CN114752574B - 一种酶催化体系、加氢酶及制备方法和应用 - Google Patents
一种酶催化体系、加氢酶及制备方法和应用 Download PDFInfo
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Abstract
本申请公开了一种酶催化体系、加氢酶及制备方法和应用。酶催化体系包括:用于催化底物经过还原反应生成产物的加氢酶、有机溶剂、辅酶和辅酶再生剂。加氢酶的氨基酸序列如SEQ ID NO:1所示,或者为与SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。通过酶催化还原反应得到可作为依折麦布中间体的(4S)‑3‑[(5s)‑5‑(4‑氟苯基)‑5‑羟基戊酰基]‑4‑苯基‑1,3‑氧氮杂环戊烷‑2‑酮。采用本申请的加氢酶制备依折麦布中间体,酶的用量少,产物转化率高。
Description
技术领域
本申请涉及酶工程及生物制药技术领域,具体涉及一种酶催化体系、加氢酶及制备方法和应用。
背景技术
依折麦布,又名依替米贝、依泽替米贝,英文名为:Ezetimibe,化学名称:1-(4-氟苯基)-(3R)-3-(4-氟苯基)-(3S-羟基丙基]-(4S(4-羟基苯基)-2-丙内酰胺,CAS:163222-33-1,分子式为C24H21F2NO3,分子量为409.43,为白色晶状粉末,是高纯化、高选择性胆固醇吸收抑制剂药物,它是第一个被美国FDA批准上市的能够选择性抑制胆固醇吸收的药物。主要用于降低人体胆固醇的含量,对治疗原发性高胆固醇血症、家族性纯合子高胆固醇血症(HoFH)、纯合子谷甾醇血症(或植物甾醇血症)都有一定作用;对动脉粥样化性疾病和冠心病等疾病的防治和治疗都有重要的意义。
依折麦布分子结构中有一个手性苄位羟基和两个修饰刚性2-氮杂环丁酮母核的立体中心,可存在8个立体异构体,因此,其绝对构型的建立和控制成为合成领域最具挑战性的工作之一。
在依折麦布合成的大部分路线中,(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮作为依折麦布的关键手性中间体,它可以由化学和酶催化动力学拆分其前体酮制备得到。其中,化学不对称还原法得到的目标产物光学纯度不够高,同时化学法成本较高,并且化学催化剂容易对环境造成严重的污染。与化学法相比,生物法表现出明显的立体专一性强、催化效率高和无污染等优点。但目前的生物法制备仍存在一些缺陷,例如酶活损失严重,导致转化率降低,需要不断增加酶量才能维持反应的进行。因此找到既能减少酶的用量又能提高产物转化率的体系对减少成本和提高产物回收有重大意义。
发明内容
本申请提供一种酶催化体系、加氢酶及制备方法和应用,可以改善目前反应中酶用量较大及转化率较低等问题。
第一方面,本申请提供一种酶催化体系,酶催化体系包括:用于催化底物经过还原反应生成产物的加氢酶、有机溶剂、辅酶和辅酶再生剂;底物为(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮,其结构如式(1)所示:
产物为(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮,其结构如式(2)所示:
进一步地,加氢酶的氨基酸序列如SEQ ID NO:1所示,或者为与SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
进一步地,底物的浓度可以为50g/L~250g/L,也可以为100g/L~200g/L,还可以为120g/L~180g/L。
进一步地,以反应体系的总体积计,有机溶剂的浓度可以为20%(v/v)~50%(v/v),也可以为25%(v/v)~45%(v/v),还可以为30%(v/v)~40%(v/v);反应体系为发生催化还原反应过程中的体系,包括酶催化体系、底物、水等。
进一步地,有机溶剂包括二甲基亚砜、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、N,N-二甲基甲酰、二氯甲烷或甲苯中的至少一种。
进一步地,辅酶选自烟酰胺腺嘌呤二核苷酸(NAD)或烟酰胺腺嘌呤二核苷酸磷酸(NADP)中的任意一种。
进一步地,辅酶的浓度可以为0.2~2g/L,也可以为0.5~1.7g/L,还可以为1.0~1.5g/L。
进一步地,辅酶再生剂包括:异丙醇和羰基还原酶。
进一步地,羰基还原酶的氨基酸序列如SEQ ID NO:2所示。
进一步地,以反应体系的总体积计,羰基还原酶的体积百分比含量可以为0.5%~1%,也可以为0.6%~0.9%,还可以为0.7%~0.8%。
进一步地,辅酶再生剂包括:葡萄糖和葡萄糖脱氢酶。
进一步地,葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
进一步地,以反应体系的总体积计,葡萄糖脱氢酶的体积百分比含量可以为0.2%~0.6%,也可以为0.3%~0.5%,还可以为0.4%。
进一步地,葡萄糖的浓度可以为30~50g/L,也可以为35~45g/L,还可以为37~43g/L。
第二方面,本申请提供一种加氢酶,加氢酶的氨基酸序列如SEQ ID NO:1所示,或者为与SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
进一步地,加氢酶的氨基酸序列是由SEQ ID NO:1所示的氨基酸序列发生一个或多个点突变得到的氨基酸序列。
进一步地,点突变的氨基酸包括SEQ ID NO:1中的第30位氨基酸F、第50位氨基酸V、第94位氨基酸D、第158位氨基酸H、第169位氨基酸A、第172位氨基酸S、第229位氨基酸D、第241位氨基酸L、第243位氨基酸R中的一个或多个。
进一步地,发生点突变的方式为F30T、V50P、D94Q、H158I、A169T、S 172R、D229V、L241I或R243Y。
进一步地,加氢酶的氨基酸序列为SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12或SEQ ID NO:13所示的氨基酸序列。
作为替代性实施方案,发生点突变的方式为以下任一所示组合:
(1)F30T和S172R;
(2)H158I、L241I和R243Y;
(3)V50P、D94Q和A169T;
(4)A169T、S172R、D229V和L241I;或
(5)F30T、D94Q、A169T、L241I和R243Y。
进一步地,加氢酶的氨基酸序列为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示的氨基酸序列。
第三方面,本申请提供一种加氢酶在催化底物经过还原反应生成产物中的应用;底物为(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮,其结构如式(1)所示:
产物为(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮,其结构如式(2)所示:
第四方面,本申请提供一种核酸分子,包括用于编码第二方面所述的加氢酶的核苷酸序列。
进一步地,核苷酸序列为下述任一项:
(a)核苷酸序列如SEQ ID NO:19所示;
(b)核苷酸序列与SEQ ID NO:19至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性;或
(c)核苷酸序列为(a)或(b)中核苷酸序列经密码子优化后获得的核苷酸序列。
进一步地,核苷酸序列为如SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ IDNO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:33所示的核苷酸序列。
第五方面,本申请提供一种(4S)-3-[(5s)-5-(4-氟苯基)5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的制备方法,包括:将底物(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮与第一方面的酶催化体系混合以产生酶催化还原反应,得到(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮。
进一步地,有机溶剂包括二甲基亚砜、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、N,N-二甲基甲酰、二氯甲烷或甲苯中的至少一种。
进一步地,底物与酶催化体系中的加氢酶的质量比可以为(120~210)∶1,也可以为(150~200)∶1,还可以为(160~180)∶1。
进一步地,底物的浓度可以为50g/L~250g/L,也可以为100g/L~200g/L,还可以为120g/L~180g/L。
进一步地,以反应体系的总体积计,酶催化体系中的有机溶剂的浓度可以为20%(v/v)~50%(v/v),也可以为25%(v/v)~45%(v/v),还可以为30%(v/v)~40%(v/v)。
进一步地,酶催化反应在辅酶和辅酶再生剂的存在下进行,其中,辅酶为NADP或NAD,辅酶再生剂为异丙醇和羰基还原酶,或葡萄糖和葡萄糖脱氢酶。
进一步地,羰基还原酶的氨基酸序列如SEQ ID NO:2所示,葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
进一步地,酶催化还原反应的pH为6.0~9.0,也可以为6.5~8.5,还可以为7.0~8.0。
进一步地,酶催化还原反应的温度为25℃~65℃,也可以为30℃~60℃,还可以为35℃~55℃。
进一步地,酶催化还原反应在振荡条件下进行:转速可以为150转每分钟~500转每分钟,也可以为200转每分钟~450转每分钟,还可以为250转每分钟~400转每分钟。
第六方面,本申请提供一种如第五方面的方法制得的(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮作为用于制备依折麦布的中间体的用途;依折麦布的结构如式(3)所示:
本申请采用加氢酶催化产生依折麦布中间体,具有如下有益效果:
(1)本申请将加氢酶与底物(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮接触而发生酶催化反应,从而制得(4S)-3-[(5s)-5-(4-氟苯基)5-羟基戊酰基]4苯基-1,3-氧氮杂环戊烷-2-酮,所用加氢酶的酶量较少,转化率较高,具有提高产物转化率和降低生产成本的优点。
(2)经实验发现,采用乙酸乙酯和乙酸丁酯作为有机溶剂,并且在包含加氢酶的预混体系中添加辅酶再生***,以使加氢酶和辅酶再生***按照特定的比例复配的技术方案有利于提高底物转化率,底物转化率至少为97%。此外,将乙酸乙酯与底物相混合获得的混合液配合葡萄糖和葡萄糖脱氢酶的辅酶循环方式加入至该预混体系中的技术手段可进一步提高底物转化率,底物转化率可达99.5%。
附图说明
为了更清楚地说明本申请实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请的一些实施例,对于本领域技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是底物与依折麦布中间体标样的HPLC图谱;
图2是实施例四中含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在1h的HPLC图谱;
图3是实施例四中含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在24h的HPLC图谱;
图4是实施例十一中含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在1h的HPLC图谱;
图5是实施例十一中含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在24h的HPLC图谱。
具体实施方式
下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
本申请提供一种酶催化体系、加氢酶及制备方法和应用。以下分别进行详细说明。需说明的是,以下实施例的描述顺序不作为对实施例优选顺序的限定。
下面实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
除非另行定义,文中所使用的所有专业与科学用语与本领域技术人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本申请中。文中所述的较佳实施方法与材料仅作示范之用,但不能限制本申请的内容。
除非另有说明,文中涉及的试剂与材料均可商购获得,或本领域技术人员可依据公知常识自行制备。
定义与说明:
如本申请所用,“重组表达载体”是指一种DNA构建体,其含有与合适的控制序列可操作地连接的核酸分子,所述控制序列能够实现核酸分子在合适的表达***中表达。在本申请实施例中,重组表达载体是指采用分子生物学技术将外源基因***至载体上,由此形成的DNA构建体,示例pET24a-1为重组表达载体。
如本申请所用,“酶制剂”是指从生物(如微生物)中提取的具有生物催化能力的物质,辅以其他成分而制得的功能化产品。在本申请实施例中,酶制剂的活性成分为酶,酶的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示,该酶制剂能够应用于依折麦布的生产工艺中。在一些实施例中,酶制剂可包括必要的赋形剂、稳定剂、保护剂等食品或药学上可接受的佐剂和/或辅料。
如本申请所用,“核苷酸序列”指来自一种核酸分子的5’至3’端的核苷酸的序列并且包括DNA或RNA分子,包括cDNA、DNA片段或部分、基因组DNA、合成(例如,化学合成)DNA、质粒DNA、mRNA以及反义RNA,其中的任一项都可以是单链或双链的。
如本申请所用,“密码子”是指规定具体氨基酸的三核苷酸序列。“密码子偏好”是指在生物体编码序列中编码氨基酸的密码子的使用频率。
如本申请所用,氨基酸由单字母或三字母代码表示,具有如下含义:A:Ala(丙氨酸);R:Arg(精氨酸);N:Asn(天冬酰胺);D:Asp(天冬氨酸);C:Cys(半胱氨酸);Q:Gln(谷氨酰胺);E:Glu(谷氨酸);G:Gly(甘氨酸);H:His(组氨酸);I:Ile(异亮氨酸);L:Leu(亮氨酸);K:Lys(赖氨酸);M:Met(甲硫氨酸);F:Phe(苯丙氨酸);P:Pro(脯氨酸);S:Ser(丝氨酸);T:Thr(苏氨酸);W:Trp(色氨酸);Y:Tyr(酪氨酸);V:Val(缬氨酸)。
本申请实施例提供一种加氢酶,加氢酶的氨基酸序列如SEQ ID NO:1所示,或者为与SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
在本申请的一些实施例中,加氢酶的氨基酸序列是由SEQ ID NO:1所示的氨基酸序列发生一个或多个点突变得到的氨基酸序列。
在本申请的一些实施例中,点突变的氨基酸包括SEQ ID NO:1中的第30位氨基酸F、第50位氨基酸V、第94位氨基酸D、第158位氨基酸H、第169位氨基酸A、第172位氨基酸S、第229位氨基酸D、第241位氨基酸L、第243位氨基酸R中的一个或多个。
在本申请的一些实施例中,发生点突变的方式为F30T、V50P、D94Q、H158I、A169T、S172R、D229V、L241I或R243Y。
在本申请的一些实施例中,加氢酶的氨基酸序列为SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12或SEQ ID NO:13所示的氨基酸序列。
在本申请的一些实施例中,发生点突变的方式为以下任一所示组合:
(1)F30T和S172R;
(2)H158I、L241I和R243Y;
(3)V50P、D94Q和A169T;
(4)A169T、S172R、D229V和L241I;或
(5)F30T、D94Q、A169T、L241I和R243Y。
在本申请的一些实施例中,加氢酶的氨基酸序列为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示的氨基酸序列。
本申请实施例还提供一种核酸分子,包括用于编码上述的加氢酶的核苷酸序列。
在本申请的一些实施例中,核苷酸序列为下述任一项:
(a)核苷酸序列如SEQ ID NO:19所示°
(b)核苷酸序列与SEQ ID NO:19至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性°或
(c)核苷酸序列为(a)或(b)中核苷酸序列经密码子优化后获得的核苷酸序列。
在本申请的一些实施例中,核苷酸序列为如SEQ ID NO:20、SEQ ID NO:21、SEQ IDNO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ IDNO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:33所示的核苷酸序列。
本申请实施例还提供一种加氢酶在催化底物经过还原反应生成产物中的应用°该加氢酶能够催化底物中的羰基形成手性的苄位羟基。底物为(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮,其结构如式(1)所示:
产物为(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮,其结构如式(2)所示:
本申请实施例还提供一种酶催化体系,酶催化体系包括:上述的加氢酶、有机溶剂、辅酶和辅酶再生剂;底物为(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮,其结构如式(1)所示;产物为(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮,其结构如式(2)所示。
在本申请的一些实施例中,底物的浓度可以为50g/L~250g/L,也可以为100g/L~200g/L,还可以为120g/L~180g/L.
在本申请的一些实施例中,以反应体系的总体积计,有机溶剂的浓度可以为20%(v/v)~50%(v/v),也可以为25%(v/v)~45%(v/v),还可以为30%(v/v)~40%(v/v)。
在本申请的一些实施例中,有机溶剂包括二甲基亚砜、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、N,N-二甲基甲酰、二氯甲烷或甲苯中的至少一种;优选的,有机溶剂为乙酸乙酯或乙酸丁酯中的至少一种。
在本申请的一些实施例中,辅酶选自烟酰胺腺嘌呤二核苷酸(NAD)或烟酰胺腺嘌呤二核苷酸磷酸(NADP)中的任意一种。当辅酶加入到酶催化体系中后,辅酶接收辅酶再生剂提供的H,从而形成还原型辅酶。该还原型辅酶能够为底物的还原反应提供H。具体而言,烟酰胺腺嘌呤二核苷酸(NAD)能够接收辅酶再生剂提供的H,形成还原型烟酰胺腺嘌呤二核苷酸(NADH)。烟酰胺腺嘌呤二核苷酸磷酸(NADP)能够接收辅酶再生剂提供的H,形成还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)。在底物的还原反应结束后,还原型辅酶失去H,又形成辅酶,后继又在辅酶再生剂的作用下形成还原型辅酶,从而实现辅酶的循环再生。
在本申请的一些实施例中,辅酶的浓度可以为0.2~2g/L,也可以为0.5~1.7g/L,还可以为1.0~1.5g/L。
在本申请的一些实施例中,辅酶再生剂包括:异丙醇和羰基还原酶。羰基还原酶能够将异丙醇的H提供给辅酶。当异丙醇中的H消耗完毕时,再加入新的异丙醇便可补充新的H,从而继续推动辅酶的循环再生过程。
在本申请的一些实施例中,羰基还原酶的氨基酸序列如SEQ ID NO:2所示。
在本申请的一些实施例中,以反应体系的总体积计,羰基还原酶的体积百分比含量可以为0.5%~1%,也可以为0.6%~0.9%,还可以为0.7%~0.8%。
在本申请的一些实施例中,辅酶再生剂包括:葡萄糖和葡萄糖脱氢酶。葡萄糖脱氢酶能够将葡萄糖的H提供给辅酶。当葡萄糖中的H消耗完毕时,再加入新的葡萄糖便可补充新的H,从而继续推动辅酶的循环再生过程。
在本申请的一些实施例中,葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
在本申请的一些实施例中,以反应体系的总体积计,葡萄糖脱氢酶的体积百分比含量可以为0.2%~0.6%,也可以为0.3%~0.5%,还可以为0.4%。
在本申请的一些实施例中,葡萄糖的浓度可以为30~50g/L,也可以为35~45g/L,还可以为37~43g/L。
本申请实施例还提供一种(4S)-3-[(5s)-5-(4-氟苯基)5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的制备方法,包括:将底物(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮与上述的酶催化体系混合以产生酶催化还原反应,得到(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮。
在本申请的一些实施例中,底物与酶催化体系中的加氢酶的质量比可以为(120~210)∶1,也可以为(150~200)∶1,还可以为(160~180)∶1。
在本申请的一些实施例中,酶催化反应在辅酶和辅酶再生剂的存在下进行,其中,辅酶为NADP或NAD,辅酶再生剂为异丙醇和羰基还原酶,或葡萄糖和葡萄糖脱氢酶;辅酶再生剂用于提供循环再生的还原型烟酰胺腺嘌呤二核苷酸(NADH)或还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH),NADH和NADPH作为供氢体参与本申请还原反应。
在本申请的一些实施例中,酶催化还原反应的pH为6.0~9.0,也可以为6.5~8.5,还可以为7.0~8.0。
在本申请的一些实施例中,酶催化还原反应的温度为25℃~65℃,也可以为30℃~60℃,还可以为35℃~55℃。
在本申请的一些实施例中,酶催化还原反应在振荡条件下进行:转速可以为150转每分钟~500转每分钟,也可以为200转每分钟~450转每分钟,还可以为250转每分钟~400转每分钟。
在本申请的一些实施例中,加氢酶的添加方式可以是:直接将加氢酶或包含加氢酶的加氢酶制剂添加于预混体系中;或者,将含有加氢酶的编码基因的转化体经发酵培养后获得的湿菌体添加于预混体系中;或者,将湿菌体经破碎处理后的细胞破碎液添加于预混体系中;或者,将细胞破碎液经分离纯化处理后的产物添加于预混体系中。
本申请实施例还提供一种如上述方法制得的(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮作为用于制备依折麦布的中间体的用途;依折麦布的结构如式(3)所示:
本申请实施例中涉及培养基的说明
(1)LB液体培养基
包含1%NaCl,1%胰蛋白胨,0.5%酵母提取物,充分混匀后,调节pH至7.0,高压蒸汽灭菌锅121℃灭菌30min。使用前添加终浓度50ug/mL卡那霉素。
(2)自诱导培养基
分别称取120g的酵母粉、32.25g蛋白胨、0.75g的硫酸镁(MgSO4)、16.5g的硫酸铵((NH4)2SO4)、32.5g的磷酸二氢钾(KH2PO4)、35.5g的磷酸氢二钠(Na2HPO4)、2.5g的葡萄糖以及10g的α-乳糖,然后将各个称取好的组分全部加入至磨粉机内搅拌均匀,称取50g自诱导培养基粉末溶于1L水,充分混匀后,调节pH至7.0,121℃灭菌30min。
本申请实施例中涉及质粒和感受态细胞的说明详见下表1:
表1
本申请实施例中涉及的基因片段及试剂说明:
本申请实施例中涉及的基因片段,包括引物、如SEQ ID NO:19所示的核苷酸序列等由生工生物工程(上海)股份有限公司合成。
本申请实施例中涉及的限制性内切酶(如:BamH I、Nde I和Dpn I)、T4连接酶、KOD高保真酶试剂盒等分子试剂均购自宝生物工程(大连)有限公司。
下面结合实施例进一步说明本申请的技术方案和有益效果。
实施例一、加氢酶的来源以及构建包含加氢酶编码基因的重组表达载体
本实施例提供了重组表达载体,其具体获得方法包括如下步骤:
通过NCBI数据库挖掘出Galactomyces reessii菌株的氨基酸序列分别如SEQIDNo.1所示。依据E.coli密码子偏好性进行密码子优化,以全基因合成的方法合成了具有SEQ ID No.1序列的加氢酶的核苷酸序列,合成的核苷酸序列如SEQ ID No.19所示。在核苷酸序列两端加入酶切位点Nde I和BamH I,将该基因克隆至pET24a对应的Nde I和BamH I位点,获得重组表达载体pET24a-1。
实施例二、构建含有加氢酶编码基因的基因工程菌
本实施例的含有加氢酶编码基因的基因工程菌的构建方法如下:
将具有如SEQ ID NO:1所示氨基酸序列的加氢酶进行一个或多个点突变,以获得多个加氢酶突变体,其中,具体突变方式详见表2。采用定点突变策略,根据待突变的氨基酸位点利用Oligo7软件来设计点突变引物,通过在上下游突变引物的5’端以***、替换或缺失碱基的方式引入突变。
(1)选择实施例一中构建的重组表达载体pET24a-1为模板,采用KOD高保真酶试剂盒进行反向PCR,从而获得加氢酶突变序列。
(2)使用Dpn I限制性内切酶处理获得加氢酶突变序列,酶切产物经T4连接酶连接后转化进大肠杆菌BL21(DE3)感受态。
(3)随后涂布含卡那霉素的LB抗性平板,置于37℃倒置培养18h,挑选单菌落转接含卡那霉素的LB液体培养基中,挑选培养液送样测序,将测序正确的克隆子保存备用,从而获得以大肠杆菌为宿主的含有加氢酶编码基因的基因工程菌,对得到的含有加氢酶编码基因的基因工程菌进行菌株活力测定,结果如表2所示:
表2
注:相对活力为该突变菌株活力相对野生型菌株的活力;
+表示活力增加1-5倍,++表示5-10倍,+++表示10-20倍,++++表示20-30倍,+++++表示30倍以上。
在表2中,SEQIDNO.5至SEQIDNO.18所示的加氢酶是在SEQIDNO.1所示加氢酶的基础上作出一个或多个位点突变而获得,相较于SEQIDNO.1所示的加氢酶,SEQIDNO.5至SEQIDNO.18所示的加氢酶的菌株活力均有显著提高。采用F30T,D94Q,A169T,L241I和R243Y组合突变方式获得的加氢酶的菌株活力最高。其次是采用A169T,S172R,D229V和L241I组合突变方式获得的加氢酶,并且采用组合突变方式获得的加氢酶的菌株活力普遍高于采用单一位点突变方式获得的加氢酶的菌株活力。
实施例三、含有加氢酶编码基因的基因工程菌的诱导表达及后处理
本实施例提供了加氢酶的酶粉,其具体获得方法包括如下步骤:
(1)将基因工程菌接种至含终浓度50ug/mL卡那霉素的LB液体培养基,在37℃,180r/min培养至OD600约0.6-0.8,获得种子菌液;将种子菌液以体积浓度1%接种量接种至新鲜的含有终浓度50ug/mL卡那霉素的自诱导培养基,于30℃培养18h。25℃、8000r/min离心10min,弃上清液,用PB pH7.0清洗两遍,并收集湿菌体备用。
(2)采用超声波破碎法对菌液进行破碎。湿菌体使用超纯水重悬,制备菌浓为20%菌液。破碎条件如下:工作1秒,间歇2s,180W,破碎10min。可也采用高压均质破碎的条件:50HZ,800bar,破碎两遍。
(3)将细胞破碎液里的细胞碎片和大分子杂质通过高速离心除去,获得粗酶液,离心条件:12000r/min,4℃,离心10-15min。
(4)菌浓为20%的粗酶液120ml,使用真空冷冻干燥-25℃将其冻干成3~4g酶粉。
实施例四、不同有机溶剂的耐受性实验
本实施例检测具有SEQ ID No.1和SEQIDNO.18序列的加氢酶在不同溶剂中的耐受性,具体实验方法包括如下步骤:
1)在30mL的反应瓶中加入1.5mL异丙醇,加入1.5mL的有机溶剂,加入0.3g底物,混匀溶解后获得混合液°
2)加入0.003g加氢酶,加入3mL水,加入10mg NADP+,调节初始pH为6.5。
3)在30℃、180r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含25%(v/v)异丙醇,25%(v/v)有机溶剂,50g/L的底物,底物:加氢酶=100:1。
有机溶剂为:二甲基亚砜、四氢呋喃、N,N-二甲基甲酰、乙酸乙酯、乙酸丁酯、乙酸丙酯、二氯甲烷或甲苯。
利用高效液相色谱(High Performance Liquid Chromatography,HPLC)法对反应产物进行检测分析。
其中,HPLC的仪器型号为Agilent 1260(安捷伦),HPLC的工作条件如下:
(1)进样液的制备:取100μL的反应产物,加入1mL乙酸乙酯萃取,12000rmp/min离心1min,再取上清用流动相稀释60倍,混匀后进样20uL°
(2)色谱柱:大赛璐IC柱,4.6*150mm,5μm;
(3)流动相的制备:将正己烷和乙醇按照92∶8的体积比混合配制而成。
(4)流速:1mL/min。
(5)检测波长:268nm。
(6)柱温:30℃。
根据底物化合物的减少量计算底物转化率(%),底物转化率(%)的测定结果详见表2,底物转化率(%)的计算公式如下式(1):
在式(1)中,A1为反应后的底物化合物峰面积,A2为反应前的底物化合物峰面积。
根据不同构型产物的量计算立体选择性(e.e值),立体选择性的测定结果详见表2,立体选择性(e.e%)的计算公式如下式(2):
在式(2)中,A3为S型产物的峰面积(本反应所需的构型),A4为R型产物的峰面积。
实验结果如表3所示:
表3
由表3中结果可知,当有机溶剂为乙酸乙酯和乙酸丁酯时,加氢酶的转化率最高,说明采用本申请的加氢酶进行催化反应时,体系中有机溶剂优选乙酸乙酯或乙酸丁酯,能够获得较高的产物转化率。且由表1结果可以得到,SEQ IDNO:1的加氢酶在不同有机溶剂中的立体选择性(e.e值)均可达到98%,SEQ ID NO:18的加氢酶在不同有机溶剂中的立体选择性(e.e值)均可达到99%,且SEQ ID NO:18的加氢酶比SEQ ID NO:18的加氢酶产物转化率更高。
如图1所示为底物与依折麦布中间体标样的HPLC图谱,如图2所示为含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在1h的HPLC图谱,如图3所示为含有乙酸乙酯和SEQID NO:18的加氢酶的反应体系在24h的HPLC图谱,由图2和图3比较可知,反应24小时后,底物的峰面积减小,依折麦布中间体峰面积显著增加,说明在乙酸乙酯溶剂中,SEQ ID NO:18的加氢酶能够较好地催化底物转化产生依折麦布中间体。
实施例五、乙酸乙酯的不同浓度
本实施例检测当有机溶剂选择为乙酸乙酯时,乙酸乙酯不同浓度时具有SEQ IDNo.1和SEQIDNO.18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在100mL反应瓶加入10mL异丙醇,加入不同浓度(v/v)的乙酸乙酯,加入5g底物,混匀后获得混合液;
2)加0.0375g加氢酶,加入0.5mL羰基还原酶,加入适量水补足总体系为50mL;再加入10mg NADP+,调节初始pH为7.5;
3)在35℃、180r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含20%(v/v)异丙醇,20%-50%(v/v)有机溶剂,100g/L的底物,底物:加氢酶=133∶1。
HPLC检测条件及转化率的计算方法同实施例一,实验结果如表4所示:
表4
由表4中结果可知,当有机溶剂乙酸乙酯浓度为40%(v/v)时,加氢酶的转化率最高,说明采用本申请的加氢酶进行催化反应时,体系中有机溶剂乙酸乙酯控制在40%(v/v)时,能够获得较高的产物转化率。
实施例六、乙酸丁酯的不同浓度
本实施例检测当有机溶剂选择为乙酸丁酯时,乙酸丁酯不同浓度时具有SEQ IDNo.1和SEQ ID NO:18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在100mL反应瓶加入10mL异丙醇,加入不同浓度(v/v)的乙酸丁酯,加入5g底物,混匀后获得混合液;
2)加入0.0375g加氢酶,加入0.5mL羰基还原酶,加入适量水补足总体系为50mL;再加入10mg NADP+,调节初始pH为7.5;
3)在35℃、180r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含20%(v/v)异丙醇,20%-50%(v/v)有机溶剂,100g/L的底物,底物:加氢酶=133∶1。
HPLC检测条件及转化率的计算方法同实施例一,实验结果如表5所示:
表5
由表5中结果可知,当有机溶剂乙酸丁酯浓度为30%(v/v)时,加氢酶的转化率最高,说明采用本申请的加氢酶进行催化反应时,体系中有机溶剂乙酸丁酯控制在30%(v/v)时,能够获得较高的产物转化率。
实施例七、用乙酸乙酯溶解不同浓度的底物
本实施例检测当溶剂选择为乙酸乙酯时,所添加底物的合适浓度,具体实验方法包括如下步骤:
1)在100mL反应瓶加入10mL异丙醇,加入15mL的乙酸乙酯,加入不同质量的底物,混匀后获得混合液;
2)加入0.0375g加氢酶,加入10mg NADP+,加入0.5mL羰基还原酶,加入24.5mL水;调节初始pH为7.0;
3)在35℃、180r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含20%(v/v)异丙醇,30%(v/v)有机溶剂。
HPLC检测条件及转化率的计算方法同实施例一,实验结果如表6所示:
表6
由表6中结果可知,对于SEQ ID NO:1的加氢酶,底物浓度越高,转化率越低,说明此反应存在底物抑制,底物浓度高会抑制酶活,导致转化率降低;而对于SEQ ID NO:18的加氢酶,当底物浓度达到150g/L时,转化率仍然可以达到98.8%,说明减轻了底物抑制,也即采用SEQ ID NO:18的加氢酶进行催化反应时,能够用相同的酶量催化更高浓度的底物,体系中有机溶剂为乙酸乙酯时,当底物浓度为150g/L时,此时底物和加氢酶的质量比为200∶1,能够获得较高的产物转化率。
实施例八、用乙酸丁酯溶解不同浓度的底物
本实施例检测当溶剂选择为乙酸丁酯时,所添加底物的合适浓度,具体实验方法包括如下步骤:
1)在100mL反应瓶加入10mL异丙醇,加入15mL的乙酸丁酯,加入不同质量的底物,混匀后获得混合液;
2)加入0.0375g加氢酶,加入10mg NADP+,加入0.5mL羰基还原酶,加入24.5mL水;调节初始pH为7.0;
3)在35℃、180r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含20%(v/v)异丙醇,30%(v/v)有机溶剂。
HPLC检测条件及转化率的计算方法同实施例一,实验结果如表7所示:
表7
由表7中结果可知,对于SEQ ID NO:1的加氢酶,底物浓度越高,转化率越低,说明此反应存在底物抑制,底物浓度高会抑制酶活,导致转化率降低;而对于SEQ ID NO:18的加氢酶,当底物浓度达到100g/L时,转化率仍然可以达到96.8%,说明减轻了底物抑制,也即采用SEQ ID NO:18的加氢酶进行催化反应时,能够用相同的酶量催化更高浓度的底物体系中有机溶剂为乙酸丁酯时,当底物浓度为100g/L时,此时底物和加氢酶的质量比为133∶1,能够获得较高的产物转化率。
实施例九、乙酸乙酯与异丙醇体系
本实施例检测当所添加的辅酶再生***为异丙醇和羰基还原酶时,有机溶剂为乙酸乙酯时具有SEQ ID No.1和SEQ ID NO:18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在500ml反应瓶加入50mL异丙醇,加入100mL的乙酸乙酯,加入37.5g的底物,获得混合液°
2)加入0.188g加氢酶,加入50mg NADP+,加入2.5mL羰基还原酶,加入97.5mL的水,反应过程中用10%NaOH调节pH为7.8-8.0。
3)在37℃、400r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含40%(v/v)乙酸乙酯,150g/L底物,底物∶加氢酶=200∶1。
HPLC检测条件及转化率的计算方法同实施例一。
实验结果:SEQ ID NO:1的加氢酶24h产物转化率为25.5%;
SEQ ID NO:18的加氢酶24h产物转化率为98.9%。
由实验结果可知,当辅酶再生***为异丙醇和羰基还原酶、有机溶剂为乙酸乙酯时,并且体系中采用前述实验结果中优选的有机溶剂和底物浓度,SEQ ID NO:18的加氢酶的产物转化率可达98.9%。
对比例一的实验方法同实施例九,区别在于,所加的酶为氨基酸序列如SEQ IDNO:4所示的酶,其24h产物转化率为97.6%。说明本申请采用的SEQ ID NO:18的加氢酶在相同的用量上可以实现相对于对比例的酶较高的产物转化率,提示本申请的加氢酶可以以较少的用量达到较高的产物转化率。
实施例十、乙酸丁酯与异丙醇体系
本实施例检测当所添加的辅酶再生***为异丙醇和羰基还原酶时,有机溶剂为乙酸丁酯时,具有SEQ ID No.1和SEQ ID NO:18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在500mL反应瓶加入50mL异丙醇,加入75mL的乙酸丁酯,加入25g的底物,获得混合液°
2)加入0.188g加氢酶,加入50mg NADP+,加入2.5mL羰基还原酶,加入122.5mL的水,反应过程中用10%NaOH调节pH为7.8-8.0。
3)在37℃、400r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含30%(v/v)乙酸丁酯,100g/L底物,底物:加氢酶=133:1。
HPLC检测条件及转化率的计算方法同实施例一。
实验结果:SEQ ID NO:1的加氢酶24h产物转化率为33%;
SEQ ID NO:18的加氢酶24h产物转化率为97%。
由实验结果可知,当辅酶再生***为异丙醇和羰基还原酶时、有机溶剂为乙酸丁酯时,并且体系中采用前述实验结果中优选的有机溶剂和底物浓度,SEQ ID NO:18的加氢酶的产物转化率可达97%。
对比例二的实验方法同实施例十,区别在于,所加的酶为氨基酸序列如SEQ IDNO:4所示的酶,其24h产物转化率为96.1%。说明本申请采用的SEQ ID NO:18的加氢酶在相同的用量上可以实现相对于对比例的酶较高的产物转化率,提示本申请的加氢酶可以以较少的用量达到较高的产物转化率。
实施例十一、乙酸乙酯与葡萄糖体系
本实施例检测当所添加的辅酶再生***为葡萄糖和葡萄糖脱氢酶时,有机溶剂为乙酸乙酯时,具有SEQ ID No.1和SEQ ID NO:18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在500mL反应瓶加入100mL的乙酸乙酯,加入37.5g的底物,37℃搅拌溶解,获得混合液;
2)称取10g葡萄糖+105mL水完全溶解,加入反应瓶中,并调节pH为7.5;
3)加入0.188g加氢酶;马上加入葡萄糖脱氢酶1mL,加入50mg NADP+;
4)在37℃、400r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含40%(v/v)乙酸乙酯,150g/L底物,底物:加氢酶=200:1,反应过程控制PH在7.5。
HPLC检测条件及转化率的计算方法同实施例一。
实验结果:SEQ ID NO:1的加氢酶24h产物转化率为38%;
SEQ ID NO:18的加氢酶24h产物转化率为99.5%。
由实验结果可知,当辅酶再生***为葡萄糖和葡萄糖脱氢酶时、有机溶剂为乙酸乙酯时,并且体系中采用前述实验结果中优选的有机溶剂和底物浓度,SEQ ID NO:18的加氢酶的产物转化率可达99.5%,转化率最高,因此本实施例体系为最优体系。
如图4所示为当辅酶再生***为葡萄糖和葡萄糖脱氢酶时,含有乙酸乙酯和SEQID NO:18的加氢酶的反应体系在1h的HPLC图谱,如图5所示为含有乙酸乙酯和SEQ ID NO:18的加氢酶的反应体系在24h的HPLC图谱,由图4和图5比较可知,反应24小时后,底物的峰面积减小,甚至底物峰基本消失,依折麦布中间体峰面积显著增加,说明当辅酶再生***为葡萄糖和葡萄糖脱氢酶时,此体系中SEQ ID NO:18的加氢酶产物转化能力非常高。
对比例三的实验方法同实施例十一,区别在于,所加的酶为氨基酸序列如SEQ IDNO:4所示的酶,其24h产物转化率为97.8%。说明本申请采用的SEQ ID NO:18的加氢酶在相同的用量上可以实现相对于对比例的酶较高的产物转化率,提示本申请的加氢酶可以以较少的用量达到较高的产物转化率。
实施例十二、乙酸丁酯与葡萄糖体系
本实施例检测当所添加的辅酶再生***为葡萄糖和葡萄糖脱氢酶时,有机溶剂为乙酸丁酯时,具有SEQ ID No.1和SEQ ID NO:18序列的加氢酶的转化率,具体实验方法包括如下步骤:
1)在500mL反应瓶加入75mL的乙酸乙酯,加入25g的底物,37℃搅拌溶解,获得混合液;
2)称取10g葡萄糖+140mL水完全溶解,加入反应瓶中,并调节pH为7.5;
3)加入0.188g加氢酶;马上加入葡萄糖脱氢酶1mL,加入50mg NADP+;
4)在37℃、400r/min的振荡条件下持续反应24h,取样后经HPLC检测产物转化率。
反应体系中包含30%(v/v)乙酸丁酯,100g/L底物,底物:加氢酶=133:1,反应过程控制PH在7.5。
HPLC检测条件及转化率的计算方法同实施例一。
实验结果:SEQ ID NO:1的加氢酶24h产物转化率为36%;
SEQ ID NO:18的加氢酶24h产物转化率为98%。
由实验结果可知,当辅酶再生***为葡萄糖和葡萄糖脱氢酶时、有机溶剂为乙酸丁酯时,并且体系中采用前述实验结果中优选的有机溶剂和底物浓度,SEQ ID NO:18的加氢酶的产物转化率可达98%。
对比例四的实验方法同实施例十二,区别在于,所加的酶为氨基酸序列如SEQ IDNO:4所示的酶,其24h产物转化率为96.8%。说明本申请采用的SEQ ID NO:18的加氢酶在相同的用量上可以实现相对于对比例的酶较高的产物转化率,提示本申请的加氢酶可以以较少的用量达到较高的产物转化率。
本申请采用加氢酶以及相应的酶催化体系,能够高效催化底物(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮还原得到可作为依折麦布中间体的(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮,本申请的加氢酶在乙酸乙酯和乙酸丁酯中耐受性和稳定性好,因此所需要的酶量更少,产物转化率高。
以上对本申请实施例所提供的一种酶催化体系、加氢酶及制备方法和应用进行了详细介绍,本文中应用了具体个例对本申请的原理及实施方式进行了阐述,以上实施例的说明只是用于帮助理解本申请的方法及其核心思想°同时,对于本领域的技术人员,依据本申请的思想,在具体实施方式及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本申请的限制。
序列表
<110> 台州酶易生物技术有限公司
<120> 一种加氢酶、酶催化体系及制备方法和应用
<141> 2021-08-17
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 279
<212> PRT
<213> Galactomyces reessii
<400> 1
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 2
<211> 283
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ala Lys Asn Phe Ser Asn Val Glu Tyr Pro Ala Pro Pro Pro Ala
1 5 10 15
His Thr Lys Asn Glu Ser Leu Gln Val Leu Asp Leu Phe Lys Leu Asn
20 25 30
Gly Lys Val Ala Ser Ile Thr Gly Ser Ser Ser Gly Ile Gly Tyr Ala
35 40 45
Leu Ala Glu Ala Phe Ala Gln Val Gly Ala Asp Val Ala Ile Trp Tyr
50 55 60
Asn Ser His Asp Ala Thr Gly Lys Ala Glu Ala Leu Ala Lys Lys Tyr
65 70 75 80
Gly Val Lys Val Lys Ala Tyr Lys Ala Asn Val Ser Ser Ser Asp Ala
85 90 95
Val Lys Gln Thr Ile Glu Gln Gln Ile Lys Asp Phe Gly His Leu Asp
100 105 110
Ile Val Val Ala Asn Ala Gly Ile Pro Trp Thr Lys Gly Ala Tyr Ile
115 120 125
Asp Gln Asp Asp Asp Lys His Phe Asp Gln Val Val Asp Val Asp Leu
130 135 140
Lys Gly Val Gly Tyr Val Ala Lys His Ala Gly Arg His Phe Arg Glu
145 150 155 160
Arg Phe Glu Lys Glu Gly Lys Lys Gly Ala Leu Val Phe Thr Ala Ser
165 170 175
Met Ser Gly His Ile Val Asn Val Pro Gln Phe Gln Ala Thr Tyr Asn
180 185 190
Ala Ala Lys Ala Gly Val Arg His Phe Ala Lys Ser Leu Ala Val Glu
195 200 205
Phe Ala Pro Phe Ala Arg Val Asn Ser Val Ser Pro Gly Tyr Ile Asn
210 215 220
Thr Glu Ile Ser Asp Phe Val Pro Gln Glu Thr Gln Asn Lys Trp Trp
225 230 235 240
Ser Leu Val Pro Leu Gly Arg Gly Gly Glu Thr Ala Glu Leu Val Gly
245 250 255
Ala Tyr Leu Phe Leu Ala Ser Asp Ala Gly Ser Tyr Ala Thr Gly Thr
260 265 270
Asp Ile Ile Val Asp Gly Gly Tyr Thr Leu Pro
275 280
<210> 3
<211> 261
<212> PRT
<213> Bacillus subtilis
<400> 3
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Val Ile Thr Gly Ser Ser
1 5 10 15
Thr Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Ala Thr Ala Lys Ala
20 25 30
Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val
35 40 45
Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly
50 55 60
Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile
65 70 75 80
Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Ile Lys Val
100 105 110
Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Ser Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Thr
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 4
<211> 352
<212> PRT
<213> Thermoanaerobacter brockii
<400> 4
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe Gln Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Ser Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> 5
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 5
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 6
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 6
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Pro Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 7
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 7
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 8
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 8
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg Ile Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 9
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 9
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 10
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 10
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 11
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 11
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Val Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 12
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 12
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 13
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 13
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 14
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 14
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 15
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 15
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg Ile Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 16
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 16
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Pro Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Leu Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 17
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 17
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Arg Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Val Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Arg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 18
<211> 279
<212> PRT
<213> Artificial Sequence
<400> 18
Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro
1 5 10 15
Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Thr Ser Leu
20 25 30
Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr
35 40 45
Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp
50 55 60
Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys
65 70 75 80
Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Gln Ser Ala
85 90 95
Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile
100 105 110
Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu
115 120 125
Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp
130 135 140
Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys
145 150 155 160
Glu Arg Gly Ser Gly Ser Phe Ile Thr Thr Ser Ser Met Ser Gly His
165 170 175
Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala
180 185 190
Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe
195 200 205
Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser
210 215 220
Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro
225 230 235 240
Ile Gly Tyr Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr
245 250 255
Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val
260 265 270
Asp Gly Gly Tyr Thr Ala Pro
275
<210> 19
<211> 837
<212> DNA
<213> Galactomyces reessii
<400> 19
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 20
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 20
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 21
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 21
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctcct gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 22
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 22
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 23
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 23
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tattttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 24
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 24
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 25
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 25
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 26
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 26
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 27
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 27
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 28
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 28
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 29
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 29
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 30
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 30
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tattttcaaa 480
gaacgtggtt ctggttcttt catcgctacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
attggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 31
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 31
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctcct gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
ctgggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 32
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 32
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgttc tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccg actctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tctcgtatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggttgaa accaaatcta aatggtggtc tttcaccccg 720
attggtcgtg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
<210> 33
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 33
atgtctgttc agaacaccac ccacttcacc ggtctgggtc cgctgccgca gccggctccg 60
aaaccggctt ctaacgttct ggacctgact tctctgaaag gtaaagttgc ttctgttacc 120
ggttcttcta ccggtatcgg ttacgctgtt gctgaagctt tcgctcaggc tggtgctgac 180
gttgctctgt ggtacaactc tcacaacgct gaagctaaag ctaaagctct gtctgaaaaa 240
tacggtatca aagctaaagc ttacaaagtt ctggttaccc aatctgctgc tgttgaagct 300
gctatcaaag aacagatcga atacttcggt aaaatcgaca tcttcgttgc taacgctggt 360
gttccgtgga ccgctggtcc gctgatcgac accgaagacg acaaagaatg gaaaaaagtt 420
atcgacctgg acttcaccgg tgtttactac tgcgctaaat acatcggtcg tcacttcaaa 480
gaacgtggtt ctggttcttt catcactacc tcttctatgt ctggtcacat cgttaacttc 540
ccgcagctgc aggctgctta caacggtgct aaagctggtg ttcgtcactt ctgcacctct 600
ctggctgttg aatgggctgg tttcgctcgt gttaacaccg tttctccggg ttacatcgct 660
accgaaatct ctgctttcgt tccggacgaa accaaatcta aatggtggtc tttcaccccg 720
attggttatg aaggtgaagc tcaggaactg gttggtgctt acctgtacct ggcttctgac 780
gcttctacct acaccaccgg tgctgacatc cgtgttgacg gtggttacac cgctccg 837
Claims (9)
2.根据权利要求1所述的酶催化体系,其特征在于,所述底物的浓度为50g/L~250g/L;
以反应体系的总体积计,所述有机溶剂的体积百分比含量为20%~50%;
所述有机溶剂选自二甲基亚砜、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、N,N-二甲基甲酰、二氯甲烷或甲苯中的至少一种;
所述辅酶选自烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸磷酸中的任意一种;
所述辅酶的浓度为0.2~2g/L;
所述辅酶再生剂包括:异丙醇和羰基还原酶,或者,葡萄糖和葡萄糖脱氢酶。
3.根据权利要求2所述的酶催化体系,其特征在于,所述羰基还原酶的氨基酸序列如SEQ ID NO:2所示;
所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示;
所述葡萄糖的浓度为30~50g/L。
4.一种加氢酶,其特征在于,所述加氢酶的氨基酸序列如SEQ IDNO:18所示。
6.一种核酸分子,其特征在于,所述核酸分子包括用于编码如权利要求4所述的加氢酶的核苷酸序列。
7.根据权利要求6所述的核酸分子,其特征在于,所述核苷酸序列为如SEQ ID NO:33所示的核苷酸序列。
8.一种(4S)-3-[(5s)-5-(4-氟苯基)5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的制备方法,其特征在于,包括:
将底物(4S)-3-[5-(4-氟苯基)-1,5-二氧代戊基]-4-苯基-2-恶唑烷酮与如权利要求1至3中任一项所述的酶催化体系混合以产生酶催化还原反应,得到(4S)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮。
9.根据权利要求8所述的制备方法,其特征在于,所述底物与所述酶催化体系中的加氢酶的质量比为(120~210):1;
所述底物的浓度为50g/L~250g/L;
以反应体系的总体积计,所述酶催化体系中的有机溶剂的体积百分比含量为20%~50%;
所述酶催化还原反应的pH为6.0~9.0;
所述酶催化还原反应的温度为25℃~65℃。
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