CN104769124A - 用于转化麦角肽碱的酶及其方法 - Google Patents
用于转化麦角肽碱的酶及其方法 Download PDFInfo
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- CN104769124A CN104769124A CN201380052664.6A CN201380052664A CN104769124A CN 104769124 A CN104769124 A CN 104769124A CN 201380052664 A CN201380052664 A CN 201380052664A CN 104769124 A CN104769124 A CN 104769124A
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- enzyme
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- ergopeptine
- nucleophilic
- ergotamine
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
- C12P17/183—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system containing an indolo[4,3-F,G]quinoleine nucleus, e.g. compound containing the lysergic acid nucleus as well as the dimeric ergot nucleus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明涉及用于转化、特别是水解裂解麦角肽碱、用于麦角肽碱的转化的酶,以及用于生产代谢麦角肽碱的酶的方法,所述麦角肽碱是α/β-水解酶在环醇环处水解裂解的麦角肽碱。
Description
本发明涉及用于转化(特别是水解裂解)麦角肽碱类(Ergopept inen)的酶,用于转化麦角肽碱类的方法,以及用于生产代谢麦角肽碱的酶的方法。
麦角肽碱类是一组麦角生物碱,并且还是由属于麦角菌(Clavicipi taceae)科麦角菌(Claviceps)属的植物相关的真菌形成的二次代谢产物。该属最突出的成员是麦角菌(Claviceps purpurea),其最主要影响如黑麦、小麦、黑小麦、大麦和玉米的谷物。另一个成员(即非洲麦角菌(Claviceps africana))被广泛发现于小米中。该科的进一步的产麦角生物碱的真菌包括香柱菌()、Neotyphodium和瘤座菌(Balansia)属的草内生菌,还有烟曲霉菌(Aspergillusfumigatus)和各种青霉属(Penicillium spp.)种也能够产生麦角生物碱。
一般情况下,麦角生物碱具有在6-位置包含甲基化氮的四环麦角灵环的特征骨架结构并且可以在C-8位置具有不同的取代基。基于这些取代基,麦角生物碱被分类为棒麦角碱(Clavine)、简单的麦角酸酰胺、麦角肽碱和麦角肽胺(Ergopeptame)。
由于它们与神经递质的结构相似性,麦角生物碱与后者的受体相互作用,引起多个效果,如中毒,但也有在医药领域的积极作用。如今,由于在磨坊方面清洗技术的改善,麦角生物碱不再构成人类领域中的问题。但是,它们仍然构成畜牧业的问题,引起多种不良症状。特别地,由麦角生物碱在动物中引起的这些症状包括坏疽、瘫瘸、减少的体重增加、增加的呼吸频率、降低的血清催乳素水平、降低的产乳量以及低繁殖率。在这方面,主要是在美国、新西兰和澳大利亚的牧草中的内生菌带来畜牧业的问题。例如,由内生菌Neotyphodium coenophialum引起的高羊茅草(Rohrschwingel)感染给畜牧生产者造成高损失。
对于上述大多数的效果或症状,麦角肽碱(其为最丰富多样的麦角生物碱类别)是负有责任的,它们自身再次被根据直接结合至D-麦角酸的氨基酸进行分类。在这方面,相应于Schardl等人所使用的命名法,麦角肽碱的特征性噁唑烷-4-酮被称为环醇环。在此,代表是麦角胺类,尤其包括麦角胺、麦角缬碱(Ergovalin)和麦角生碱,其中第一个氨基酸为L-丙氨酸。还有一类是麦角毒碱类,其中结合至D-麦角酸的第一个氨基酸是L-缬氨酸。其中的代表是麦角克碱、麦角隐亭碱或麦角柯宁碱。最后还有一个代表是Ergoxin类,其中结合至麦角酸的第一个氨基酸是α-氨基丁酸。其中的代表是麦角亭(Ergostin)和麦角宁(Ergonin)。
其中,麦角缬碱是在牧草中内生生长的Neotyphodium和香柱菌种的主要生物碱之一,并且具有兽医毒理学意义,例如在羊茅草中毒中。9,10-二氢麦角肽碱极少天然存在并且迄今为止仅在Sphacelia sorghi中被检测到。部分合成所得的二氢麦角肽碱(如二氢麦角胺和二氢麦角毒碱)在偏头痛和心血管疾病的治疗中具有治疗意义。但是,除了上述积极效果,特别是麦角肽碱的治疗意义,它们的毒性作用具有不可忽视的意义,因为,特别是它们的毒性(由于例如被污染的谷物或毒性内生菌的饮食),会导致许多生理系统(如动物或人体内的生殖器官、生长导向系统和心血管结构)的破坏或损害或损伤。另外,目前没有任何疑问的是,被麦角胺或麦角肽碱污染的谷物的摄入会直接负面影响胃肠系统并因此不仅严重损害动物的健康,而且严重损害它们的能力。
本发明目的在于提供酶和酶制剂以及衍生出这种酶和酶制剂的基因,其能够降解麦角肽碱至较低毒性的代谢物,特别是麦角酰胺(Ergin)。
为了达到这个目的,本发明主要特征在于所述酶是将麦角肽碱在环醇环处水解裂解的α/β-水解酶。所述麦角肽碱的噁唑烷-4-酮环被定义为环醇环。其中麦角肽碱在环醇环处通过麦角肽碱特异性α/β-水解酶酶促裂解,以多步反应(其部分自发地进行)实现麦角肽碱至麦角酰胺的降解。在此,α/β-水解酶是一类具有不同催化功能的酶的成员,其尤其能够攻击天然麦角肽碱的环醇环并将麦角肽碱经二级麦角酸酰胺(麦角羟基酸)降解成麦角酰胺。
根据本发明的另一个实施方案,所述酶主要特征在于它们包含由亲核氨基酸和组氨酸和酸性氨基酸组成的催化三联体,并且所述三联体被包含于具有α/β-水解酶折叠的肽链中。特别完全的酶促裂解经如下实现,即所述催化三联体由亲核氨基酸丝氨酸、组氨酸和酸性氨基酸天冬氨酸或谷氨酸之一组成,并且所述三联体被包含于具有麦角肽碱特异性α/β-水解酶折叠的肽链中。使用具有上述催化三联体的酶,令人惊讶地能够将麦角肽碱完全酶促裂解至麦角酰胺。在此,所述酶促裂解发生于麦角肽碱的所述环醇环的3′位点,在该水解裂解过程中三个环(即环醇环、内酰胺环和吡咯烷环)的组以几个步骤被裂解最终形成麦角酰胺,如从下面的反应方案可知的。
根据本发明的另一个实施方案,所述酶主要特征在于所述α/β-水解酶包含具有序列Gly-Gln-Ser-Arg-Asn-Gly的亲核肘(Ellbogen)。如果所述α/β-水解酶包含具有序列Gly-Gln-Ser-Arg-Asn-Gly的亲核肘,麦角肽碱至麦角羟基酸的酶促降解会特别快速,后所述麦角羟基酸自发转化成麦角酰胺。在这种情况下,所述亲核肘是α/β-水解酶的核心部分。其中存在具有亲核侧链的催化活性氨基酸,所述亲核侧链呈具有位于拉曼图(Ramachandran-Plots)的不利区域中非寻常键角的结构(Ollis et al.,1992)。所述亲核肘的氨基酸序列(在本发明的情况下是Gly-Gln-Ser-Arg-Asn-Gly)是保守的,并且还能例如被用于对α/β-水解酶进行分类(Kourist et al.,2010)。
根据本发明的另一个优选的实施方案,能通过具有序列ID No.1的酶实现完全降解。具有序列ID No.1的酶已被证明在麦角肽碱至麦角酰胺的酶促裂解中特别有效。亲核氨基酸丝氨酸在保守结构(即所述亲核肘)中具有中心作用。所述亲核肘位于β5-链和随后的α-螺旋之间,并且包含Sm-X-Nu-X-Sm的共有序列,其中Sm为小氨基酸,X是任何氨基酸,以及Nu代表亲核氨基酸。序列ID No.1为G-Q-S-R-N。序列ID No.1的α/β-水解酶属于就它们的作用方式而言不需要任何辅因子的酶。
根据本发明的另一个优选的实施方案,所述酶特征在于它与序列IDNo.1具有至少96%的序列同一性,其中所述酶的催化性质被基本上保持。可以令人惊讶地证实除了具有序列ID No.1的酶之外,还可以采用其修饰,并且经修饰的酶仍然可能有好结果,如在具有序列ID No.5的酶的情况下所示。
根据本发明的另一个优选的实施方案,所述酶特征在于它包含不同于序列ID No.1的N-或C-末端序列,尤其是延长的N-或C-末端序列,特别是具有序列ID No.5的酶并且它显示与序列ID No.1具有至少96%的序列同一性。可以令人惊讶地证实除了具有序列ID No.1的酶之外,还可以采用其宽度修饰,其中特别是所述N-末端可以被修饰。如果具有改变的起始序列的酶显示与序列ID No.1具有至少96%的序列同一性,那么会实现特别好的结果。
具有不同于序列ID No.1的N-末端的酶同样能完全降解麦角胺。
为了将麦角肽碱完全降解和解毒,本发明目的还在于提供用于酶促转化麦角肽碱的方法。
为了实现这个目的,根据本发明的方法主要特征在于所述麦角肽碱在环醇环处被水解裂解为初级代谢物。
令人惊讶地表明,在麦角肽碱于环醇环处水解裂解至麦角羟基酸和麦角脯氨酸(Egoprolin)环二肽之后,这些中间产物进行自发性反应生成特别是麦角酰胺和丙酮酸。由此所形成的反应产物显示相对于起始产物的显著降低(如果不是可以忽略不计的)的毒性。
优选根据本发明的方法是基本上这样进行的:所述裂解是通过环醇环的C3′-原子上的亲核攻击实现的。特别有利和完全的结果可以这样实现:所述环醇环的C3′-原子上的亲核攻击是通过催化三联体实现的,所述催化三联体被包含于具有α/β-水解酶折叠的肽链中并由亲核氨基酸丝氨酸、组氨酸和酸性氨基酸天冬氨酸或谷氨酸之一组成。实施这样的方法实现了麦角肽碱至初级代谢物的快速和完全降解,其相应于如根据本发明的另一个优选的实施方案,所述初级代谢物被进一步转换成麦角酰胺。根据本发明的另一个优选的实施方案,这样的反应是通过自发反应实现的,其中为此选择环境条件使得所述降解的中间产物被直接并完全转化成麦角酰胺。
如根据本发明的另一个优选的实施方案,所述方法是这样进行的:由用所述α/β-水解酶的水解裂解所形成的初级代谢物的进一步反应是通过在所述反应反应介质中存在的酶实现的。实施这样的方法采用总是存在于天然环境中的酶,其令人惊讶地能够将所述初级代谢物完全降解为麦角酰胺。
此外,本发明目的在于提供用于生产代谢麦角肽碱的酶的方法。为了达到这个目的,根据本发明的所述方法是这样进行的:将编码根据本发明的酶的基因克隆入表达载体、转化入原核和/或真核宿主细胞,并且在宿主细胞中表达。这样的步骤能够提供高的酶浓度,其能够将六个麦角肽碱(即麦角胺、麦角缬碱、麦角柯宁碱、麦角克碱、麦角隐亭碱或麦角生碱)和它们各自的异构体形式(即麦角异胺(Ergotaminin)、麦角异缬碱(Ergovalinin)、麦角异柯宁碱(Ergocorninin)、麦角异克碱(Ergocristinin)、麦角异隐亭碱(Ergocryptinin)和麦角异生碱(Ergosinin))完全转换成麦角酰胺。在此优选使用具有序列IDNo.2、4或6的基因,经此能够进一步提高使所形成的酶组合。
当如下实施所述方法,则根据本发明实现特别高的酶活性:将所述基因转化入作为宿主细胞的选自巴斯德毕赤酵母(Pichia pastoris)、大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)的微生物中并进行表达。在本申请中所使用的名称巴斯德毕赤酵母是名称Komagataella pastoris的同义词,巴斯德毕赤酵母更旧而Komagataella pastoris是系统是上更新的名称(Yamada et al.,1995)。
如下实施所述方法,可经此进一步提高酶活性:所述序列ID No.1的酶(特别是具有序列ID No.5的带有his标签的酶)用亲和层析进行纯化。具有序列ID No.5的纯化的酶不仅使得麦角肽碱完全转换成麦角酰胺,而且这样的纯化的酶会特别显示尤其高的催化活性,特别是在大约6和大约9之间的pH范围。
根据本发明的另一个优选的实施方案,在此如下进行所述反应的第一步:用序列ID No.1的酶裂解所述环醇环。在这样的方法实施中,麦角肽碱能够几乎完全转换成具有低血管收缩活性的代谢物。
根据本发明的酶制剂优选被应用于饲料或青贮饲料添加剂。在这样的使用情况下,仅通过混合所述酶制剂能够对饲料或青贮饲料添加剂上存在的麦角肽碱部分在喂养之前并且部分在动物的胃肠道中进行解毒。
在下文中,通过示例性实施方案和附图的方式对本发明进行更详细的说明。其中,
图1示出了用序列ID No.1的酶进行麦角胺至麦角酰胺的反应的动力学;
图2示出了由序列ID No.1的酶进行麦角肽碱麦角柯宁碱、麦角隐亭碱、麦角生碱、麦角缬碱和麦角胺的反应,和针对麦角隐亭碱、麦角生碱的示例性的阴性对照;
图3呈现带有基因序列ID No.2的毕赤酵母表达载体pGAPZαC的图;以及
图4呈现带有基因序列ID No.2的枯草芽孢杆菌表达载体pET43的图。
实施例1:
序列ID No.1的酶的催化活性的确定
具有序列ID No.2(其编码包含S94-D234-H270的催化三联体的α/β-水解酶)的基因通过采用标准方法被克隆入表达载体pET28a(+)中,在大肠杆菌中被转化和表达。在大肠杆菌BL21(DE3)中表达之后,带有his标签的酶通过亲和层析进行纯化。采用Pierce BSA蛋白质测定试剂盒确定所述酶浓度,并且所述酶被用于活性测定中。在50mM磷酸钠缓冲液(pH 7.0)中在25℃进行所述测定。
在解毒实验的范围内,采用0.079μg/ml的酶浓度和5mg/kg的麦角胺浓度。
用于反应六个麦角肽碱(即麦角胺、麦角缬碱、麦角柯宁碱、麦角克碱、麦角隐亭碱或麦角生碱)和它们各自的异构体形式(即麦角异胺、麦角异缬碱、麦角异柯宁碱、麦角异克碱、麦角异隐亭碱和麦角异生碱)的进一步的实验采用1.58μg/ml序列ID No.1的酶和10mg/kg的麦角胺或等摩尔(合计)浓度的剩余的麦角肽碱或它们的差向异构体。所述结果示于图2中。
采用HPLC-FLD或HPLC-MS/MS对所述样品进行分析,其中分别解析确定各差向异构体的总和的浓度。平行于酶促反应过程中的麦角肽碱浓度的降低,观察到麦角羟基酸(代谢物1)和麦角脯氨酸环二肽(代谢物2)的形成。在进一步的反应过程中,检测到代谢物1至麦角酰胺的转换。
图1示例性地显示了麦角胺与序列ID No.1进行反应的动力学。在所述反应过程中,检测到微量的不稳定的中间产物,并且所述反应的最终产物是麦角酰胺。从图1可以明显看出序列ID No.1麦角胺至麦角酰胺的几乎完全降解在4小时内发生。所有其它麦角肽碱(即麦角缬碱、麦角柯宁碱、麦角克碱、麦角隐亭碱或麦角生碱)以及它们各自的异构体形式(即麦角异缬碱、麦角异柯宁碱、麦角异克碱、麦角异隐亭碱和麦角异生碱)的反应过程具有可比性。
实施例2:
序列ID No.1的酶的N-末端的鉴定
为了鉴定序列ID No.1的酶的N-末端,采用标准方法将具有序列ID No.2和ID No.6的基因克隆入pET28a(+)并转化入大肠杆菌。
表达之后,细菌细胞被溶解于50mM磷酸钠缓冲液并采用法式压滤(French press)(20,000psi)进行裂解。裂解物以1:10、1:100和1:1000稀释被用于5mg/kg麦角胺的降解批次中。所述批次在25℃进行孵育并且所述样品采用HPLC-FLD进行分析。
降解测试的结果表明两种酶均能够转化麦角胺。然而,具有较短的核苷酸序列的酶显示出显著更高的活性,因而该变体已经甚至能够以1:1000稀释完全转化麦角胺,较长的变体已经以1:100稀释仅仅显示很低的活性。
实施例3:
序列ID No.1的酶的活性温度范围和温度稳定性的确定
为了确定序列ID No.1的酶的活性的最佳温度,0.1μg/ml的酶与5mg/kg麦角胺在Teorel l-Stenhagen通用缓冲液(pH 9.0)中在范围从10℃至50℃的不同温度下进行孵育。在此,所述酶在10℃至35℃的范围中显示活性,基于起始速率的最佳为35℃。
为了确定温度稳定性,所述酶在范围从10℃至60℃的不同温度下孵育1h。这之后,所述酶溶液以0.1μg/ml的浓度在Teorell-Stenhagen通用缓冲液(pH 7.0)中与0.1mg/ml BSA和5mg/kg麦角胺在25℃进行孵育。结果表明所述酶在直至30℃的温度下是稳定的,在40℃孵育之后仍然显示一定的活性,然而在35℃和40℃之间出现活性的降低。综上所述,序列ID No.1的酶基本上在胃肠道中的常规温度条件下具有最佳温度。
实施例4:
序列ID No.1的酶的活性最佳pH和pH稳定性的确定
为了确定ErgA活性的最佳pH范围,将0.1μg/ml的酶与5mg/kg麦角胺采用Teorell-Stenhagen通用缓冲液于25℃在不同的pH值下进行孵育。选择这种缓冲液是因为通过柠檬酸盐、磷酸盐和硼酸盐的组合借助盐酸可在pH 2至pH 12的范围中调整相同缓冲能力。在此显示,所述酶在pH 6至pH 11的范围中显示活性,在pH 8至pH 9具有小的活性平台。
为了确定pH稳定性,所述酶于25℃在范围从pH 2至pH 12的不同pH值下孵育1h。随后,所述酶溶液以0.1μg/ml的浓度在Teorell-Stenhagen通用缓冲液(pH 7.0)中与0.1mg/ml BSA和5mg/kg麦角胺在25℃下进行孵育。在此也显示出一个活性平台,这次在pH 6至pH 9的范围中,超出这个范围活性急剧降低。在这个范围中的活性确保序列ID No.1的酶作为饲料添加剂的技术应用。
实施例5:
序列ID No.1的酶在巴斯德毕赤酵母中的表达
将序列ID No.2的基因克隆入pGAPZαC、转化入巴斯德毕赤酵母并采用标准方法表达。带有序列ID No.2的基因的表达载体pGAPZαC被示于图3中。在50mM磷酸钠缓冲液(pH 7.0)中用5mg/kg麦角胺在25℃下进行降解实验。培养物上清液以1:100稀释进行使用。采用HPLC-FLD对所述样品进行分析。基于来自SDS-PAGE和降解实验的结果,可以在培养物上清液中确认序列ID No.1的酶的表达。
实施例6:
序列ID No.1的酶在枯草芽孢杆菌中的表达
将序列ID No.2的基因克隆入pHT43、转化入枯草芽孢杆菌并采用标准方法表达。带有序列ID No.2的基因的表达载体pHT43被示于图4中。在50mM磷酸钠缓冲液(pH 7.0)中用5mg/kg麦角胺在25℃进行降解实验。培养物上清液以1:10稀释进行使用。采用HPLC-FLD对所述样品进行分析。基于来自SDS-PAGE和降解实验的结果,可以在培养物上清液中确认ErgA的表达。
实施例7:
在瘤胃模型中的降解实验
在体外瘤胃模型中测试了序列ID No.1的酶的降解麦角生物碱的酶活。为此,采用由合成瘤胃液、干草和小麦、玉米和大豆的谷物混合物组成的溶液1:1稀释新鲜瘤胃液。为了证实麦角肽碱的反应,使用序列ID No.1的酶(1μg/ml)和5mg/kg的麦角胺的起始物。在隔膜上使用发酵管,并且所述批次在39℃的水浴中进行孵育。借助HPLC/ESI-MS/MS的分析显示,麦角胺在所述瘤胃模型中完全被转换成麦角酰胺和麦角酸。
文献
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SCHARDL C.L.,PANACCIONE D.G.&TUDZYNSKI P.2006.Ergot Alkaloids-Biologyand Molecular Biology.The Alkaloids,63,45-86.
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Claims (18)
1.用于转化、特别是水解裂解麦角肽碱的酶,其特征在于,所述酶是在环醇环处水解裂解麦角肽碱的α/β-水解酶。
2.根据权利要求1的酶,其特征在于,所述酶包含由亲核氨基酸、组氨酸和酸性氨基酸组成的催化三联体,并且所述三联体被包含于具有α/β-水解酶折叠的肽链中。
3.根据权利要求2的酶,其特征在于,所述催化三联体由亲核氨基酸丝氨酸、组氨酸和酸性氨基酸天冬氨酸或谷氨酸之一组成,并且所述三联体被包含于具有α/β-水解酶折叠的肽链中。
4.根据权利要求1、2或3的酶,其特征在于,所述α/β-水解酶包含具有序列Gly-Gln-Ser-Arg-Asn-Gly的亲核肘。
5.根据权利要求1-4中任一项的酶,其特征在于,与序列ID No.1具有至少96%的序列同一性。
6.根据权利要求5的酶,其特征在于,特别是具有序列ID No.3的酶。
7.根据权利要求5的酶,其特征在于,特别是具有序列ID No.1的酶。
8.根据权利要求5的酶,其特征在于,是具有不同于序列ID No.1的N-或C-末端的序列、特别是延长的N-或C-末端的序列,特别是序列ID No.3或序列ID No.5的酶。
9.用于酶促转化麦角肽碱的方法,其特征在于,所述麦角肽碱在环醇环处被水解裂解为初级代谢物。
10.根据权利要求9的方法,其特征在于,所述裂解是通过环醇环的C3’-原子上的亲核攻击实现的。
11.根据权利要求9或10的方法,其特征在于,所述环醇环的C3’-原子上的亲核攻击是通过催化三联体实现的,所述催化三联体被包含于具有α/β-水解酶折叠的肽链中并由亲核氨基酸丝氨酸、组氨酸和酸性氨基酸天冬氨酸或谷氨酸之一组成。
12.根据权利要求9、10或11的方法,其特征在于,由所述麦角肽碱的环醇环的水解裂解形成的初级代谢物被进一步发生反应。
13.根据权利要求12的方法,其特征在于,所述初级代谢物的进一步反应是通过自发化学反应实现的。
14.根据权利要求12或13的方法,其特征在于,在所述初级代谢物的进一步反应中形成麦角酰胺。
15.根据权利要求12、13或14的方法,其特征在于,所述初级代谢物的进一步反应是通过在反应介质中存在的酶实现的。
16.用于生产对麦角肽碱进行代谢、特别是对麦角肽碱进行解毒的酶的方法,其特征在于,将编码根据权利要求1-8任一项的基因克隆入表达载体、转化入原核和/或真核宿主细胞并且在宿主细胞中表达。
17.根据权利要求16的方法,其特征在于,具有序列ID No.2、4或6的基因被用作所述基因。
18.根据权利要求16或17的方法,其特征在于,将所述基因转化入作为宿主细胞的选自巴斯德毕赤酵母(Pichia pastoris)、大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)的微生物中并在其中进行表达。
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DAVID L. OLLIS ET AL.: "The α/β hydrolase fold", 《PROTEIN ENGINEERING》 * |
DEBORAH A RATHBONE ET AL.: "Microbial transformation of alkaloids", 《ECOLOGY AND INDUSTRIAL MICROBIOLOGY》 * |
LUDMILA MARTI´NKOVA ET AL.: ""Hydrolysis of lysergamide to lysergic acid by Rhodococcus equi A4", 《JOURNAL OF BIOTECHNOLOGY》 * |
ROBERT KOURIST ET AL.: "The a/b-Hydrolase Fold 3DM Database (ABHDB) as a Tool for Protein Engineering", 《CHEMBIOCHEM》 * |
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