CN111041019A - 一种天冬氨酸酶突变体及其应用 - Google Patents

一种天冬氨酸酶突变体及其应用 Download PDF

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CN111041019A
CN111041019A CN201910423712.7A CN201910423712A CN111041019A CN 111041019 A CN111041019 A CN 111041019A CN 201910423712 A CN201910423712 A CN 201910423712A CN 111041019 A CN111041019 A CN 111041019A
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田振华
程占冰
秦丽军
徐艳冰
刘巧
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Yikelai Biotechnology Group Co ltd
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Abstract

本发明公开了一种天冬氨酸酶突变体，其在如SEQ ID NO.2所示的氨基酸序列上包括以下突变：M321V、K324T和N326S，所述天冬氨酸酶突变体具有比野生型天冬氨酸酶催化(E)‑4‑(2,4,5‑三氟苯基)丁‑2‑烯酸的比酶活高的酶活性。本发明还公开了该天冬氨酸酶突变体在制备(R)‑3‑氨基‑4‑(2,4,5‑三氟苯基)‑丁酸中的用途。采用本发明的天冬氨酸酶突变体进行酶催化反应制备西他列汀手性中间体(R)‑3‑氨基‑4‑(2,4,5‑三氟苯基)‑丁酸时，转化率更高，立体选择性更高，收率更高，生产成本更低，环境更友好，有利于工业化大生产。

Description

一种天冬氨酸酶突变体及其应用
技术领域
本发明涉及一种天冬氨酸酶突变体及其应用。
背景技术
糖尿病是由于胰岛素分泌改变，导致胰岛素缺乏，或者胰岛素活性降低，或者在两者共同影响下，出现的代谢性疾病。该疾病以高血糖为特征，并同时伴有蛋白质、糖及脂肪的代谢紊乱。糖尿病及其并发症对人类健康的危害程度居心血管疾病、肿瘤之后的第三位，成为危害人类健康的重要疾病。国际糖尿病联合会预计，到2030年，患病总人数将超过4.35亿人。而我国已成为世界糖尿病患病率增长速度最快的国家之一，目前约有4000万糖尿病患者，人数仅次于印度，居世界第二位。在糖尿病的四种类型中，II型糖尿病占90％以上，多见于30岁以上的中老年人，病因主要是由于机体对胰岛素不敏感。
西他列汀磷酸盐(Sitagliptin phosphate)，其结构式如式A所示
Figure BDA0002066806410000011
是2006年FDA批准上市的第一个二肽基酶-IV(DPP-4)抑制剂，用于治疗II型糖尿病，其单用或与二甲双胍、吡格列酮合用都有明显的降血糖作用，且服用安全，耐受性好，不良反应少。商品名为捷诺维(Januvia)，目前已经在全世界60多个国家批准使用，2012年销售额已达40.86亿美元，同比增长23％。因此，西他列汀磷酸盐是属于国际最新且附加值极高的“重磅炸弹”。
制备西他列汀磷酸盐的核心步骤在于构建与氨基相连的C原子的手性中心。2018年Imre Papai等在J.Am.Chem.Soc.2018,140,12216-12225公开了一种合成西他列汀中间体的技术路线。该技术路线使用(E)-4-(2,4,5-三氟苯基)丁-2-烯酸为原料，经过一种硫脲类催化剂不对称催化，构建得到手性化合物(R)-3-(苄氧氨基)-4-(2,4,5-三氟苯基)丁酸甲酯，ee值91％，收率83％，再经多步合成得到西他列汀磷酸盐。此方法中催化剂比较昂贵，且收率，ee值不高，不利用工业化大生产。
Figure BDA0002066806410000021
文献Chemcatchem，2014，966-968公开了在BSASP的基础上突变T187、M321、K324、N326四个位点，并将其用于催化丁烯酸，其中催化效果最好的突变体为BSASP-C6，但是本发明人发现该突变体并不能用于以(E)-4-(2,4,5-三氟苯基)丁-2-烯酸为底物的催化反应当中。因此迫切需要寻找一种可用于催化底物(E)-4-(2,4,5-三氟苯基)丁-2-烯酸从而合成西他列汀中间体，且能够使得反应时转化率更高、收率更高、立体选择性更高而成本较低的一种催化剂。
发明内容
本发明所要解决的技术问题是为了解决现有技术中催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的酶催化活性不高的缺陷，故而，本发明提供了一种天冬氨酸酶突变体及其应用。采用本发明的天冬氨酸酶突变体进行酶催化反应制备西他列汀手性中间体(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的方法，比现有技术中化学不对称催化制得西他列汀手性中间体(R)-3-(苄氧氨基)-4-(2,4,5-三氟苯基)丁酸甲酯的方法，转化率更高，立体选择性更高，收率更高，生产成本更低，环境更友好，有利于工业化大生产。
为了解决上述技术问题，本发明提供了一种天冬氨酸酶突变体，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上包括以下突变：M321V、K324T和N326S，所述天冬氨酸酶突变体具有比野生型天冬氨酸酶催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的比酶活高的酶活性。
本发明中，所述的野生型天冬氨酸酶可为本领域常规，一般情况下其氨基酸序列如SEQ ID NO.2所示，和/或，其核苷酸序列如SEQ ID NO.1所示。
较佳地，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上还包括第187位氨基酸残基T的突变；所述第187位氨基酸残基T的突变优选为C、F、D、S、N或V，更优选为C或F。
更佳地，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上还包括L358Y。
较佳地，所述天冬氨酸酶突变体的氨基酸序列如SEQ ID NO.4、SEQ ID NO.6、SEQID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.16或SEQ ID NO.18所示。
较佳地，所述天冬氨酸酶突变体的核苷酸序列如SEQ ID NO.3、SEQ ID NO.5、SEQID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15或SEQ ID NO.17所示。
为了解决上述技术问题，本发明提供了一种分离的核酸，所述核酸编码上述的天冬氨酸酶突变体。
较佳地，编码所述核酸的核苷酸序列如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15或SEQ ID NO.17所示。
为了解决上述技术问题，本发明提供了一种重组表达载体，其包含上述的核酸。
为了解决上述技术问题，本发明提供了一种转化体，其包含上述的核酸或上述的重组表达载体。
为了解决上述技术问题，本发明提供了一种(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的制备方法，其包括以下步骤：在上述天冬氨酸酶突变体的存在下，将(E)-4-(2,4,5-三氟苯基)丁-2-烯酸和氨基供体进行氢胺化反应，即得(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸；反应过程具体可以是如下所示：
Figure BDA0002066806410000041
较佳地，所述天冬氨酸酶突变体可以是以天冬氨酸酶突变体菌泥的形式存在，所述天冬氨酸酶突变体菌泥的浓度可以为20g/L-200g/L，例如100g/L。
较佳地，所述(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的浓度0.05mol/L-0.5mol/L，优选0.1mol/L。
较佳地，所述氨基供体为NH4Cl、氨水、甲酸铵、乙酸铵、碳酸铵、碳酸氢铵、硫酸铵、硫酸氢铵、磷酸铵、磷酸氢二铵中、磷酸二氢铵中的一种或多种。
较佳地，所述氨基供体与底物(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的摩尔比为1:1-20:1，例如10:1。
较佳地，所述氢胺化反应的反应溶剂为含有DMSO(二甲基亚砜)的水，优选含5％DMSO的水，所述％为DMSO占水的体积百分比。
较佳地，所述氢胺化反应的反应体系的pH为7-9，较佳地为8.5。
较佳地，所述氢胺化反应的反应体系的温度为30-60℃，较佳地为45℃。
较佳地，所述氢胺化反应的反应时间为6-24小时，较佳地为12小时。
较佳地，进行所述氢胺化反应时摇床的转速可为本领域常规，例如为220rpm。一般转速不宜过快，过快容易造成设备机械部分损坏。
较佳地，所述氢胺化反应中一般情况下是包括缓冲液的，例如Tris碱，以控制溶液的pH值。
本发明中，化合物所述浓度若无特殊说明，均为反应前所述化合物占整个反应体系的浓度。
为了解决上述技术问题，本发明提供了一种上述的天冬氨酸酶突变体在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸中的用途。
较佳地，所述的天冬氨酸酶突变体通过催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸制备得到所述的(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规，具体简写符号对应的氨基酸如表1所示。
表1
氨基酸名称 三字母符号 单字母符号 氨基酸名称 三字母符号 单字母符号
丙氨酸(alanine) Ala A 亮氨酸(leucine) Leu L
精氨酸(arginine) Arg R 赖氨酸(lysine) Lys K
天冬酰胺(asparagine) Asn N 甲硫氨酸(methionine) Met M
天冬氨酸(asparticacid) Asp D 苯丙氨酸(phenylalanine) Phe F
半胱氨酸(cysteine) Cys C 脯氨酸(proline) Pro P
谷氨酰胺(glutanine) Gln Q 丝胺酸(serine) Ser S
谷氨酸(glutamicacid) Glu E 苏氨酸(threonine) Thr T
甘氨酸(Glicine) Gly G 色氨酸(tryptophan) Trp W
组氨酸(histidine) His H 酪氨酸(tyrosine) Tyr Y
异亮氨酸(isoleucine) Ile I 颉氨酸(valine) Val V
所述氨基酸对应的密码子也为本领域常规，具体氨基酸与密码子的对应关系如表2所示。
表2
Figure BDA0002066806410000051
Figure BDA0002066806410000061
本发明的积极进步效果在于：采用本发明的天冬氨酸酶突变体进行酶催化反应制备西他列汀手性中间体(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸，比现有技术中化学不对称催化制得(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的方法，转化率更高，立体选择性更高，收率更高，生产成本更低，环境更友好，有利于工业化大生产。
附图说明
图1为(E)-4-(2,4,5-三氟苯基)丁-2-烯酸标准品的HPLC图谱。
图2为(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品的HPLC图谱。
图3为实施例3中天冬氨酸酶突变体2催化反应结束后测转化率HPLC图谱。
图4为N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸消旋体的手性HPLC图谱。
图5为(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品的手性HPLC图谱。
图6为实施例3中天冬氨酸酶突变体2催化反应结束后反应液手性HPLC图谱。
图7为(E)-4-(2,4,5-三氟苯基)丁-2-烯酸标准品的鉴定图谱。
图8为(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的HNMR图谱。
图9为(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸的HNMR图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
pET21a和bugbuster protein extraction reagent购买自Novagen公司；DpnI酶购买自英潍捷基(上海)贸易有限公司；NdeI酶、HindIII酶购买自Thermo Fisher公司，E.coli C2566感受态细胞购买自北京鼎国昌盛生物技术有限责任公司。
测转化率的HPLC方法：
色谱柱：Agilent Eclipse plus C18(3.5μm，150×4.6mm)；梯度洗脱：流动相A为0.1％高氯酸水溶液，流动相B为乙腈，30％B(0.00min)，90％B(15min)，90％B(30min)，30％B(33min)，30％B(40min)；检测波长：210nm；流速：0.5ml/min；进样体积：5μl；柱温：30℃。
测ee值的HPLC方法：
色谱柱：Daicel Chiralpak AD-H(4.6mm*250mm,5μm)；流动相：正己烷：异丙醇＝90:10；检测波长：210nm；流速：1.0ml/min；进样体积：10μl；柱温：25℃；运行时间：40min。
实施例1 AspB(aspartase，天冬氨酸酶)突变体文库的构建
从NCBI检索到来源于Bacillus sp.YM55-1的AspB酶，PDB号1J3U_A，氨基酸序列为SEQ ID NO.2，基因序列为SEQ ID NO.1，针对SEQ ID NO.2的第187位、321位、324位、326位、358位进行突变的突变体文库构建所设计的引物序列如表3所示：
表3引物序列表
Figure BDA0002066806410000081
其中，N代表A、G、C、T中任何一种核苷酸，M代表A或C，K代表G或T；其根据所述位点需突变成的氨基酸的编码核苷酸来选择，如C187NNK正向引物中的NNK可以代表AAG(赖氨酸)、AAT(天冬氨酸)、AGG(精氨酸)或AGT(丝氨酸)等，具体氨基酸所对应的核苷酸可参见表2。
根据序列表中SEQ ID NO.1的序列合成基因AspB，基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)，AspB的PDB号为1J3U，然后以NdeI和HindIII酶切位点引入质粒pET21a，构建质粒pET21a-AspB。以质粒pET21a-AspB为模板，PCR扩增目的条带。
PCR扩增体系为：
表4 PCR扩增体系
试剂 用量(μL)
2X PCR buffer(含高保真酶) 25
正向引物 1
反向引物 1
模板 1
去离子水 22
PCR扩增程序如下：
表5 PCR扩增程序
Figure BDA0002066806410000091
对PCR产物进行DpnI消化，37℃，2小时。反应完成转化至C2566感受态细胞，涂布在含有100μg/mL氨苄青霉素的LB培养基，37℃培养过夜，收菌，得到包含突变体文库的转化子。
实施例2高通量筛选突变体文库
按照如下实验步骤进行筛选
第一步初筛，采用尼龙膜转印的筛选方法进行。待实施例1中平板长出转化子，使用灭菌处理过的尼龙膜印迹平板上的菌落，再将膜上的菌落用500μL含80μg/mL氨苄青霉素，0.1mM IPTG，和0.01mM MgCl2的TB培养基洗涤菌落，将洗涤后所得菌液转移至96孔板中诱导，25℃，200rpm过夜诱导后，4000rpm离心20min收菌，弃掉培养液，最后加入60μLbugbuster裂解，30℃裂解2h，将所得酶裂解液按照表6反应体系投反应，在45℃，220rpm，反应3天，HPLC检测反应效果。
表6初筛反应体系
Figure BDA0002066806410000101
第二步复筛，将上述HPLC检测到有产物生成的平板，挑单菌落至96孔板诱导。将菌落挑取至含80μg/mL氨苄青霉素和0.01mM MgCl2的500μL TB培养基中37℃培养至OD600值1.0左右，加IPTG终浓度至0.1mM，于25℃，200rpm过夜诱导后，4000rpm离心20min收菌，弃掉培养液，最后加入100μL bugbuster裂解，30℃裂解2h，将酶裂解液按照表7复筛反应体系投反应，在45℃，220rpm，反应3天，HPLC检测反应效果，筛选出阳性突变体如表8所示(在初筛和复筛阶段，都是通过HPLC来判断的，后续实施例3中有验证使用最终筛选所得的突变体的菌泥催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸底物时，所得产物确为(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸，为正确的产物)。
表7复筛反应体系
Figure BDA0002066806410000102
表8阳性突变体
Figure BDA0002066806410000111
第三步摇瓶发酵，收集阳性突变体菌泥。接种阳性突变体至200mL TB培养基，加氨苄青霉素至终浓度100μg/mL，37℃，250rpm摇床生长至OD600大约在1.0左右，降温至25℃，加IPTG至终浓度0.1mM，250rpm摇床诱导过夜，4000rpm离心20min，弃培养液，收菌，称重，按照1g菌泥加3mL pH8.5Tris盐酸盐缓冲液的比例重悬细胞，冰水浴超声裂解，12000rpm离心收集上清酶液得重组AspB突变体粗酶液，用于测酶活实验。
AspB酶活的定义：pH8.5，45℃条件下，1mL反应体系中每分钟生成1μmol(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸所需要的酶量。
比酶活为每毫克酶蛋白所含的活力单位(即，每mg上述阳性突变体菌泥所具有的活力单位)，计算公式：酶活/蛋白含量，单位是U/mg或U/g。
酶活检测方法为：
5mL反应体系中，加入4mL pH8.5的50mM Tris盐酸缓冲液(该缓冲液含5mM的(E)-4-(2,4,5-三氟苯基)丁-2-烯酸，50mM NH4Cl，2mM MgCl2)，1mL的上述重组AspB突变体粗酶液，于45℃摇床250rpm震荡反应，反应5小时，取反应液1mL进液相，测产物峰面积，根据产物峰面积比例计算酶活。酶活数据如表9。
表9阳性AspB突变体酶活
Figure BDA0002066806410000121
其中，*代表0.05U/mg以下，**代表酶活在0.05-0.1U/mg之间；***代表酶活在0.1-1U/mg之间；****代表酶活在1U/mg以上。
实施例3 AspB突变体在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸中的应用
Figure BDA0002066806410000122
准确称量60.6mg的Tris碱，216mg的(E)-4-(2,4,5-三氟苯基)丁-2-烯酸，0.5gNH4Cl，加入到含5％DMSO(二甲基亚砜)的7mL水中，使用氨水调pH 8.5，最后加入按实施例2方法制备的菌泥1g至反应体系，最后补2～3mL水至终体积10mL。在45℃，220rpm，过夜12h反应，HPLC分析转化率(之后本实施例没有直接检测产物(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的ee值，而是将产物继续反应，得到(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸后检测的ee值)，之后加入二碳酸二叔丁酯(Boc酸酐)218mg反应48小时，取反应液测ee值，乙酸乙酯萃取，旋干后，所得(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸的HNMR图谱见图9。HPLC结果见表10。从表10中可以看出，转化率和ee值均较高。
催化反应结束后分析转化率的HPLC图谱分析结果见图3(附图中均以表9中的天冬氨酸酶突变体2为例进行说明)，其中，7.827min为(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的出峰位置，4.896min为(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的出峰位置。(E)-4-(2,4,5-三氟苯基)丁-2-烯酸标准品(为发明人自行合成，鉴定图谱如图7所示)的HPLC图谱如图1所示，其中，(E)-4-(2,4,5-三氟苯基)丁-2-烯酸标准品的保留时间为7.865min。(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品(制备方法主要参考PCT/CN2018/095148，其HNMR图谱见图8)的HPLC图谱如图2所示，其中，(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品的保留时间为4.932min。可见，该实施例中底物(E)-4-(2,4,5-三氟苯基)丁-2-烯酸和产物(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的出峰时间与各自标准品的出峰时间基本一致。
催化反应完全结束后反应液的手性HPLC图谱分析结果见图6(附图中均以天冬氨酸酶突变体2为例进行说明)，其中，保留时间为8.972min的为(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸，15.872min为(3S)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸；N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸消旋体的手性HPLC图谱如图4，(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品(购买自凯瑞斯德生化(苏州)有限公司)的手性HPLC图谱如图5所示((3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸的保留时间为8.692min)。该实施例所制备得到的产物的成分与标准品中(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸的出峰时间基本一致，说明该实施例制备得到了(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸。
虽然上述结果图均以天冬氨酸酶突变体2为例，但是发明人进行了所有其他突变体的实验，也均验证了本发明的这些突变在参与上述反应时能够催化所述底物(E)-4-(2,4,5-三氟苯基)丁-2-烯酸，并且均生成了正确的产物。
表10反应转化率和ee值
酶编号 反应时间 转化率 ee值
1 12h 0 0
2 12h 91％ 95％
3 12h 56％ 96％
4 12h 15％ 98％
5 12h 20％ 97％
6 12h 10％ 98％
7 12h 5％ 94％
8 12h 60％ 97％
9 12h 30％ 96％
10 12h 0 0
11 12h 0 0
12 12h 0 0
SEQUENCE LISTING
<110> 上海弈柯莱生物医药科技有限公司
<120> 一种天冬氨酸酶突变体及其应用
<130> P180117514C
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 1404
<212> DNA
<213> Bacillus sp. YM55-1
<400> 1
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgcac ccatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
atgcctggta aggtgaaccc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gctgaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 2
<211> 468
<212> PRT
<213> Bacillus sp. YM55-1
<400> 2
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Thr His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Met Pro Gly Lys Val Asn Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 3
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324T-N326S-L358L
<400> 3
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accaacgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg ccacctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
gttccgggta ccgtttctcc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgatc 1380
cacccgggta tcgctggtcg taaa 1404
<210> 4
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324T-N326S-L358L
<400> 4
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 5
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187F-M321V- K324T-N326S-L358L
<400> 5
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgctt tcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 6
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187F-M321V- K324T-N326S-L358L
<400> 6
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Phe His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 7
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187D-M321V-K324T-N326S-L358L
<400> 7
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgcga tcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 8
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187D-M321V-K324T-N326S-L358L
<400> 8
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Asp His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 9
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187S-M321V-K324T-N326S-L358L
<400> 9
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgctc gcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 10
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187S-M321V-K324T-N326S-L358L
<400> 10
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Ser His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 11
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187N-M321V-K324T-N326S-L358L
<400> 11
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgcaa tcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 12
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187N-M321V-K324T-N326S-L358L
<400> 12
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Asn His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 13
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187V-M321V-K324T-N326S-L358L
<400> 13
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgcgt tcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 14
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187V-M321V-K324T-N326S-L358L
<400> 14
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Val His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 15
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187T-M321V-K324T-N326S-L358L
<400> 15
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgcac tcatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gttaaacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 16
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187T-M321V-K324T-N326S-L358L
<400> 16
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Thr His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 17
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324T-N326S-L358Y
<400> 17
atgaacaccg atgtgcgcat tgagaaggac ttcctgggtg aaaaggaaat cccgaaggat 60
gcctattacg gcgtgcagac catccgtgcc acagagaact ttcctatcac cggctaccgc 120
atccatccgg aactgattaa gagcctgggc attgtgaaga aaagcgccgc actggcaaac 180
atggaggtgg gtctgctgga taaggaagtg ggtcagtaca tcgtgaaggc cgccgacgaa 240
gttattgaag gtaagtggaa cgatcagttt atcgtggacc cgattcaggg cggcgcaggt 300
acaagcatta atatgaacgc caacgaagtg atcgcaaacc gcgccctgga actgatgggt 360
gaggaaaagg gcaactatag caagatcagc ccgaacagcc acgttaacat gagccagagc 420
accaatgatg catttccgac cgcaacccat attgccgtgc tgagtctgct gaatcagctg 480
atcgagacca ccaagtacat gcagcaggag tttatgaaga aggccgacga attcgccggc 540
gttattaaaa tgggccgctg ccatctgcaa gacgccgttc cgattctgct gggtcaggag 600
tttgaggctt atgctcgtgt gatcgcacgt gacattgagc gcatcgccaa tacccgtaac 660
aacctgtatg atatcaacat gggcgcaacc gccgttggca caggcctgaa tgcagacccg 720
gagtacatta gcatcgttac cgagcacctg gccaaattta gcggtcatcc gctgcgtagt 780
gcccagcatc tggttgatgc cacccagaat acagattgct acaccgaggt gagcagtgcc 840
ctgaaagtgt gcatgatcaa tatgagtaag attgccaacg acctgcgctt aatggcaagt 900
ggcccgcgcg caggcctgag cgaaattgtt ctgcctgcac gccaaccggg cagcagcatc 960
gtccctggta cggtgtcgcc ggtgatgccg gaagtgatga accaggttgc cttccaggtg 1020
ttcggtaacg acctgaccat cacaagcgca agcgaagcag gccagttcga gtataacgtg 1080
atggaacctg tgctgttttt taacttaatt cagagcatca gtattatgac aaatgttttt 1140
aagtctttta ccgaaaactg tctgaaaggt atcaaggcca acgaggaacg catgaaagag 1200
tatgtggaaa aaagcattgg catcatcacc gccatcaacc cgcatgtggg ctatgagaca 1260
gccgccaaac tggcccgcga agcctattta accggcgaga gtattcgcga gctgtgtatc 1320
aagtacggcg tgctgaccga agagcagctg aacgagatcc tgaatccgta cgagatgatc 1380
catcctggca ttgcaggtcg caaa 1404
<210> 18
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324T-N326S-L358Y
<400> 18
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Thr Val Ser Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Tyr Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 19
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187C-M321I-K324M-N326C-L358L
<400> 19
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accaacgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg ccacctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
atcccgggta tggtttgccc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgatc 1380
cacccgggta tcgctggtcg taaa 1404
<210> 20
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187C-M321I-K324M-N326C-L358L
<400> 20
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Ile Pro Gly Met Val Cys Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 21
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187C-M321I-K324L-N326C-L358L
<400> 21
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accaacgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg ccacctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
atcccgggtc tggtttgccc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgatc 1380
cacccgggta tcgctggtcg taaa 1404
<210> 22
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187C-M321I-K324L-N326C-L358L
<400> 22
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Ile Pro Gly Leu Val Cys Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 23
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324I-N326C-L358L
<400> 23
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accaacgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg ccacctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
gttccgggta tcgtttgccc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgatc 1380
cacccgggta tcgctggtcg taaa 1404
<210> 24
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> T187C-M321V-K324I-N326C-L358L
<400> 24
Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp
165 170 175
Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp
210 215 220
Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro
225 230 235 240
Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Val Pro Gly Ile Val Cys Pro Val Met Pro Glu Val Met Asn Gln Val
325 330 335
Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Ile His Pro Gly Ile
450 455 460
Ala Gly Arg Lys
465
<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> C187NNK正向引物
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21)
<223> k is g or t
<400> 25
gttattaaaa tgggccgcnn kcatctgcaa gacgccgttc 40
<210> 26
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> C187NNK反向引物
<220>
<221> misc_feature
<222> (20)
<223> m is a or c
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 26
gaacggcgtc ttgcagatgm nngcggccca ttttaataac 40
<210> 27
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> L358NNK正向引物
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (18)
<223> k is g or t
<400> 27
gcaggccagt tcgagnnkaa cgtgatggaa cctg 34
<210> 28
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> L358NNK反向引物
<220>
<221> misc_feature
<222> (17)
<223> m is a or c
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 28
caggttccat cacgttmnnc tcgaactggc ctgc 34
<210> 29
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> 321nnk-324nnk-326nnk正向引物
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (18)
<223> k is g or t
<220>
<221> misc_feature
<222> (25)..(26)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27)
<223> k is g or t
<220>
<221> misc_feature
<222> (31)..(32)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (33)
<223> k is g or t
<400> 29
ccgggcagca gcatcnnkcc tggtnnkgtg nnkccggtga tgccggaag 49
<210> 30
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> 321nnk-324nnk-326nnk 反向引物
<220>
<221> misc_feature
<222> (17)
<223> m is a or c
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (23)
<223> m is a or c
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (32)
<223> m is a or c
<220>
<221> misc_feature
<222> (33)..(34)
<223> n is a, c, g, or t
<400> 30
cttccggcat caccggmnnc acmnnaccag gmnngatgct gctgcccgg 49

Claims (10)

1.一种天冬氨酸酶突变体，其特征在于，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上包括以下突变：M321V、K324T和N326S，所述天冬氨酸酶突变体具有比野生型天冬氨酸酶催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的比酶活高的酶活性。
2.如权利要求1所述的天冬氨酸酶突变体，其特征在于，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上还包括第187位氨基酸残基T的突变；
较佳地，所述第187位氨基酸残基T的突变为C、F、D、S、N或V，优选C或F。
3.如权利要求2所述的天冬氨酸酶突变体，其特征在于，所述天冬氨酸酶突变体在如SEQ ID NO.2所示的氨基酸序列上还包括L358Y。
4.如权利要求1～3任一项所述的天冬氨酸酶突变体，其特征在于，所述天冬氨酸酶突变体的氨基酸序列如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ IDNO.12、SEQ ID NO.14、SEQ ID NO.16或SEQ ID NO.18所示；较佳地，所述天冬氨酸酶突变体的核苷酸序列如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15或SEQ ID NO.17所示。
5.一种分离的核酸，其特征在于，所述核酸编码如权利要求1～4任一项所述的天冬氨酸酶突变体。
6.一种重组表达载体，其包含如权利要求5所述的核酸。
7.一种转化体，其包含如权利要求5所述的核酸或如权利要求6所述的重组表达载体。
8.一种(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸的制备方法，其特征在于，所述制备方法包括以下步骤：在如权利要求1～4任一项所述的天冬氨酸酶突变体的存在下，将(E)-4-(2,4,5-三氟苯基)丁-2-烯酸和氨基供体进行氢胺化反应，即得(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸。
9.如权利要求8所述的制备方法，其特征在于，所述天冬氨酸酶突变体以天冬氨酸酶突变体菌泥的形式存在，所述天冬氨酸酶突变体菌泥的浓度为20g/L-200g/L，例如100g/L；
和/或，所述(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的浓度0.05mol/L-0.5mol/L，优选0.1mol/L；
和/或，所述氨基供体为NH4Cl、氨水、甲酸铵、乙酸铵、碳酸铵、碳酸氢铵、硫酸铵、硫酸氢铵、磷酸铵、磷酸氢二铵、磷酸二氢铵中的一种或多种；
和/或，所述氨基供体与底物(E)-4-(2,4,5-三氟苯基)丁-2-烯酸的摩尔比为1:1-20:1，例如10:1；
和/或，所述氢胺化反应的反应溶剂为含有DMSO的水，优选含5％DMSO的水；
和/或，所述氢胺化反应的反应体系的pH为7-9，较佳地为8.5；
和/或，所述氢胺化反应的反应体系的温度为30-60℃，较佳地为45℃；
和/或，所述氢胺化反应的反应时间为6-24小时，较佳地为12小时。
10.一种如权利要求1～4任一项所述的天冬氨酸酶突变体在制备(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸中的用途；
较佳地，所述的天冬氨酸酶突变体通过催化(E)-4-(2,4,5-三氟苯基)丁-2-烯酸制备得到所述的(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸。
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CN111593039A (zh) * 2020-06-17 2020-08-28 台州酶易生物技术有限公司 重组天冬氨酸酶突变体、编码基因及其应用
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CN110791494A (zh) * 2019-11-28 2020-02-14 江南大学 一种天冬氨酸酶突变体、包含天冬氨酸酶突变体的重组表达载体和重组菌及应用
CN111593039A (zh) * 2020-06-17 2020-08-28 台州酶易生物技术有限公司 重组天冬氨酸酶突变体、编码基因及其应用
CN112921023B (zh) * 2021-03-30 2022-11-11 长兴制药股份有限公司 一种重组天冬氨酸裂解酶及高重复利用率用于制备r-3-氨基丁酸的方法
CN112921023A (zh) * 2021-03-30 2021-06-08 长兴制药股份有限公司 一种重组天冬氨酸裂解酶及高重复利用率用于制备r-3-氨基丁酸的方法
CN115197928B (zh) * 2021-04-09 2023-08-08 山东新和成精化科技有限公司 天冬氨酸酶突变体及其制备方法和用途
CN115197928A (zh) * 2021-04-09 2022-10-18 山东新和成精化科技有限公司 天冬氨酸酶突变体及其制备方法和用途
CN113122527B (zh) * 2021-04-25 2022-08-09 江南大学 一种酶活提高及最适pH改变的天冬氨酸酶突变体
CN113122527A (zh) * 2021-04-25 2021-07-16 江南大学 一种酶活提高及最适pH改变的天冬氨酸酶突变体
CN114934038A (zh) * 2022-05-05 2022-08-23 安徽丰原发酵技术工程研究有限公司 天冬氨酸酶突变体及其应用
CN114934038B (zh) * 2022-05-05 2023-09-12 安徽丰原发酵技术工程研究有限公司 天冬氨酸酶突变体及其应用
CN116217440A (zh) * 2022-12-30 2023-06-06 浙江永太手心医药科技有限公司 一种西他列汀关键中间体的制备方法
CN116836963A (zh) * 2023-06-30 2023-10-03 秦皇岛华恒生物工程有限公司 一种天冬氨酸酶突变体及其应用
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