CN114214293B - 一种恶臭假单胞菌细胞色素p450环氧酶突变体及其应用 - Google Patents
一种恶臭假单胞菌细胞色素p450环氧酶突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种恶臭假单胞菌细胞色素P450环氧酶突变体及其应用,属于生物工程技术领域。本发明通过对来源于恶臭假单胞菌的细胞色素P450环氧酶进行改造,克服了之前野生型的酶因为过氧化氢逸散和酸醇对质子转化率低而导致的催化效率低的局限,最终得到最佳突变体Q6(PpCytPA250V/T255E/S296V/A297P/P298N),以此突变体为催化剂,以降冰片烯为底物,转化后10mL规模摇瓶产量为72.05g/L,摩尔产率为61.59%,单位菌体生产环氧降冰片烷的能力为3.6g/g,几乎不产生羟基化副产物。因此大大降低了之前的高菌体量以及繁复的后期分离纯化过程,为环氧降冰片烷的工业化生产和绿色生产奠定了基础。
Description
技术领域
本发明涉及一种恶臭假单胞菌细胞色素P450环氧酶突变体及其应用,属于生物工程技术领域。
背景技术
环氧降冰片烷,又名3-环氧丙基[3,2,1,02,4]辛烷,属于降茨烷的衍生物之一。作为一个饱和的桥环化合物,其化学式为C7H10O,熔点为126℃。它是由环己烷的碳骨架在1,4位桥连一个亚甲基,同时2,3位与一个氧原子构成环氧结构形成的。
目前,环氧降冰片烷的主要生产方法为化学合成法,主要为过氧化氢法,但是这种方法虽然得率高,能耗却较大,同时会对环境产生毒害作用,不符合绿色生产、安全生产和可持续发展的要求。通过生物法制备环氧降冰片烷具有产品质量稳定安全、工艺条件温和、高效、环保等特点,可以减轻环境和资源压力,因此迫切需要一种有效的生物法高效制备环氧降冰片烷。
目前,微生物法生产环氧降冰片烷涉及到一个关键的酶细胞色素P450环氧酶(CytP,Cytochrome P450 epoxidase),它具有广泛的底物杂泛性,可以催化降冰片烯,使其C=C双键环氧化,形成环氧降冰片烷。而微生物法制备环氧降冰片烷主要为酶转化法。目前,由于酶转化法具备环境有利、反应温和及操作简便等优点而更具备工业应用价值。但是,酶转化法大规模制备环氧降冰片烷受到以下限制:(1)在O2介导的经典催化循环中,CytP主要催化含有C=C双键的底物羟基化,环氧化反应作为副反应催化效率极低且伴随着大量的羟基化产物的生成。催化特异性的降低极大程度限制了环氧降冰片烷的产率及后期分离纯化;(2)在H2O2介导的分流途径中,H2O2作为环氧化反应中的氧原子来源和电子供体,可实现对环氧化反应的专一性催化。但酶活性中心无法有效阻止H2O2的逸散,致使发生大量无效催化,环氧化效率低;(3)不论是经典循环途径还是H2O2分流途径,促使中间体Cpd 0转化为关键中间体Cpd I的质子传递都尤为重要,该质子传递由酸醇对介导,如何增强酸醇对传递质子的效率以增强Cpd I的生成效率是目前研究的重点问题。因此,从工业角度来看,在保证特异性催化环氧化的H2O2分流途径中,迫切需要解决CytP的H2O2逸散和酸醇对传递效率低的问题,以提高利用H2O2催化时CytP的环氧化酶活,使得通过酶转化法制备环氧降冰片烷实现规模化应用。
近几十年来,蛋白质工程已经成为在分子水平改善酶的性质的有效策略,如扩大底物范围、提高酶的活性和改善酶的稳定性的最有效的方法。因此,通过蛋白质工程设计CytP,可能解决其催化酶活低的问题。蛋白质工程改造主要可以分四类:传统的定向进化(即非理性设计)、半理性设计、理性设计(基于结构与计算机技术)和多种策略的组合应用。目前,关于利用蛋白质工程改造CytP已经取得了一定的研究进展。一方面,通过活性位点附近大体积侧链残基的置换,在能接纳底物分子的基础上,适当收缩底物口袋以阻止H2O2的大量逸散,保证活性位点处的H2O2浓度足够CytP利用以催化环氧化反应;另一方面,通过人工构建酸醇对,增强酸醇对对质子的转移效率从而增强关键中间体Cpd I的生成效率,促进整个催化循环效率的提升。两个蛋白质工程策略的组合对催化酶活的改善效果十分可观,可以满足实际的工业化需求。
发明内容
本发明提供了一种可以高效制备环氧降冰片烷的PpCytP突变体及其改造方法,并利用该突变体蛋白催化降冰片烯制备环氧降冰片烷。本发明所构建的菌株在环氧降冰片烷制备中具有低环境损害、高催化特异性、高酶活等优点,极大提高了工业化的生产效率。
由于过氧化氢作为强氧化剂会造成PpCytP活性中心血红素铁的降解,损失酶活甚至加速酶的失活,所以在两相反应体系中过氧化氢的浓度必须维持在既能满足催化环氧化要求又不能过高导致酶失活的范围内。一般解决的方法有过氧化氢的原位添加或者低量添加,所以本发明采用过氧化氢的分批添加,并对分批添加的方式进行优化,以达到PpCytP突变体催化环氧化的最佳效果。
本发明提供了一种恶臭假单胞菌细胞色素P450环氧酶PpCytP突变体,所述恶臭假单胞菌细胞色素P450环氧酶PpCytP的氨基酸序列如SEQ ID NO.1所示,所述突变体是将亲本第250位、第255位、第296位、第297位、第298位的氨基酸中的一个或多个进行突变。
在一种实施方式中,所述恶臭假单胞菌细胞色素P450环氧酶的编码基因的核酸序列如SEQ ID NO.2所示。
在一种实施方式中,所述突变体相对于PpCytP亲本,将其第250位氨基酸突变为缬氨酸,获得突变体A250V。
在一种实施方式中,所述突变体相对于PpCytP亲本,将其第255位氨基酸突变为谷氨酸,获得突变体T255E。
在一种实施方式中,所述突变体相对于PpCytP亲本,将其第250位和第255位氨基酸同时突变,分别突变为缬氨酸和谷氨酸,获得突变体A250V/T255E。
在一种实施方式中,所述突变体相对于PpCytP亲本,将其第250位、第255位和第296位氨基酸同时突变,分别突变为缬氨酸、谷氨酸和缬氨酸,获得突变体A250V/T255E/S296V。
在本发明的一种实施方式中,所述突变体相对于PpCytP亲本,将其第250位、第255位、第296位和第297位氨基酸同时突变,分别突变为缬氨酸、谷氨酸、缬氨酸和脯氨酸,获得突变体A250V/T255E/S296V/A297P。
在本发明的一种实施方式中,所述突变体相对于PpCytP亲本,将其第250位、第255位、第296位、第297位和第298位氨基酸同时突变,分别突变为缬氨酸、谷氨酸、缬氨酸、脯氨酸和天冬酰胺,获得突变体A250V/T255E/S296V/A297P/P298N。
在一种实施方式中,所述突变体A250V、T255E、A250V/T255E、A250V/T255E/S296V、250V/T255E/S296V/A297P、A250V/T255E/S296V/A297P/P298N的氨基酸序列分别如SEQ IDNO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13所示。
本发明提供了编码所述突变体的基因。
在一种实施方式中,编码所述突变体A250V、T255E、A250V/T255E、A250V/T255E/S296V、250V/T255E/S296V/A297P、A250V/T255E/S296V/A297P/P298N的基因的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ IDNO.14所示。
本发明提供了含有所述基因的载体。
在一种实施方式中,所述载体包括但不限于pET系列、Duet系列、pGEX系列、pHY300、pHY300PLK、pPIC3K或pPIC9K系列载体。
优选地,所述载体为pET28a(+)。
本发明提供了一种获得所述PpCytP突变体的方法,所述方法包括以下步骤:
(1)在恶臭假单胞菌细胞色素P450环氧酶PpCytP氨基酸序列的基础上确定突变位点;设计定点突变的突变引物,以携带细胞色素P450环氧酶PpCytP基因的载体为模板进行定点突变;构建含突变体的质粒载体;
(2)将突变体质粒转化进宿主细胞;
(3)挑选阳性克隆进行发酵培养,并纯化细胞色素P450环氧酶突变体PpCytP。
在一种实施方式中,所述宿主细胞为细菌和真菌细胞。
优选地,所述宿主细胞为大肠杆菌,更优选地,所述大肠杆菌为大肠杆菌BL21(DE3)。
本发明提供了一种制备环氧降冰片烷的方法,所述方法是以降冰片烯为反应底物,利用所述突变体,或所述重组微生物细胞转化生产环氧降冰片烷。
在一种实施方式中,将所述重组微生物细胞添加至含有将冰片烷的反应体系中,使得重组微生物细胞在反应体系中的浓度为10~60g/L。
在一种实施方式中,反应体系由液相和水相组成,有机相与水相体积比为2:1,有机相为乙酸乙酯,水相为pH8.0~9.0的磷酸缓冲液和过氧化氢。
在一种实施方式中,所述反应体系中的底物量为1~5mol/L,过氧化氢的量为0.5~1mol/L或是反应体系体积的10%,优选地,过氧化氢的量为反应体系体积的5%~10%,更优选的,过氧化氢的添加量为反应体系体积的10%。
在一种实施方式中,分批次添加过氧化氢,过氧化氢的添加方式如(a)~(d)任一所示:
(a)匀速分批添加:每隔12h添加一次过氧化氢;
(b)前期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应12h、24h时分别添加1/4的过氧化氢;
(c)中期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应24h、36h时分别添加1/4的过氧化氢;
(d)中期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应36h、48h时分别添加1/4的过氧化氢。
在一种实施方式中,在pH5.5~9.0、25~40℃条件下反应24~80h。
本发明提供了所述突变体在制备环氧降冰片烷中的应用。
本发明提供了所述重组微生物细胞在制备环氧降冰片烷中的应用。
本发明的有益效果:
(1)本发明通过对来源于恶臭假单胞菌细胞色素P450环氧酶进行改造,构建得到了环氧酶突变体,用于催化生产环氧降冰片烷。
(2)本发明构建得到的突变体不仅大大提高了PpCytP的环氧化活性,同时对底物具有很高的催化特异性,提高了单位催化剂的生产能力,有效降低了生产成本。
(3)本发明得到的突变体在10mL两相催化体系中以降冰片烯为底物,采用过氧化氢后期分批添加方式时环氧降冰片烷的产量可达72.05g/L,摩尔产率可达61.59%,单位菌体生产环氧降冰片烷的能力为3.6g/g,羟基化副产物产量<0.01g/L,摩尔产率<0.0084%,加快了酶转化法生产环氧降冰片烷的工业化进程。
附图说明
图1为本发明PpCytP野生型诱导表达的SDS-PAGE图;泳道1~3分别是诱导表达后全细胞、上清液和沉淀中所目的蛋白条带大小。
图2为PpCytP野生型于两相体系中转化结束后的有机层GC图。
图3为转化反应液中降冰片烯的质谱图。
图4为转化反应液中生成的环氧降冰片烷质谱图。
图5为突变体Q5催化时过氧化氢分批添加方式和环氧降冰片烷产量的关系。横坐标轴中A(All)代表一次性添加方式;C(Constant)代表匀速分批添加方式;E(Early)代表前期分批添加方式;M(Medium)代表中期分批添加方式;L(Later)代表后期分批添加方式。
图6为突变体Q6催化时过氧化氢分批添加方式和环氧降冰片烷产量的关系。
具体实施方式
基因来源:本专利所涉及到的生物酶PpCytP基因来源于Pseudomonas putidaKT2440,pET28a(+)质粒购自Novagen(Madison,WI,U.S.A.),限制性内切酶、T4 DNA连接酶、pri meSTAR等购自TaKaRa(Dalian,China)。O-diacetylbenzene和标样购自SIGMA。PpCytP突变体均为分子改造所得,其余试剂均为市场购买所得。
配制LB培养基:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L,在121℃灭菌20min。
配制TB培养基:胰蛋白胨12g/L,酵母提取物(安琪酵母粉802)24g/L,甘油4mL/L,KH2PO4 2.31g/L和K2HPO4 12.31g/L。
配制pH8.5的磷酸缓冲液:过量添加100mmol/L KCL,具体配方见《工业微生物实验技术手册》(中国轻工业出版社,诸葛健主编)。
GC法测定环氧降冰片烷:样品分析采用气相色谱仪Agilent GC-7890B,手性气相色谱柱DB-5;进样口温度为250℃;初始柱温45℃2min,以10℃/min升温至250℃;载气为氦气,流速1.0mL/min,分流比10:1。
环氧降冰片烷含量的测定:反应产物采用气相色谱法(GC)测定。气相检测条件参见上述GC法测定环氧降冰片烷。在此检测条件下,乙酸乙酯、降冰片烯、环氧降冰片烷(EPO-NBE)的保留时间分别为2.255min、3.315min和6.195min。
摩尔产率测定方法:采用GC法测环氧降冰片烷的含量。摩尔产率被定义为1mol降冰片烯底物所能转化的环氧降冰片烷的摩尔量,一般用生成产物和初始底物的摩尔(浓度)量比表示。通过测定环氧降冰片烷的含量即可计算摩尔产率。
EPO-NBE摩尔产率=(P/S0)×100%;
其中:P代表EPO-NBE的最终摩尔浓度,S0代表NBE的初始摩尔浓度。
实施例1:单突突变体的构建和筛选
(1)单突突变体构建:将野生型PpCytP酶的核苷酸序列(如SEQ ID NO.2所示)连接至pET28a(+)质粒的多克隆酶切位点处,设计PpCytPA250V和PpCytPT255E突变位点的引物,如表1所示,通过全质粒PCR进行突变体构建。
表1单突变体突变引物序列
构建反应PCR扩增体系:PrimeSTAR酶0.5μL、5×PrimeSTAR Buffer 10μL、dNTP 4μL每个突变位点的两条引物各1μL、模板(含有野生型PpCytPWT酶的重组质粒)4μL、水32.5μL;反应条件为:94℃3min;98℃10s;55℃30s;68℃4min;将2~4三个步骤循环29次;68℃5min;4℃保温,扩增得到PCR产物。
将上述反应体系在37℃孵育3h以消化质粒模板(消化体系为:DpnI 0.5μL、上述反应PCR产物45μL、10×T Buffer 5μL),消化结束后得到的消化产物通过化学转化方法导入大肠杆菌BL21感受态细胞,化学转化法具体步骤:
(1)将10μL同源重组产物导入100μL BL21感受态细胞;
(2)冰浴15-30min;
(3)42℃水浴热激90s,取出后迅速放入冰中静置冰浴3-5min;
(4)加入800μL无抗性LB培养基混匀,于37℃,200rpm培养1h;
(5)5000rpm离心2min收菌;
(6)移去上清,剩余100-200μL吹吸混匀涂布至含0.05mg/mL卡那霉素抗性平板上,37℃恒温培养12h左右。
(7)挑取单克隆于含0.05mg/mL卡那霉素抗性LB中,200rpm、37℃恒温培养12h后,送去公司测序,测序正确的即为阳性转化子,从阳性转化子中提取质粒,得到分别带有突变体PpCytPA250V和PpCytPT255E的重组质粒。
实施例2:双突、三突、四突及五突突变体的构建和筛选
(1)双突突变体构建:在突变体PpCytPA250V的基础上,利用突变引物T255E-S和T255E-A(表2),通过全质粒PCR进行双突突变体构建,具体实施方式参见实施例1中步骤(1),所用引物如SEQ ID NO.17和SEQ ID NO.18所示,制备得到双突突变体PpCytPA250V/T255E。
(2)三突突变体的构建:在突变体PpCytPA250V/T255E的基础上,底物口袋残基的大体积置换策略突变体利用表2突变引物,通过全质粒PCR进行三突变体的构建,具体实施方式参见实施例1,分别对第253位、254位、296位、297位、298位和299位残基进行PpCytPA250V/T255E基础上的叠加饱和突变。
表2三突变体突变引物序列
(4)多突变体的筛选:将测序正确的突变体菌株接种至LB种子培养基,200rpm、37℃培养培养10h左右,分别以2%(v/v)接种量接种至摇瓶发酵培养基,在200rpm、37℃培养至OD600=0.8左右,加入终浓度为0.2mmol/L的IPTG,诱导条件为200rpm、25℃诱导16h。将诱导表达后的菌液经6000r/min离心15min收集菌体。
转化条件为:10mL两相体系,底物(降冰片烯,纯度>99%)浓度为1mol/L,全细胞添加量为20g/L,转化温度30℃,两相体系中有机相为乙酸乙酯,水相为pH8.5的磷酸缓冲液和533μL过氧化氢(0.5mol/L),有机相与水相体积之比为2,转化时间为72h,转速为200rpm。
将转化结束后的转化液5000r/min离心10min,吸取上层有机相,经无水硫酸镁干燥,过0.22μm有机膜,用GC方法进行测定环氧降冰片烷的产量,结果如表3所示,最终突变体PpCytPA250V/T255E/S296V,PpCytPA250V/T255E/A297P和PpCytPA250V/T255E/P298N效果最好,三者的环氧化产物生成量相差无几。
表3不同位点大体积置换突变体摇瓶筛选结果
(5)四突突变体(在突变体PpCytPA250V/T255E/S296V基础上对A297P进行突变):以PpCytPA250V/T255E/S296V为模板,利用突变引物A297P-S和A297P-A进行全质粒PCR,PCR产物进行消化,PCR体系与消化体系和实施例1相同。
表4四突变体突变引物序列
(6)五突突变体(在突变体PpCytPA250V/T255E/S296V/A297P基础上对P298N进行突变):以PpCytPA250V/T255E/S296V/A297P为模板,利用突变引物P298N-S和P298N-A进行全质粒PCR,PCR产物进行消化,PCR体系与消化体系和实施例1相同。
表5五突变体突变引物序列
实施例3:过氧化氢的分批添加
具体实施方式参见实施例2步骤(4),区别在于,10mL两相转化体系中过氧化氢的添加总量增加到1mL(水相总体积保持不变),添加方式分为一次性添加、匀速分批添加、前期分批添加、中期分批添加和后期分批添加。具体添加方式如表6所示。
表6过氧化氢分批添加方式
针对突变体Q5(PpCytPA250V/T255E/S296V/A297P)和Q6(PpCytPA250V/T255E/S296V/A297P/P298N)菌株分别采用表6中的五种过氧化氢分批添加方式,于10mL两相体系中转化72h后采用GC法测定环氧降冰片烷产量。
结果如图4和图5所示,与533μL的H2O2总添加量相比,一次性添加1mL的H2O2会导致突变体酶活受损,摩尔产率下降;后期分批添加方式的效果明显优于其他分批添加方式。在后期分批添加H2O2的方式下,突变体Q5在转化72h时的环氧降冰片烷的产量可达65.07g/L,摩尔产率为55.62%;突变体Q6在转化72h时的环氧降冰片烷的产量可达72.05g/L,摩尔产率为61.59%。
对比例1
具体实施方式参见实施例3,不同之处在于,将突变体Q6菌株替换为野生型Q0菌株去进行发酵与转化实验。转化结束后,将转化结束后的转化液5000r/min离心10min,吸取上层有机相,经无水硫酸镁干燥,过0.22μm有机膜,用GC方法进行测定环氧降冰片烷的产量。GC色谱图结果显示:在过氧化氢后期分批添加的情况下效果最佳,环氧降冰片烷产量为35.02g/L,转化率为29.93%。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 无锡阿科力科技股份有限公司
江南大学
<120> 一种恶臭假单胞菌细胞色素P450环氧酶突变体及其应用
<130> BAA211716A
<160> 14
<170> PatentIn version 3.3
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Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
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Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
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35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
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Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
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Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
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Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
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Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
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Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Ala Gly Thr Asp Thr Thr Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Ser Ala Pro Phe Ala Gly Arg Ile Thr
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Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
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Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
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Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
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Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
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atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
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gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgcc ggcactgata ctacggccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccgtccgc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
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Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
50 55 60
Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
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Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
85 90 95
Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
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Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
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Ala Leu His Leu Val Glu Ile Ala Phe Val Gly Thr Asp Thr Thr Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
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atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
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gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
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ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgtt ggcactgata ctacggccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccgtccgc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
<210> 5
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<212> PRT
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Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
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50 55 60
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85 90 95
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100 105 110
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115 120 125
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145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
195 200 205
Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Ala Gly Thr Asp Thr Glu Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Ser Ala Pro Phe Ala Gly Arg Ile Thr
290 295 300
Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
305 310 315 320
Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
325 330 335
Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
340 345 350
Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
355 360 365
Leu Gln Gly Ala Thr Gly Leu Arg Val Leu Phe Glu Arg Leu Pro Glu
370 375 380
Leu Lys Val Val Pro Asp Gln Pro Leu Asn Phe Ala Pro Met Ala Leu
385 390 395 400
Leu Pro Val Arg Leu Ser Leu Gln Val Ile Trp
405 410
<210> 6
<211> 1236
<212> DNA
<213> 人工序列
<400> 6
atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
tttcgtgatc cggccagcat ttgccagcgt gcgcgtgagg aaacgccggt ctttttctac 120
gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgcc ggcactgata ctgaagccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccgtccgc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
<210> 7
<211> 411
<212> PRT
<213> 人工序列
<400> 7
Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
50 55 60
Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
65 70 75 80
Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
85 90 95
Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
195 200 205
Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Val Gly Thr Asp Thr Glu Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Ser Ala Pro Phe Ala Gly Arg Ile Thr
290 295 300
Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
305 310 315 320
Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
325 330 335
Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
340 345 350
Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
355 360 365
Leu Gln Gly Ala Thr Gly Leu Arg Val Leu Phe Glu Arg Leu Pro Glu
370 375 380
Leu Lys Val Val Pro Asp Gln Pro Leu Asn Phe Ala Pro Met Ala Leu
385 390 395 400
Leu Pro Val Arg Leu Ser Leu Gln Val Ile Trp
405 410
<210> 8
<211> 1236
<212> DNA
<213> 人工序列
<400> 8
atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
tttcgtgatc cggccagcat ttgccagcgt gcgcgtgagg aaacgccggt ctttttctac 120
gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgtt ggcactgata ctgaagccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccgtccgc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
<210> 9
<211> 411
<212> PRT
<213> 人工序列
<400> 9
Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
50 55 60
Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
65 70 75 80
Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
85 90 95
Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
195 200 205
Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Val Gly Thr Asp Thr Glu Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Val Ala Pro Phe Ala Gly Arg Ile Thr
290 295 300
Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
305 310 315 320
Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
325 330 335
Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
340 345 350
Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
355 360 365
Leu Gln Gly Ala Thr Gly Leu Arg Val Leu Phe Glu Arg Leu Pro Glu
370 375 380
Leu Lys Val Val Pro Asp Gln Pro Leu Asn Phe Ala Pro Met Ala Leu
385 390 395 400
Leu Pro Val Arg Leu Ser Leu Gln Val Ile Trp
405 410
<210> 10
<211> 1236
<212> DNA
<213> 人工序列
<400> 10
atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
tttcgtgatc cggccagcat ttgccagcgt gcgcgtgagg aaacgccggt ctttttctac 120
gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgtt ggcactgata ctgaagccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccggttgc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
<210> 11
<211> 411
<212> PRT
<213> 人工序列
<400> 11
Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
50 55 60
Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
65 70 75 80
Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
85 90 95
Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
195 200 205
Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Val Gly Thr Asp Thr Glu Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Val Pro Pro Phe Ala Gly Arg Ile Thr
290 295 300
Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
305 310 315 320
Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
325 330 335
Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
340 345 350
Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
355 360 365
Leu Gln Gly Ala Thr Gly Leu Arg Val Leu Phe Glu Arg Leu Pro Glu
370 375 380
Leu Lys Val Val Pro Asp Gln Pro Leu Asn Phe Ala Pro Met Ala Leu
385 390 395 400
Leu Pro Val Arg Leu Ser Leu Gln Val Ile Trp
405 410
<210> 12
<211> 1236
<212> DNA
<213> 人工序列
<400> 12
atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
tttcgtgatc cggccagcat ttgccagcgt gcgcgtgagg aaacgccggt ctttttctac 120
gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgtt ggcactgata ctgaagccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccggttcc tccttttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
<210> 13
<211> 411
<212> PRT
<213> 人工序列
<400> 13
Met Glu Ile Leu Asp Arg Pro Gln Ala Pro Ser Asp Phe Asn Pro Met
1 5 10 15
Ser Glu Gln Ser Phe Arg Asp Pro Ala Ser Ile Cys Gln Arg Ala Arg
20 25 30
Glu Glu Thr Pro Val Phe Phe Tyr Ala Pro Leu Gly Val Trp Met Val
35 40 45
Thr Arg Arg Glu Asp Ala Glu Arg Val Leu Ser Glu Trp Glu Thr Phe
50 55 60
Ser Ser Leu Ala Asn Ser Pro Asn Val Pro Glu Glu Phe Arg Ser Arg
65 70 75 80
Phe Ala Pro Ser Val Met Ala Asp Ser Ile Val Ala Ile Asp Pro Pro
85 90 95
Arg His Thr Gln Ala Arg Asn Val Ile Gln Arg Gly Phe Met Lys Pro
100 105 110
Lys Ile Asp Pro Leu Glu Pro Ile Ile Glu Gln Arg Ala His Glu Ile
115 120 125
Ile Asp Arg Phe Ala Gly Glu Ser Gly Thr Glu Ile Met Asn Asn Tyr
130 135 140
Cys Leu Glu Leu Thr Thr Arg Thr Leu Met Ala Leu Tyr Asp Leu Pro
145 150 155 160
Leu Glu Asp Arg Pro Met Phe Glu Arg Ile Arg Asp Val Ser Ile Lys
165 170 175
Val Leu Ala Ser Val Tyr Glu Pro Met Gln Glu Pro Glu Lys Ser Arg
180 185 190
Val Trp Asn Glu Tyr Val Ser Gly Tyr Glu Tyr Phe Tyr Gln Leu Val
195 200 205
Glu Gln Arg Arg Asn Ser Asp Ala Arg Asp Ile Ile Ser Thr Met Ala
210 215 220
Ser Gln Lys Asp Asn Gln Gly Asn Pro Ala Leu Ser Thr Glu Arg Ile
225 230 235 240
Ala Leu His Leu Val Glu Ile Ala Phe Val Gly Thr Asp Thr Glu Ala
245 250 255
Gln Met Met Ala Asn Ala Ile Leu Phe Leu Asp Ser His Pro Glu Ala
260 265 270
Leu Ala Ala Ala Lys Ala Asp Lys Thr Leu Trp Ser Arg Val Phe Glu
275 280 285
Glu Thr Val Arg Arg Arg Pro Val Pro Asn Phe Ala Gly Arg Ile Thr
290 295 300
Thr Thr Glu Val Glu Ile Gln Gly Val Lys Ile Pro Ala Gly Ser Pro
305 310 315 320
Val Trp Val Ser Leu Ala Ala Ala Asn Thr Asp Pro Arg His Val Gly
325 330 335
Cys Pro Met Asn Phe Asp Ile Asn Arg Glu Ala Pro Gln Asp His Leu
340 345 350
Ala Phe Thr Lys Gly Arg His Thr Cys Pro Gly Ala Pro Leu Ala Arg
355 360 365
Leu Gln Gly Ala Thr Gly Leu Arg Val Leu Phe Glu Arg Leu Pro Glu
370 375 380
Leu Lys Val Val Pro Asp Gln Pro Leu Asn Phe Ala Pro Met Ala Leu
385 390 395 400
Leu Pro Val Arg Leu Ser Leu Gln Val Ile Trp
405 410
<210> 14
<211> 1236
<212> DNA
<213> 人工序列
<400> 14
atggagatcc ttgatcgccc acaggcccct agtgacttca atcccatgtc tgaacagtcg 60
tttcgtgatc cggccagcat ttgccagcgt gcgcgtgagg aaacgccggt ctttttctac 120
gcgcctttag gggtgtggat ggtcactcgt cgtgaagacg ctgaacgcgt cctttcagaa 180
tgggaaacat tctcgagtct tgccaactcc cctaacgtcc cggaagagtt tcgcagccgc 240
ttcgccccca gtgtgatggc cgacagcatc gtcgctatcg accccccgcg tcacactcaa 300
gcccgtaacg taattcagcg tggtttcatg aaaccgaaga ttgatcctct ggaacctatt 360
attgaacagc gcgcgcacga gattattgat cgctttgctg gagagtccgg caccgaaatc 420
atgaataact actgtttgga attaaccacg cgtactttaa tggccttata cgacttgccc 480
ttggaagatc gtcctatgtt tgagcgtatt cgcgatgtat caattaaagt gttagctagt 540
gtgtatgagc ccatgcaaga gcctgaaaaa agccgcgtct ggaacgagta cgtctcggga 600
tatgaatatt tctaccaatt ggtggagcag cgccgcaact ccgacgcgcg cgatattatc 660
agcacaatgg catcgcagaa agacaaccaa ggcaatccgg ccttatccac cgaacgtatt 720
gctcttcacc tggtggagat tgcattcgtt ggcactgata ctgaagccca aatgatggca 780
aacgcgatcc tgtttttgga ttctcacccc gaggccttag cagctgccaa agcagacaaa 840
accctttggt ctcgtgtttt tgaggagaca gttcgccgtc gcccggttcc taactttgcc 900
gggcgcatta caacgacaga agtcgagatt cagggggtga aaatcccagc gggtagtcca 960
gtttgggtat ctttagcagc agcgaataca gacccacgtc atgtcggatg tccgatgaat 1020
tttgacatca atcgtgaagc cccacaagac cacttagctt tcacaaaagg tcgccataca 1080
tgccctgggg ctccattggc ccgtttacaa ggggcgaccg ggttacgtgt cctgtttgaa 1140
cgcctgccag agctgaaagt agtgcctgat cagccgttga acttcgcacc aatggcgctg 1200
cttccagttc gtttgagttt acaggtgatt tggtaa 1236
Claims (10)
1. 一种细胞色素P450环氧酶突变体,所述突变体的氨基酸序列如SEQ ID NO.11或SEQID NO.13所示。
2.编码权利要求1所述的突变体的基因。
3.含有权利要求2所述基因的载体。
4.表达权利要求1所述突变体的重组微生物细胞。
5.含有权利要求2所述基因的重组微生物细胞。
6.根据权利要求4或5所述的重组微生物细胞,其特征在于,以大肠杆菌为宿主细胞。
7.一种制备环氧降冰片烷的方法,其特征在于,所述方法是以降冰片烯为底物,利用权利要求1所述突变体,或权利要求4~6任一所述重组微生物细胞转化生产环氧降冰片烷。
8. 根据权利要求7所述的方法,其特征在于,将权利要求4~6任一所述重组微生物细胞添加至含有降冰片烯的反应体系中,使得重组微生物细胞在反应体系中的浓度为10~60 g/L;在pH5.5~9.0、25~40℃条件下反应24~80 h。
9. 根据权利要求8所述的方法,其特征在于,反应体系由液相和水相组成,有机相与水相体积比为2:1,有机相为乙酸乙酯,水相为pH8.0~9.0的磷酸缓冲液和过氧化氢;反应体系中底物量为1~5 mol/L,过氧化氢的添加量为反应体系体积的5%~10%,分批次添加过氧化氢,过氧化氢的添加方式如(a)~(d)任一所示:
(a)匀速分批添加:每隔10~12h添加一次过氧化氢;
(b)前期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应第10~12 h、20~24 h时分别添加1/4的过氧化氢;
(c)中期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应20~24h、32~36 h时分别添加1/4的过氧化氢;
(d)中期分批添加:反应开始时将1/2的过氧化氢添加至反应体系中,随后在反应32~36h、44~48 h时分别添加1/4的过氧化氢。
10.权利要求1所述突变体或权利要求4~6任一所述重组微生物细胞在制备环氧降冰片烷中的应用。
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