaacccgc 2280
catcaggctg aataccaaaa ccgcggcacc gaaaacgaca tgatcgtttt ttccccgact 2340
acctctgatc gtccggtcct ggcttgggac gtcgtagctc cgggtcagag cggttttatt 2400
gcaccggatg gtaccgtcga taagcactat gaagatcagc tgaagatgta cgagaacttt 2460
ggccgcaagt ctctgtggct gaccaaacag gacgtggagg cccacaaaga atctcaggaa 2520
gttctgcacg ttcagcgt 2538
<210> 6
<211> 2538
<212> DNA
<213> 人工序列
<220>
<223>
<400> 6
atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60
agcttacctg cactggctga acagtctagc tctgagatta agattgtgcg tgacgaatac 120
ggcatgcctc atatctacgc caacgacacc tggcacctgt tctacggcta tggctacgtg 180
gtagcacagg accgtctgtt tcagatggaa atggctcgtc gtagcaccca gggcaccgta 240
gcagaagtgc tgggcaaaga cttcgtgaag ttcgacaaag acattcgtcg caactactgg 300
ccggacgcga tccgtgcgca gattgcggcg ctgagcccgg aagacatgag catcctgcaa 360
ggttacgctg atggtatgaa cgcatggatc gataaagtga acacgaaccc tgaaaccctg 420
ctgccgaaac agttcaacac ctttggcttc accccgaaac gctgggaacc gttcgatgtg 480
gcgatgatct tcgtgggcac tatggccaat cgcttctctg attctacctc cgagatcgac 540
aatctggccc tgctgaccgc actgaaagac aagtatggtg tcagccaggg catggcggtg 600
ttcaaccagc tgaaatggct ggtcaacccg tccgcgccga ctacgatcgc ggtgcaggag 660
tctaactacc cgctgaaatt caaccaacag aacagccaga cggctgcact gctgccgcgt 720
tatgatctgc cagcgccaat gctggatcgc ccggctaaag gtgcagacgg tgctctgctg 780
gcgctgactg ctggcaaaaa tcgcgaaacc atcgttgctc aattcgcaca gggcggtgcg 840
aatggtctgg ctggctatcc gaccacctct aacatgtggg tgatcggtaa atctaaagcg 900
caggacgcga aagcgatcat ggttaacggt ccgcaggcgg gctggtacgc tccggcctat 960
acctacggta tcggcctgca tggtgcaggc tatgacgtca ctggtaacac tccgttcgcg 1020
tatcctggtc tggttttcgg tcacaacggt gttatcagct ggggttccac cgcaggcttt 1080
ggtgatgatg ttgacatttt tgctgaacgt ctgagcgcag aaaaaccggg ctactacctg 1140
cacaacggta aatgggtaaa aatgctgtct cgcgaagaga ccatcacggt taaaaacggt 1200
caggcggaaa ctttcactgt gtggcgcacc gtacacggca acatcctgca gaccgaccag 1260
actactcaga ctgcttacgc taaatcccgt gcctgggacg gtaaggaagt agcatccctg 1320
ctggcgtgga cgcaccagat gaaagccaaa aactggcagg agtggaccca gcaagcggcc 1380
aaacaggcac tgacgattaa ctggtattac gcagacgtga acggtaacat cggttatgtt 1440
cacaccggcg catacccgga ccgtcagtct ggccatgatc cgcgtctgcc ggtgccaggc 1500
actggcaaat gggattggaa aggtctgctg ccgttcgaaa tgaatccaaa agtatacaac 1560
ccgcagtccg gttacattgc caactggaac aactccccgc agaaagacta cccggcatct 1620
gatctgtttg cgttcctgtg gggtggtgcc gatcgtgtta ccgagattga ccgcctgctg 1680
gaacagaaac cgcgcctgac ggccgatcag gcatgggacg ttatccgtca aacttcccgt 1740
caggacctga acctgcgtct gttcctgccg actctgcaag cagcaacgtc cggtctgact 1800
cagagcgatc ctcgtcgtca actggttgag acgctgactc gttgggatgg catcaacctg 1860
ctgaacgacg acggtaaaac ctggcaacaa ccaggttctg ctatcctgaa cgtttggctg 1920
acctccatgc tgaaacgtac cgtcgttgcg gctgtaccga tgccgtttga taagtggtac 1980
tctgctagcg gctatgaaac cacccaggat ggcccaaccg gctccctgaa catttctgtt 2040
ggcgcgaaaa tcctgtatga agcggtacag ggtgataaat cccctatccc acaggctgtt 2100
gatctgttcg ccggcaaacc gcagcaggaa gtagttctgg ctgcgctgga agacacctgg 2160
gaaactctgt ctaagcgtta cggtaacaac gttagcaact ggaaaacccc ggccatggct 2220
ctgaccttcc gtgcgaataa tttcttcggt gttccgcagg ctgcggcgga agaaacccgc 2280
catcaggctg aataccaaaa ccgcggcacc gaaaacgaca tgatcgtttt ttccccgact 2340
acctctgatc gtccggtcct ggcttgggac gtcgtagctc cgggtcagag cggttttatt 2400
gcaccggatg gtaccgtcga taagcactat gaagatcagc tgaagatgta cgagaacttt 2460
ggccgcaagt ctctgtggct gaccaaacag gacgtggagg cccacaaaga atctcaggaa 2520
gttctgcacg ttcagcgt 2538
Claims (11)
1.蛋白质,为如下(a)或(b):
(a)由序列表中序列2所示的氨基酸序列组成的蛋白质;
(b)由序列表中序列4所示的氨基酸序列组成的蛋白质。
2.编码权利要求1所述蛋白质的核酸分子。
3.根据权利要求2所述的核酸分子,其特征在于:所述核酸分子为编码权利要求1所述蛋白质的基因,所述基因为如下任一所示的DNA分子:
1)序列表中序列1所示的DNA分子;
2)序列表中序列3所示的DNA分子。
4.含有权利要求2或3所述核酸分子的重组载体。
5.含有权利要求2或3所述核酸分子的表达盒。
6.含有权利要求2或3所述核酸分子的重组菌。
7.权利要求1所述的蛋白质在作为青霉素G酰化酶中的应用。
8.权利要求1所述的蛋白质在如下任一中的应用:
(a1)作为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中提高Vs/Vh;
(a2)作为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中降低α。
9.权利要求1所述的蛋白质或权利要求2或3所述的核酸分子或权利要求4所述的重组载体或权利要求5所述的表达盒或权利要求6所述的重组菌在如下任一中的应用:
(b1)制备具有青霉素G酰化酶活性的产品;
(b2)制备头孢拉定或其他β-内酰胺类抗生素。
10.一种制备头孢拉定的方法,包括步骤:制备权利要求1所述的蛋白质;以所述蛋白质为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定。
11.一种在青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定的动力学控制合成反应中提高Vs/Vh和/或降低α的方法,其特征在于:所述方法中以权利要求1所述蛋白质为青霉素G酰化酶催化二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸缩合生成头孢拉定。
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