CN1228812A - 微生物、由其获得的内酰胺酶、以及它们的应用 - Google Patents
微生物、由其获得的内酰胺酶、以及它们的应用 Download PDFInfo
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- CN1228812A CN1228812A CN97197622A CN97197622A CN1228812A CN 1228812 A CN1228812 A CN 1228812A CN 97197622 A CN97197622 A CN 97197622A CN 97197622 A CN97197622 A CN 97197622A CN 1228812 A CN1228812 A CN 1228812A
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- hydrolysis
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Classifications
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
在食酸丛毛单胞菌的一个菌株中发现了一种内酰胺酶,它具有良好的稳定性,能够水解二环内酰胺即2-氮杂二环[2.2.1]庚-5-烯-3-酮的一个对映体,得到(-)内酰胺和(+)氨基酸。对该酶已进行分离和克隆并且鉴定了其结构。
Description
本发明涉及一种微生物、由其获得的内酰胺酶、以及它们的应用。
二环γ-内酰胺即2-氮杂二环[2.2.1]庚-5-烯-3-酮是可用于生产碳环核苷的有用的合成纤维,所述碳环核苷在作为治疗剂方面的重要性日益增加。这类核苷所针对的公开的领域包括抗病毒剂(例如,Vince和Hua,医学化学杂志(J.Med.Chem.),33:17-21(1990),抗例如HIV)和心血管舒张药(腺苷兴奋剂)。这类药剂中碳环的主要益处在于其对体内酶分解的抗性。通过比较,天然存在的核糖基核苷更容易被核酸酶裂解,因而丧失其生物活性。
尽管自然界中的碳环核苷类是已知的,例如得自桔色链霉菌(Streptomyces citricolor)的芒霉素,但天然产率低,分离到的产物还必须进一步处理以获得更有用的化合物。一种更为经济的途径是从γ-内酰胺开始用化学方法合成所需化合物。然而,由于是化学方法合成的,γ-内酰胺是外消旋的。通过常规合成方法,最终药物也将是对映体的混合物;如果对映体之一不是很有活性或者引起不希望有的副作用,这就会导致有关调节的问题。因此,需要在合成中实施一个步骤,其中可以分离外消旋合成纤维的两个对映体之一,然后在它的基础上构建药物的其它部分。
这样做的一个有效途径是使用一种酶通过酰胺键来选择性水解外消旋γ-内酰胺的一个对映体,得到环状氨基酸化合物并留下另一个对映体。然后,通过萃取到二氯甲烷中可以轻易地将残留的内酰胺与氨基酸产物分离开,通过结晶法纯化并且用于随后的下游化学合成,制成所需药物。通过仔细选择适当的酶可能发现一种只对内酰胺对映体之一具有高选择性的酶,这样就以或多或少大于50%的转化率,保留了高对映体过量(>90%)的内酰胺。已经发现了对两种对映体中任一种具有选择性的酶。
EP-A-0424064公开了进行上述拆开的方法,提供了能产生具有不同选择性的酶的两种生物体。一种红球菌属(Rhodococcus)菌株产生一种酶,该酶水解(-)内酰胺而使(+)内酰胺得以分离供进一步应用;而一个假单胞菌属(Pseudomonad)产生一种酶,该酶水解(+)内酰胺而使(-)内酰胺得以分离。
文献中还描述了进行这些选择性水解的其它酶。Taylor等在四面体(Tetrahedron):不对称现象,4(6):1117-1128(1993)中描述了一种得自荧光假单胞菌(Pseudomonas fiuorescens)的对(+)内酰胺水解有选择性的酶和一种得自金杆菌属(Aureobacterium)的一个菌株的对(-)内酰胺水解有选择性的酶。Brabban等在工业微生物学杂志(J.Ind.Microbiology),16:8-14(1996)中描述了对(+)内酰胺水解具有选择性的另一种酶。
为了开发一种先进的工业生物转化方法,希望使用较稳定的酶或完整细胞生物催化剂。通过固定化可以使生物催化剂得以回收和再利用,这就大大降低了生物催化剂成本并能大规模处理生物催化剂而没有活性的明显损失。还经常发现,更稳定的生物催化剂更能耐受高的底物和/或产物浓度而不失活。于是,在给定的动力学和处理条件下,这就能使生物转化在可能的最高反应物浓度下操作。这具有两个优点:它只需最小的反应器体积,还使产物处理过程中液体处理体积达到最低程度。
Taylor等(同上)描述了一种得自金杆菌属菌种的内酰胺酶,该酶在提高的温度下很稳定,它选择性地水解(-)γ-内酰胺,给出(+)γ-内酰胺和作为一种产物的(-)氨基酸。已经把得自该生物体的酶固定化,在为期数月的操作中该酶保持其稳定性。具有良好稳定性和相反选择性的酶还是未知的,尽管Brabban等(同上)筛选了一些不同的有潜力的分离物。以前对显示所需内酰胺酶活性的假单胞菌属类型的生物体的研究表明这些生物体稳定性差。这是令人遗憾的,因为更有用的合成纤维是(-)γ-内酰胺,与例如通过金杆菌属内酰胺酶的作用形成的(-)氨基酸相比,(-)γ-内酰胺具有更为天然的立体化学并且使其更易于产生功能。因此,需要一种对(+)二环γ-内酰胺水解具有高选择性的稳定的γ-内酰胺酶。
已令人惊奇地发现,从环境中分离到的一个食酸丛毛单胞菌(Comamonas acidovorans)菌株能产生一种酶,该酶在拆开所需γ-内酰胺的工业方法中有很大应用潜力。该酶不仅比以前鉴定的(+)γ-内酰胺酶具有大得多的温度稳定性,而且能够使生物拆开在很高的底物/产物浓度下进行。按照布达佩斯条约的条款,该生物体已于1996年8月30日保藏于NCIMB,23St.Machar Street,Aberdeen,UK,保藏号为NCIMB40827。
编码该γ-内酰胺酶的基因已被分离和测序(参见SEQ ID NO:1),并且推断出该酶的氨基酸序列(参见SEQ ID NO:2)。本发明涉及具有这种结构的化合物,及其具有相同活性的片断,这种片断对于本领域普通技术人员而言是显而易见的。该新型酶的特征在于其稳定性,即下列一种或多种:
在40℃保持4小时后活性保留大于85%或者在60℃保持4小时后活性大于30%;
在100g外消旋内酰胺加300ml缓冲剂的初始浓度下水解,进行到(+)内酰胺水解至少90%而(-)内酰胺水解小于5%。
该新型酶可用于所需γ-内酰胺的对映体的混合物例如外消旋混合物的对映体专一性水解(enantiospecific hydrolysis)。反应后,残留(-)内酰胺可轻易地与水解所形成的(+)氨基酸分离。这两个反应都能在本领域普通技术人员已知的条件下进行。
该酶可以完整细胞或分离的形式使用。如需要,可通过本领域普通技术人员已知的方法将该酶固定化。
该酶可由所述保藏的生物体产生。或者可以通过重组技术产生。
采用本文提供的DNA和氨基酸序列,本领域技术人员可轻易地构建本文公开的基因和酶的片断或突变。保留了列举的酶的活性的这些片断和突变在本发明范围之内。而且,由于遗传密码的丰余性,多种不同的DNA序列可编码本文公开的氨基酸序列。构建编码相同或相似酶的这些替代性DNA序列在本领域普通技术人员的能力范围内。这些DNA序列在本发明范围内。本文所用的表述“基本上相同的”序列是指那些具有不显著影响活性的氨基酸替换、缺失、添加或插入的序列。保留了活性的片断也包括于该定义中。
本发明的基因可用已知方法分离并可导入多种微生物宿主中。该基因的表达直接或间接导致酶的胞内产生和维持。该基因可借助于合适的载体导入微生物宿主。
有多种途径可用于在允许基因稳定维持和表达的条件下将该基因导入微生物宿主。一个DNA构建体可以包括用于基因表达的转录和翻译调节信号,在其调节控制之下的基因以及一个与宿主生物体中的一个序列同源的DNA序列,由此将发生整合,和/或一个在该宿主中挥发作用的复制系统,由此将发生整合或稳定维持。
沿转录方向,即编码或有义序列的5’→3’方向,该构建体可包括转录调节区,如果有的话,以及启动子,此时调节区可在启动子的5’或3’,核糖体结合位点,起始密码子,具有与起始密码子同相的开放读框的结构基因,终止密码子,聚腺苷酸化信号,如果有的话,以及终止区。呈双链形式的该序列可单独地用于微生物宿主的转化,但通常将与涉及标记物的一个DNA序列一起被含于其中。
该基因可被导入转录/翻译起始和终止区之间,以便在起始区的调节控制之下。该构建体可含于一个质粒中,它可包括至少一个复制系统,但可包括一个以上,此时,一个复制系统被用于质粒形成期间的克隆化,而第二个复制系统是在最终宿主中发挥作用所必需的。此外,如上所述,可以存在一个或多个标记物。当需要整合时,该质粒将合乎需要地包括一个与宿主基因组同源的序列。
可按常规方式分离转化体,通常采用一种选择技术,它能对照未修饰生物体或转移生物体选择出所需生物体(当存在时)。然后可以检验转化体的活性。
合适的宿主细胞包括原核生物和真核生物。一个例子是大肠杆菌(E.coli)。
下列实施例阐述了本发明。1.分离有潜力的γ-内酰胺酶生产菌株
将得自沟的大约1g土壤与20ml 50mM磷酸钾缓冲剂(pH7)混合,混合良好并在室温下振荡30分钟。然后将该悬浮液的0.4%接种物置于锥形瓶中的25ml富集培养基中,在30℃下振荡41小时。采用如下富集培养基:
(g.l-1)
酵母提取物 0.1
NH4Cl 2.0
KH2PO4 7.0
Na2HPO4 2.0
MgSO4 0.4
CaCl2 0.2
痕量元素溶液 0.2
外消旋的二环γ-内酰胺 2.0
5M NaOH 至pH7
痕量元素溶液含有:
(g.l-1)
CaCl2.2H2O 3.6
ZnO 2.0
CuCl.2H2O 0.85
Na2MoO.2H2O 4.8
MnCl2.4H2O 2.0
FeCl3.6H2O 5.4
H3BO3 0.3
CoCl2.6H2O 2.4
浓HCl 250ml
然后将0.5%接种物转入同种培养基的第二个富集瓶(25ml)中,再生长94小时。此时,从瓶中取样,在10mM磷酸盐缓冲剂(pH7)中稀释,铺在如下培养基上:
(g.l-1)
酵母提取物 0.1
NH4Cl 2.0
KH2PO4 7.0
Na2HPO4 2.0
MgSO4 0.4
CaCl2 0.2
痕量元素溶液 0.2
纯净琼脂 15.0
5M NaOH 至pH7
然后在浇平板之前,在冷却下将2.0g.l-1的N-乙酰基-L-苯丙氨酸过滤灭菌至上述高压灭菌器培养基中。在30℃下培养6天后,挑出菌落,在其它琼脂平板上纯化,接着用于筛选研究。2.筛选分离出的分离物:
让分离到的菌落生长于如下培养基中:
(g.l-1)
酵母提取物 5.0
NH4Cl 2.0
KH2PO4 7.0
Na2HPO4 2.0
MgSO4 0.4
CaCl2 0.2
痕量元素溶液 1.0
外消旋的二环γ-内酰胺 2.0
葡萄糖 10.0
5M NaOH 至pH7
将一个菌落接种入灭菌塑料容器中的4ml过滤灭菌的培养基中,于30℃在振荡器中生长约24小时。
然后对培养物进行离心,将沉淀物重悬于1ml的50mM磷酸盐缓冲剂(pH7)中。再向其中添加在类似缓冲剂中的1ml 100g.l-1外消旋二环γ-内酰胺。在30℃、振荡下进行反应。在之后的7天内取样,通过HPLC分析内酰胺的转化。对于那些显示明显水解的反应,通过GC测定对映体过量(ee)。
分离出的一个菌株显示出所需特性。在初始筛选中,144小时生物转化后,该菌株获得了添加底物的52%转化率,残留内酰胺为(-)对映体,ee>99%。由NCIMB鉴定出该生物体是食酸丛毛单胞菌的一个菌株。如上所述,该菌株已保藏于NCIMB。
采用了如下分析方法:
水解程度(HPLC)。适当稀释样品,将20μl注到15cm KromasilC-8柱上。洗脱缓冲剂是10mM磷酸盐缓冲剂(pH7)中的50%甲醇;流速为1ml.min-1;运行时间5分钟。检测在λ=225nm下进行。
反应产物的ee(GC)。将样品萃取到乙酸乙酯中,用无水硫酸镁干燥,注到50m CP Cyclodextrin毛细管柱上。分析过程中,将炉温由初始的140℃提高到200℃。3.发酵
用如下培养基制备种子瓶:
(g.l-1)
酵母提取物 10
(NH4)2SO4 1
KH2PO4 5
MgSO4.7H2O 0.1
CaCl2.2H2O 0.05
痕量元素 0.1
NaOH 至pH7
痕量元素溶液如上述定义,所不同的是浓HCl的量是333ml.l-1。
在500ml锥形管中制备75ml培养基。用生物体接种锥形管,在25℃、振荡下培养,直至吸收度(520nm)达到3.5~7为止。然后将细胞以0.1%的浓度接种到盛有1.5L如下(灭菌的)培养基的发酵器中:
(g.l-1)
酵母提取物 20
(NH4)2SO4 2
KH2PO4 5
MgSO4.7H2 0.5
CaCl2.2H2O 0.1
痕量元素 1.0
琥珀酸 10
PPG2025 2ml
NaOH 至pH7
初始温度为25℃,pH控制在7.1。保持大约0.5vvm的恒定空气流量,搅拌在500-1000rpm变化以保持需氧条件。18.6小时后,开始缓慢添加浓缩的酵母提取物,速率相当于每小时每初始升添2g酵母提取物,即3g/h。24小时后完成发酵,通过离心收集细胞,在冰箱中以细胞糊形式贮存备用。收集大约为82g湿细胞的总生物量,最终发酵活性产率为0.45U.ml-1(其中,1U是每分钟水解1μmolγ-内酰胺)。4.温度稳定性
将35.8g细胞糊解冻并加700ml裂解缓冲剂中,该缓冲剂含有10mM磷酸钠(pH7)、10mM EDTA、0.1%Triton X-100、5mM二硫苏糖醇和1mg.ml-1溶菌酶。在室温下搅拌裂解缓冲剂5.5小时,然后添加用HCl调至pH7的37ml 5%聚乙烯亚胺溶液,在离心回收上清液之前再搅拌1小时。
在良好混合下向500ml上清液中缓慢添加174g硫酸铵以溶解该盐。20分钟后,离心收集沉淀物,重悬于100ml 10mM磷酸钠(pH7)中。再将其对2倍的5L 10mM磷酸钠(pH7.1)进行渗析,然后在冰箱中贮存。
为进行温度稳定性试验,把冷冻的渗析液解冻,用微型SephadexG-25凝胶过滤柱把2×2.5ml样品缓冲剂换成3.5ml磷酸盐缓冲盐水(PBS)或10mM Tris缓冲剂,pH8.0。将缓冲剂换成10mM Tris缓冲剂就产生了沉淀物(它含有一些活性),该沉淀物用离心法回收。接着,将每次制备的样品置于60℃热块、40℃水浴或25℃培养器中。在1、2和4.3小时取样,分析残余内酰胺酶活性。经4.3小时培养之后获得如下结果:
缓冲剂 温度(℃) 残余活性(开始时的%)
PBS 25 97
PBS 40 87
PBS 60 32Tris(pH8) 25 110Tris(pH8) 40 105Tris(pH8) 60 45
通过对比,Brabban等(同上)所述的荧光假单胞菌γ-内酰胺酶在37℃下保持4小时后其活性损失达70-80%。新型酶的温度稳定性明显更高。这开创了把该酶固定化于固体支持体上并在许多生物转化中重复利用的可能性,从而大大降低了其成本对过程的影响。5.完整细胞生物转化
将冷冻的细胞糊(25g)解冻并在50mM KH2PO4(300ml,pH7)中搅拌,所述细胞糊是按类似于实施例3中所述的发酵法获得的,所不同的是此情形中测得最终酶产率为0.67U.ml-1。向其中添加固体形式的γ-内酰胺(100g),反应物在25℃下搅拌24小时。添加硅藻土(28g),然后是聚乙烯亚胺(28ml在水中的5%溶液),接着是异丙醇(175ml)。再搅拌10分钟后,过滤除去固形物,然后真空蒸发滤液至体积为200ml。用二氯甲烷(200ml)萃取水相5次,然后用无水MgSO4干燥有机提取物。用丙酮(150ml)洗涤滤饼,(用无水MgSO4)干燥提取物,然后将全部合并的有机级分真空蒸发至干。这产生44.3g灰白色固体,经分析,它是对映体过量>99%的(-)内酰胺。
该生物转化过程可以在很高的底物浓度(1g底物/3ml缓冲剂)下进行,仍能提供(+)内酰胺对映体的完全水解。因此,这具有高度的体积有效性,它能以最小的体积生产(-)内酸胺,从而降低了液体处理要求并降低了间歇式生物转化反应器的体积要求。6.基因的鉴定与分离
通过添加补充了溶菌酶(1.5mg.ml-1)的TESS缓冲剂(50mMTris.HCl[pH 8.0],10mM EDTA,25mM NaCl,25%w/v蔗糖)来处理一定量的细胞糊(500mg)。该处理在37℃下进行1小时,所得原生质球通过添加10%SDS(1.5%最终浓度)来裂解。向该细胞裂解液中以1g.ml-1添加固体氯化铯。一旦溶解,以80μg.ml-1的最终浓度添加溴化乙锭。然后将该悬浮液加入Sorvall Ultracrimp超速离心管,通过在20℃、30000rpm下离心72小时建立起梯度。一旦分辨并由浓的溴化乙锭带显现,用注射器移除基因组DNA。通过用氯化铯-饱和丁醇萃取来除去溴化乙锭。最后,将基因组DNA在10000体积TE缓冲剂(10mM Tris.HCl,1mM EDTA[pH8.0])中渗析,有两个交替。
通过用Sau3AI(Promega Corp.)限制酶进行时程部分限制酶切消化来制备基因组文库。水平琼脂糖凝胶电泳分析出这些DNA片断在1.0-4.0kb的范围内。通过在25mA电流下在TBE(16mM Tris.HCl[pH8.0],8mM硼酸,400μM EDTA)中电洗脱来切除这些片断。洗脱的DNA片断通过用等体积的Tris-缓冲苯酚:氯仿萃取以及乙醇沉淀来进行纯化。将Sau3AⅠ部分基因组DNA片断连接到pUC19上;参见Yanish-Peron等,基因(Gene)33:103-119(1985)。该克隆用载体pUC19事先通过BamHI(Promega Corp.)限制酶切消化进行过线性化,5’-磷酸基因用小牛小肠碱性磷酸酶(Promega Corp.)除去以防重新连接。使用T4 DNA连接酶(Boehringer Mannheim Ltd)、以载体和基因组片断的各种比例、在14℃下进行连接。将连接反应物转化入Max Efficiency大肠杆菌DH5α(Gibco BRL Life Sciences),将转化过的大肠杆菌铺到补充有氨苄青霉素(100μg.ml-1)、X-Gal(50μg.ml-1)和1mM IPTG的胰化蛋白胨大豆琼脂(Oxoid Ltd)上。37℃下过夜培养后,将转化过的大肠杆菌菌落吸附到用甲醇中的20mg.ml-1(+)-内酰胺浸渍过的Whatman2滤纸盘上。在室温下培养滤纸4小时,用丙酮中的2%w/v茚三酮显色。在60℃下显色后,在单菌落周围可清晰地看到在紫色背景上的一个醒目的褐色晕圈,这是产生氨基酸的标志。分离出该单一的内酰胺酶表达克隆,通过Achiral和Chiral HPLC分析证实内酰胺酶活性。7.内酰胺酶基因的鉴定和测序
从内酰胺酶表达克隆制备质粒DNA。限制酶切消化分析表明存在1.9kb的Sau3AⅠ限制片断。对插入片断的DNA序列分析表明该片断掺入了一个1.6kb的开放读框(ORF),该读框被pUC19的上游lac启动子驱动时翻译成575个残基(61KDa.)的蛋白质;参见序列表。被翻译的ORF的推断氨基酸序列表明与得自耻垢分枝杆菌(Mycobacteriumsmegmatis)和食甲基嗜甲基菌(Methylophilus methylotrophus)的乙酰胺酶有大于65%的同源性。已显示这些酶能水解短链脂族酰胺;参见Draper,遗传微生物学杂志(J.Gen.Microbiol.)46:111-123(1969)。
参照序列表,该1.9kb内酰胺酶片断存在于两个保留的BamHⅠ限制位点之内。位于插入片断5’的序列掺入了lac启动子和pUC19的核糖体结合位点。
接着,通过插入得自野生型大肠杆菌质粒ColE1的cer元件来修饰携带内酰胺酶基因的pUC19构建体。该构建体被称为pPET1。
要理解的是,大肠杆菌质粒pPET1衍生自pUC19,它携带得自食酸丛毛单胞菌、连接到BamHⅠ限制位点上的1.9kb Sau3AⅠ基因组片断。把野生型质粒ColE1的cer稳定性元件借助于BamHⅠ(部分的)和NdeⅠ限制插入到内酰胺酶片断的3’。8.重组内酰胺酶的生长。
将重组体大肠杆菌菌株接种入1升的带挡板的摇瓶中,该摇瓶中含有补充了氨苄青霉素(100μg.ml-1)的100ml TSB培养基(OxoidLtd.)。将摇瓶和接种物在37℃、以300rpm在定轨摇床(25mm摆幅)中振荡下,培养16小时。把种子培养物接种(1%)入含1.5升TSB培养基的2.8升实验室生物反应器中。温度保持在25℃,pH为7.0,溶解氧程度(tension)>50%。对照TSB培养基空白,在520nm光密度下监视生长情况。生长24小时后,离心(5000g,4℃,10分钟)收集细胞。将细胞贮存于-20℃下备用。9.重组细胞的应用
如上所述,使携带重组质粒pPET1的大肠杆菌菌株生长并将其贮存。把细胞以10%w/v重悬于100mM Tris.HCl,pH7.5。将外消旋的内酰胺以100mg.ml-1重悬于100mM Tris.HCl,pH7.5。(+)-内酰胺生物转化的反应条件为:10mg.ml-1外消旋内酰胺,与在100mM Tris.HCl,pH7.5中的0.1%w/v重组细胞混合。在25℃、225rpm振荡下,让悬浮液反应1小时。反应1小时后,HPLC分析显示,30%的(+)-内酰胺转化为酸,选择性大于95%对映体过量。
序列表(1)一般信息:
(ⅰ)申请人:
(A)名称:Chiroseience Limited
(B)街道:Cambridge Science Park,Milton Road
(C)城市:Cambridge
(D)郡:N/A
(E)国家:英国
(F)邮编(EIP):CB4 4WE
(ⅱ)发明名称:微生物、由其获得的内酰胺酶,及它们的应用
(ⅲ)序列数目:2
(ⅳ)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,Version#1.30(EPO)
(ⅴ)当前申请资料:
申请号:(2)SEQ ID NO:1的信息:
(ⅰ)序列特征:
(A)长度:1951个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线型
(ⅱ)分子类型:DNA(基因组的)
(ⅲ)假设:无
(ⅳ)反义:无
(ⅵ)原始来源:
(A)生物体:食酸丛毛单胞菌(ⅸ)特征:
(A)名称/键:CDS
(B)位置:49..1773(ⅹⅰ)序列描述:SEQ ID NO:1CGCTCGTATG TTGGGATGTG AGCGATACAA TTTCACACAG GAACAGCT ATG ACC ATG 57
Met Thr Met
1ATA ACG CCA AGC TTG CAT GCC TCG GCA GGT CGG ACT CTA GAG GAT CCG 105Ile Thr Pro Ser Leu His Ala Ser Ala Gly Arg Thr Leu Glu Asp Pro
5 10 15TTT TTT CCC ACT GCC ATC GCA AGG AGC ACA CCA TGG CCG GAA ACC CTG 153Phe Phe Pro Thr Ala Ile Ala Arg Ser Thr Pro Trp Pro Glu Thr Leu20 25 30 35ATC AAG GTC GAT CTC AAC CAG TCC CCC TAC GAC AAC CCG CAG GTG CAC 201Ile Lys Val Asp Leu Asn Gln Ser Pro Tyr Asp Asn Pro Gln Val His
40 45 50AAC CGC TGG CAT CCC GAC ATT CCC ATG GCG GTC TGG GTG GAG CCG GGC 249Asn Arg Trp His Pro Asp Ile Pro Met Ala Val Trp Val Glu Pro Gly
55 60 65GCG GAG TTC AAG CTG GAG ACC TAT GAC TGG ACC GGC GGC GCC ATC AAG 297Ala Glu Phe Lys Leu Glu Thr Tyr Asp Trp Thr Gly Gly Ala Ile Lys
70 75 80AAC GAC GAC AGC GCC GAA GAC GTG CGC GAC GTG GAT CTG TCC ACC GTC 345Asn Asp Asp Ser Ala Glu Asp Val Arg Asp Val Asp Leu Ser Thr Val
85 90 95CAC TTC CTG TCC GGC CCC GTG GGC GTG AAG GGC GCG CAG CCC GGC GAC 393His Phe Leu Ser Gly Pro Val Gly Val Lys Gly Ala Gln Pro Gly Asp100 105 110 115CTG CTG GTG GTG GAC CTG CTG GAC ATC GGC GCG CGC GAC GAC AGC CTC 441Leu Leu Val Val Asp Leu Leu Asp Ile Gly Ala Arg Asp Asp Ser Leu
120 125 130TGG GGC TTC AAC GGC TTT TTC TCC AAG CAG AAT GGC GGC GGC TTC CTG 489Trp Gly Phe Asn Gly Phe Phe Ser Lys Gln Asn Gly Gly Gly Phe Leu
135 140 145GAC GAG CAT TTC CCG CTG GCC CAG AAG TCC ATC TGG GAC TTC CAC GGC 537Asp Glu His Phe Pro Leu Ala Gln Lys Ser Ile Trp Asp Phe His Gly
150 155 160ATG TTC ACC AAG AGC CGC CAC ATC CCC GGC GTC AAC TTC GCA GGC CTC 585Met Phe Thr Lys Ser Arg His Ile Pro Gly Val Asn Phe Ala Gly Leu
165 170 175ATC CAC CCG GGC CTG ATC GGC TGC CTG CCC GAC CCC AAG ATG CTG GCC 633Ile His Pro Gly Leu Ile Gly Cys Leu Pro Asp Pro Lys Met Leu Ala180 185 190 195AGC TGG AAT GAG CGC GAG ACC GGC CTC ATC GCC ACC GAC CCC GAC CGC 681Ser Trp Ash Glu Arg Glu Thr Gly Leu Ile Ala Thr Asp Pro Asp Arg
200 205 210ATT CCC GGC CTG GCC AAC CCG CCC AAC GCC ACC ACC GCC CAC ATG GGC 729Ile Pro Gly Leu Ala Asn Pro Pro Asn Ala Thr Thr Ala His Met Gly
215 220 225CAG ATG CAG GGC GAG GCG CGC GAC AAG GCC GCC GCC GAA GGC GCA CGC 777Gln Met Gln Gly Glu Ala Arg Asp Lys Ala Ala Ala Glu Gly Ala Arg
230 235 240ACC GTG CCG CCG CGC GAG CAC GGC GGC AAC TGC GAC ATC AAG GAC CTC 825Thr Val Pro Pro Arg Glu His Gly Gly Asn Cys Asp Ile Lys Asp Leu
245 250 255TCG CGC GGC TCG CGC GTG TTC TTC CCC GTC TAC GTG GAC GGC GCG GGC 873Ser Arg Gly Ser Arg Val Phe Phe Pro Val Tyr Val Asp Gly Ala Gly260 265 270 275CTG AGC GTG GGC GAC CTG CAC TTC AGC CAG GGT GAT GGC GAG ATC ACC 921Leu Ser Val Gly Asp Leu His Phe Ser Gln Gly Asp Gly Glu Ile Thr
280 285 290TTC TGG GGG CCC ATC GAG ATG CCC GGC TGG GTG CAC ATG AAG GTC TCG 969Phe Trp Gly Pro Ile Glu Met Pro Gly Trp Val His Met Lys Val Ser
295 300 305CTG ATC AAG GGC GGC ATG GCC AAG TAC GGC ATC AAG AAC CCC ATC TTC 1017Leu Ile Lys Gly Gly Met Ala Lys Tyr Gly Ile Lys Asn Pro Ile Phe
310 315 320AAG CCC AGC CCC ATG ACG CCC AAC TAC CAA GGA CTA CCT GAT CTT CGA 1065Lys Pro Ser Pro Met Thr Pro Asn Tyr Gln Gly Leu Pro Asp Leu Arg
325 330 335AGG CAT CTC GGT GGA CGA AAA GGG CAA GCA GCA CTA CCT GGA CGT GAC 1113Arg His Leu Gly Gly Arg Lys Gly Gln Ala Ala Leu Pro Gly Arg Asp340 345 350 355CGT GGC CTA CCG CCA GGC CTG CCT GAA CGC CAT CGA GTA CCT GAA GAA 1161Arg Gly Leu Pro Pro Gly Leu Pro Glu Arg His Arg Val Pro Glu Glu
360 365 370ATT CGG CTA CAG CGG CGC CCA GGC CTA CTC GCT GCT GGG CAC GGC GCC 1209Ile Arg Leu Gln Arg Arg Pro Gly Leu Leu Ala Ala Gly His Gly Ala
375 380 385CGT GCA GGG CCA CAT CAG CGG CGT GGT GGA CGT GCC CAA TGC CTG CGC 1257Arg Ala Gly Pro His Gln Arg Arg Gly Gly Arg Ala Gln Cys Leu Arg
390 395 400CAC GCT GTG GCT GCC CAC GGA GAT CTT CGA CTT CGA CAT CAA TCC CAC 1305His Ala Val Ala Ala His Gly Asp Leu Arg Leu Arg His Gln Ser His
405 410 415GGC CGA GGG ACC ACA GAA GAT CAT CAC GGG CGG GGT GGA TCT GCC CAT 1353Gly Arg Gly Thr Thr Glu Asp His His Gly Arg Gly Gly Ser Ala His420 425 430 435CGC CCA GGA CAA GTA AGC CCG GCA TAC GAC ACC CGC CAT CCA CCA TTC 1401Arg Pro Gly Gln Val Ser Pro Ala Tyr Asp Thr Arg His Pro Pro Phe
440 445 450GCC AGA GGC CGC CCA TGC CCA CCT ATG ACT ACC ACT GCA CCG CAT GCG 1449Ala Arg Gly Arg Pro Cys Pro Pro Met Thr Thr Thr Ala Pro His Ala
455 460 465GCG GCT TCG ACG CGC TGC GCA GCC TCT CGC AGC GCA ACG AGC CCG CGC 1497Ala Ala Ser Thr Arg Cys Ala Ala Ser Arg Ser Ala Thr Ser Pro Arg
470 475 480CCT GCC CCA GCT GCG AGG CGG CCT CGC CCC GCG TCT TCG TCA GCG CGC 1545Pro Ala Pro Ala Ala Arg Arg Pro Arg Pro Ala Ser Ser Ser Ala Arg
485 490 495CGC GCC TGG CCT GCA CCA GCC CCG AAC AGC GCC GCG CCC ACG ACA CCA 1593Arg Ala Trp Pro Ala Pro Ala Pro Asn Ser Ala Ala Pro Thr Thr Pro500 505 510 515ACG AGC GCG CCC GGC ACG AGC CCA GGC GCT CAC GCG ATG TGG CCG AGG 1641Thr Ser Ala Pro Gly Thr Ser Pro Gly Ala His Ala Met Trp Pro Arg
520 525 530GCA GCT ACG CGC GCA TGC GCC ACC CCA TCG GGC TGC GGC TGC TGC AGC 1689Ala Ala Thr Arg Ala Cys Ala Thr Pro Ser Gly Cys Gly Cys Cys Ser
535 540 545GGC GCC AGC AAG CGC GGC TCC ACG GTC ACG GCG CCC AAC GGC GCC AAG 1737Gly Ala Ser Lys Arg Gly Ser Thr Val Thr Ala Pro Asn Gly Ala Lys
550 555 560ACC TTC CCG ACC AAG CGG CCC TGG ATG ATC AGC CAC TGACCGCGGA 1783Thr Phe Pro Thr Lys Arg Pro Trp Met Ile Ser His
565 570 575CCCTGCGCCG CACCAATGAC AAGGGCCCGC GACGCGGGCC TTTGTCCTGC CTGGCCGTAC 1843CGCTCAGTGC ACGGCGCCGA TGAAGCCGGC CAGCTCCGGC GTCTGCGGGT TGGCGAACAG 1903CTGCTTGGCC CGGGGCCGCT TTCGTGGATC CCCGGTACCG AATCGATC 1951(2)SEQ ID NO:2的信息:
(ⅰ)序列特征:
(A)长度:575个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(ⅹⅰ)序列描述:SEQ ID NO:2Met Thr Met Ile Thr Pro Ser Leu His Ala Ser Ala Gly Arg Thr Leu1 5 10 15Glu Asp Pro Phe Phe Pro Thr Ala Ile Ala Arg Ser Thr Pro Trp Pro
20 25 30Glu Thr Leu Ile Lys Val Asp Leu Asn Gln Ser Pro Tyr Asp Asn Pro
35 40 45Gln Val His Asn Arg Trp His Pro Asp Ile Pro Met Ala Val Trp Val
50 55 60Glu Pro Gly Ala Glu Phe Lys Leu Glu Thr Tyr Asp Trp Thr Gly Gly65 70 75 80Ala Ile Lys Asn Asp Asp Ser Ala Glu Asp Val Arg Asp Val Asp Leu
85 90 95Ser Thr Val His Phe Leu Ser Gly Pro Val Gly Val Lys Gly Ala Gln
100 105 110Pro Gly Asp Leu Leu Val Val Asp Leu Leu Asp Ile Gly Ala Arg Asp
115 120 125Asp Ser Leu Trp Gly Phe Asn Gly Phe Phe Ser Lys Gln Asn Gly Gly
130 135 140Gly Phe Leu Asp Glu His Phe Pro Leu Ala Gln Lys Ser Ile Trp Asp145 150 155 150Phe His Gly Met Phe Thr Lys Ser Arg His Ile Pro Gly Val Asn Phe
165 170 175Ala Gly Leu Ile His Pro Gly Leu Ile Gly Cys Leu Pro Asp Pro Lys
180 185 190Met Leu Ala Ser Trp Asn Glu Arg Glu Thr Gly Leu Ile Ala Thr Asp
195 200 205Pro Asp Arg Ile Pro Gly Leu Ala Asn Pro Pro Asn Ala Thr Thr Ala
210 215 220His Met Gly Gln Met Gln Gly Glu Ala Arg Asp Lys Ala Ala Ala Glu225 230 235 240Gly Ala Arg Thr Val Pro Pro Arg Glu His Gly Gly Asn Cys Asp Ile
245 250 255Lys Asp Leu Ser Arg Gly Ser Arg Val Phe Phe Pro Val Tyr Val Asp
260 265 270Gly Ala Gly Leu Ser Val Gly Asp Leu His Phe Ser Gln Gly Asp Gly
275 280 285Glu Ile Thr Phe Trp Gly Pro Ile Glu Met Pro Gly Trp Val His Met
290 295 300Lys Val Ser Leu Ile Lys Gly Gly Met Ala Lys Tyr Gly Ile Lys Asn305 310 315 320Pro Ile Phe Lys Pro Ser Pro Met Thr Pro Asn Tyr Gln Gly Leu Pro
325 330 335Asp Leu Arg Arg His Leu Gly Gly Arg Lys Gly Gln Ala Ala Leu Pro
340 345 350Gly Arg Asp Arg Gly Leu Pro Pro Gly Leu Pro Glu Arg His Arg Val
355 360 365Pro Glu Glu Ile Arg Leu Gln Arg Arg Pro Gly Leu Leu Ala Ala Gly
370 375 380His Gly Ala Arg Ala Gly Pro His Gln Arg Arg Gly Gly Arg Ala Gln385 390 395 400Cys Leu Arg His Ala Val Ala Ala His Gly Asp Leu Arg Leu Arg His
405 410 415Gln Ser His Gly Arg Gly Thr Thr Glu Asp His His Gly Arg Gly Gly
420 425 430Ser Ala His Arg Pro Gly Gln Val Ser Pro Ala Tyr Asp Thr Arg His
435 440 445Pro Pro Phe Ala Arg Gly Arg Pro Cys Pro Pro Met Thr Thr Thr Ala450 455 460Pro His Ala Ala Ala Ser Thr Arg Cys Ala Ala Ser Arg Ser Ala Thr465 470 475 480Ser Pro Arg Pro Ala Pro Ala Ala Arg Arg Pro Arg Pro Ala Ser Ser
485 490 495Ser Ala Arg Arg Ala Trp Pro Ala Pro Ala Pro Asn Ser Ala Ala Pro
500 505 510Thr Thr Pro Thr Ser Ala Pro Gly Thr Ser Pro Gly Ala His Ala Met
515 520 525Trp Pro Arg Ala Ala Thr Arg Ala Cys Ala Thr Pro Ser Gly Cys Gly
530 535 540Cys Cys Ser Gly Ala Ser Lys Arg Gly Ser Thr Val Thr Ala Pro Asn545 550 555 560Gly Ala Lys Thr Phe Pro Thr Lys Arg Pro Trp Met Ile Ser His
565 570 575
Claims (13)
1.一种能够水解二环内酰胺即2-氮杂二环[2.2.1]庚-5-烯-3-酮的一个对映体的酶,该酶具有以下列一个或多个为特征的稳定性:
在40℃下保持4小时后活性保留大于85%或者在60℃下保持4小时后活性大于30%;
在100g外消旋内酰胺加300ml缓冲剂的初始浓度下水解,进行到(+)内酰胺水解至少90%而(-)内酰胺水解小于5%。
2.根据权利要求1的酶,具有的特征是水解在所述初始浓度下发生,进行到(+)内酰胺水解大于98%而(-)内酰胺水解小于2%。
3.一种能够水解二环内酰胺即2-氮杂二环[2.2.1]庚-5-烯-3-酮的一个对映体的酶,它得自食酸丛毛单胞菌。
4.根据权利要求3的酶,得自食酸丛毛单胞菌NCIMB40827。
5.包括SEQ ID NO:2中所示氨基酸序列的酶,或其片断,能够水解二环内酰胺即2-氮杂二环[2.2.1]庚-5-烯-3-酮的一个对映体。
6.根据前述任意权利要求的酶,其呈固定化形式。
7.编码权利要求5的酶的分离的核苷酸分子。
8.根据权利要求7的核苷酸分子,具有SEQ ID NO:1所示序列。
9.一种能够表达权利要求5的酶的微生物。
10.根据权利要求9的微生物,具有食酸丛毛单胞菌NCIMB40827的基本特征。
11.生产权利要求1-5中任一项的酶的方法,它包括培养权利要求9或权利要求10的微生物。
12.立体选择性水解2-氮杂二环[2.2.1]庚-5-烯-3-酮的对映体混合物的方法,它包括将该混合物与权利要求1-5中任一项的酶或者权利要求9或10的微生物接触。
13.根据权利要求12的方法,它还包括将残留的(-)内酰胺与水解所形成的(+)氨基酸分离开。
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EP1095160B1 (de) * | 1998-07-09 | 2004-01-28 | Lonza AG | Verfahren zur herstellung von (1r,4s)-2-azabicyclo 2.2.1 hept-5-en-3-on-derivaten |
GB9907082D0 (en) * | 1999-03-26 | 1999-05-19 | Chirotech Technology Ltd | The preparation of carboxylic acid derivatives |
KR101082030B1 (ko) * | 2004-02-10 | 2011-11-10 | 에스케이바이오팜 주식회사 | (-)-감마-락탐의 제조방법 및 이에 이용되는 신규한미생물 균주 |
JP5704763B2 (ja) | 2009-07-02 | 2015-04-22 | ドクター・レディーズ・ラボラトリーズ・リミテッド | トランス−4−アミノシクロペンタ−2−エン−1−カルボン酸誘導体の製造 |
CN112442474B (zh) * | 2020-12-09 | 2022-08-23 | 江南大学 | 一种(-)γ-内酰胺的制备方法 |
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DE69016739T2 (de) * | 1989-10-16 | 1995-06-14 | Chiroscience Ltd | Chirale Azabicycloheptanone und Verfahren zu ihrer Herstellung. |
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- 1997-09-01 AT AT97939003T patent/ATE451463T1/de active
- 1997-09-01 DE DE69739691T patent/DE69739691D1/de not_active Expired - Lifetime
- 1997-09-01 KR KR1019997001807A patent/KR20000068439A/ko not_active Application Discontinuation
- 1997-09-01 AU AU41238/97A patent/AU719066B2/en not_active Ceased
- 1997-09-01 ES ES97939003T patent/ES2338074T3/es not_active Expired - Lifetime
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- 1997-09-01 JP JP51233598A patent/JP4437170B2/ja not_active Expired - Fee Related
- 1997-09-01 PT PT97939003T patent/PT925361E/pt unknown
- 1997-09-01 CN CN97197622A patent/CN1228812A/zh active Pending
- 1997-09-01 DK DK97939003.6T patent/DK0925361T3/da active
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105586289A (zh) * | 2015-12-11 | 2016-05-18 | 江西省科学院微生物研究所 | 一种能拆分(+/-)γ-内酰胺得到(-)γ-内酰胺的假单胞菌及其筛选和应用 |
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CA2264651A1 (en) | 1998-03-12 |
US6423522B1 (en) | 2002-07-23 |
AU719066B2 (en) | 2000-05-04 |
AU4123897A (en) | 1998-03-26 |
EP0925361A1 (en) | 1999-06-30 |
KR20000068439A (ko) | 2000-11-25 |
US6090616A (en) | 2000-07-18 |
CA2264651C (en) | 2012-08-07 |
JP4437170B2 (ja) | 2010-03-24 |
GB9618340D0 (en) | 1996-10-16 |
BR9711648A (pt) | 1999-08-24 |
DE69739691D1 (de) | 2010-01-21 |
EP0925361B1 (en) | 2009-12-09 |
ATE451463T1 (de) | 2009-12-15 |
WO1998010075A1 (en) | 1998-03-12 |
PT925361E (pt) | 2010-03-08 |
ZA977912B (en) | 1998-09-03 |
ES2338074T3 (es) | 2010-05-03 |
JP2001524805A (ja) | 2001-12-04 |
DK0925361T3 (da) | 2010-04-12 |
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