CN116042599A - 一种用于生产5-羟基色氨酸的体外多酶体系 - Google Patents
一种用于生产5-羟基色氨酸的体外多酶体系 Download PDFInfo
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- CN116042599A CN116042599A CN202211163898.5A CN202211163898A CN116042599A CN 116042599 A CN116042599 A CN 116042599A CN 202211163898 A CN202211163898 A CN 202211163898A CN 116042599 A CN116042599 A CN 116042599A
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- Prior art keywords
- dihydrobiopterin
- reductase
- enzyme
- pterin
- dehydratase
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Abstract
本发明提供了一种用于生产5‑羟基色氨酸的体外(固定化)多酶体系,多酶体系包括:苯丙氨酸羟化酶和构成辅因子循环系统的四氢生物蝶呤的二氢生物蝶呤还原酶和喋呤‑4α‑甲醇胺脱水酶。本发明多酶体系能够进行5‑羟基色氨酸的体外高效酶法合成,在合成的同时实现了辅因子的体外循环,显著降低了生产成本,提高了催化效率;以大孔载体作为酶载体的共固定化多酶催化体系实现了酶的循环利用,共固定化多酶体系兼具很高的催化效率和稳定性,可在连续多批次反应后依然保持较高的酶转化率,酶活性无明显衰减。
Description
技术领域:
本发明属于5-羟基色氨酸生产领域,特别涉及一种用于生产5-羟基色氨酸的体外多酶催化体系,以及将该多酶体系固定化的方法。
背景技术:
5-羟基色氨酸是一种氨基酸类似物。它在人体内可作为血清素的前体物质继而作为褪黑素的前体物质。5-羟基色氨酸具有一定的生理作用,根据临床研究发现,服用5-羟基色氨酸能明显的改善忧郁症患者的情绪。另外,5-羟基色氨酸也能促进睡眠品质,改善轻度失眠。另外也有临床研究证据显示,5-羟基色氨酸具有止痛作用,能够改善部分肌纤维痛症病患的不适,对于慢性压力性头痛的改善也有作用。有临床研究发现,5-羟基色氨酸能够运用于肥胖者食欲控制的辅助上。5-羟基色氨酸在某些国家被归为药物成分,在美国、加拿大和英国,5-羟基色氨酸被视作膳食补充剂销售。
利用生物合成法生产5-羟基色氨酸,是利用微生物所产的特异性酶,专一性地催化底物合成5-羟基色氨酸。苯丙氨酸羟化酶(Phenylalanine hydroxylase,PAH)可以催化芳香族氨基酸的苯环上添加一个羟基,是生产羟基化芳香族氨基酸的良好催化剂,可以色氨酸为底物生产附加产值高的5-羟基色氨酸。随着5-羟基色氨酸市场需求量的日益增加,高效生物催化剂的开发对其工业应用至关重要。然而,苯丙氨酸羟化酶催化色氨酸转化为5-羟基色氨酸的反应过程中,需要四氢生物蝶呤(tetrahydrobiopterin,BH4)作为辅因子。四氢生物蝶呤的价格高昂,因此需要利用辅因子循环系统循环利用四氢生物蝶呤。辅因子循环系统由喋呤-4α-甲醇胺脱水酶(PCD)和二氢生物蝶呤还原酶(DHPR)组成。在反应的过程中,由于酶无法重复利用从而使得工艺成本较高,且水溶性的酶不易从反应系统中分离出来,不利于控制和自动化生产。因此,开发将苯丙氨酸羟化酶及其辅因子循环系统的固定化技术,增加酶的稳定性、降低生产成本的同时使酶可以反复多次使用,在降低成本的基础上可进行5-羟基色氨酸的连续生产,对5-羟基色氨酸的产业发展具有十分重要的意义。
发明内容:
本发明的目的在于提供一种用于生产5-羟基色氨酸的体外多酶(催化合成)体系和固定化多酶体系;固定化多酶体系性能稳定,制备过程中不使用有毒害物质,可以实现5-羟基色氨酸的连续转化,有效提高了酶的利用率。
本发明技术方案具体如下:
一种用于生产5-羟基色氨酸的体外多酶体系,其特征在于包括:苯丙氨酸羟化酶和构成辅因子循环系统的四氢生物蝶呤的二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶。
进一步地,上述体外多酶体系中,所述苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的质量比为:1~3:1~2:1~2。
进一步地,上述体外多酶体系为固定在大孔载体上的固定化多酶体系。
进一步地,上述固定化多酶体系通过如下步骤制备获得:
1)将大孔载体置于磷酸钾缓冲液中,于25℃、150rpm振荡1h,振荡处理重复2~3次,之后用超纯水清洗大孔载体1~2次;
2)将苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶加入磷酸钠缓冲液中,之后再加入适量经步骤1处理后的大孔载体,于25℃、150rpm振荡反应12h;
3)反应产物用pH7.5的HEPES-NaOH缓冲液清洗至少3次后得到固定化多酶体系。
进一步地,所述步骤2)中,苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶是以浓度为5~8mg/ml的酶液的形式逐滴加入2mol/L,pH6.4的磷酸钠缓冲液中,苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的加入量按质量比为:1~3:1~2:1~2,滴加过程中对反应容器辅以振荡;大孔载体与酶的加量比例为:每20~35mg酶对应加入1g大孔载体。
优选地,所述大孔载体为大孔吸附树脂、环氧树脂、氨基树脂、硅藻土或大孔冻凝胶中的任意一种。
进一步地,本发明提供一种利用上述固定化多酶体系制备5-羟基色氨酸的方法,具体包括如下步骤:
(1)向浓度为100mM的色氨酸溶液中加入微量的四氢生物蝶呤;
(2)继续向色氨酸溶液中加入固定化多酶体系,之后于30℃下反应1.5~2.5h;该步骤中,固定化多酶体系的加入量参照如下比例:每50mL色氨酸溶液中加入1g固定化多酶体系;
(3)将反应后的溶液过滤去除固定化多酶体系,即获得含有5-羟基色氨酸的粗溶液产品。
进一步地,所述制备5-羟基色氨酸的方法还包括步骤(4):将所述步骤(3)中过滤得到的固定化多酶体系用pH7.5的HEPES-NaOH缓冲液洗涤,洗涤后的固定化多酶体系继续放置在pH7.5的HEPES-NaOH缓冲液中,于4℃保存备用。
进一步地,本发明提供一种用于共表达上述体外或固定化多酶体系中的喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的工程菌株,该工程菌株通过如下步骤构建获得:
1)分别以喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的核苷酸序列为模板设计引物并进行PCR扩增反应;
2)反应结束后,取少量反应产物进行琼脂糖凝胶电泳,观察结果并纯化回收喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的目的基因产物;
3)采用NcoⅠ和BamHⅠ两种限制性内切酶分别对纯化回收的二氢生物蝶呤还原酶的目的基因产物和pETDuet-1载体进行双酶切;
4)对双酶切产物进行琼脂糖凝胶电泳的鉴定、纯化回收并确定二氢生物蝶呤还原酶的目的基因产物与pETDuet-1载体的相对浓度;
5)将酶切后的二氢生物蝶呤还原酶的目的基因产物与pETDuet-1载体进行连接;连接前调整两者的浓度比例,使pETDuet-1载体与二氢生物蝶呤还原酶的目的基因产物在连接反应体系中的摩尔比为1:1~3:1;
6)将连接产物转化大肠杆菌,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为表达二氢生物蝶呤还原酶的工程菌株;
7)对表达二氢生物蝶呤还原酶的工程菌株进行扩大培养后收集菌细胞,从中提取可表达二氢生物蝶呤还原酶基因的pETDuet-1载体,采用NdeⅠ和XhoⅠ两种限制性内切酶分别对载体和喋呤-4α-甲醇胺脱水酶的目的基因产物进行双酶切;
8)将步骤7)双酶切后的目的基因产物和载体进行连接;将连接产物转化大肠杆菌,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为共表达二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶的工程菌株,其菌细胞内包含可同时表达二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶基因的载体。
进一步地,本发明提供用于在PCR反应中扩增上述体外或固定化多酶体系中苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的基因序列的引物组合,其特征在于包括:
用于扩增苯丙氨酸羟化酶基因序列的上游引物PAH-NdeI和下游引物PAH-EcoRI,核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;
用于扩增喋呤-4α-甲醇胺脱水酶基因序列的上游引物PCD-NdeⅠ和下游引物PCD-XhoⅠ,核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示;
用于扩增二氢生物蝶呤还原酶基因序列的上游引物DHPR-NcoⅠ和下游引物DHPR-BamHⅠ,核苷酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。
本发明提供的体外多酶体系能够进行5-羟基色氨酸的体外高效酶法合成,在合成的同时实现了辅因子的体外循环,显著降低了生产成本,提高了催化效率;以大孔载体作为酶载体的共固定化多酶催化体系实现了酶的循环利用,共固定化多酶体系兼具很高的催化效率和稳定性,可在连续多批次反应后依然保持较高的酶转化率,酶活性无明显衰减。
附图说明:
图1本发明固定化多酶体系的酶转化率随反应批次的变化趋势。
图2色氨酸和5-羟基色氨酸的HPLC鉴定图。
具体实施方式:
下面通过具体的实施方案叙述本发明方法。除非特别说明,本发明具体实施方式中所用的技术手段均为本领域技术人员所公知的方法。另外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
实施例1
苯丙氨酸羟化酶、二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶(纯酶液)的制备。
1.表达苯丙氨酸羟化酶的工程菌株的制备
(1)以苯丙氨酸羟化酶的核苷酸序列(genebank序列号:AAD37774.1)为模板设计上游引物PAH-NdeI(SEQ ID NO:1所示)和下游引物PAH-EcoRI(SEQ ID NO:2所示),进行PCR扩增反应,。PCR反应体系为50μL体系,组分如表1所示。
表1苯丙氨酸羟化酶基因的PCR扩增反应体系
PCR反应条件及过程具体如下:
反应结束后,取2μL扩增产物进行0.8%琼脂糖凝胶电泳,观察结果并纯化回收约1000bp处的PCR产物(目的基因产物)。DNA的纯化回收采用OMEGA公司的小量DNA纯化回收试剂盒(货号:D2000-02),按照试剂盒操作说明进行纯化回收。
(2)采用NdeI和EcoRI两种限制性内切酶分别对纯化回收的目的基因产物和pET28a载体进行双酶切,将酶切后的目的基因产物和载体进行连接。
其中,目的基因产物的酶切反应体系为50μL,体系中各组分如表2所示:
表2目的基因产物的酶切反应体系
载体的酶切反应体系为50μL,体系中各组分如表3所示:
表3 pET28a载体的酶切反应体系
双酶切反应体系于37℃反应2h,琼脂糖凝胶电泳鉴定酶切产物。酶切产物的纯化回收采用OMEGA公司的胶回收纯化试剂盒(货号:D2500-02),参照试剂盒说明书进行酶切产物的纯化回收。
(3)通过琼脂糖电泳确定酶切后的目的基因产物与载体的相对浓度,调整两者的倍数使载体与目的基因产物在连接反应体系中的摩尔比为1:1~3:1;之后采用DNA连接试剂盒进行目的基因产物和载体的连接,连接反应体系如表4所示:
表4载体与目的基因产物的连接反应体系
连接体系于16℃反应6h或过夜,得到连接产物。
(4)将连接产物转化大肠杆菌BL21,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为表达苯丙氨酸羟化酶的工程菌株。具体操作如下:
将连接产物加入到大肠杆菌BL21感受态细胞中,混匀后冰浴30min,42℃热激90s,立即冰浴5min,再加入LB复苏液,于37℃、220r/min振荡复苏培养40~60min,之后4000r/min离心5min;离心得到的菌体用LB培养基悬浮,混合涂布于含有卡那霉素的LB平板上,待菌液充分吸收后,于37℃倒置培养12~16h,直至出现单菌落;挑取单菌落进行测序验证,验证无误的阳性转化子即为表达苯丙氨酸羟化酶的工程菌株。
2.共表达喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的工程菌株的制备
(1)分别以喋呤-4α-甲醇胺脱水酶的核苷酸序列(genebank序列号:QDW01644.1)为模板设计上游引物PCD-NdeⅠ(SEQ ID NO:3所示)和下游引物PCD-XhoⅠ(SEQ ID NO:4所示);以二氢生物蝶呤还原酶的核苷酸序列(genebank序列号:URT17505.1)为模板设计上游引物DHPR-NcoⅠ(SEQ ID NO:5所示)和下游引物DHPR-BamHⅠ(SEQ ID NO:6所示);之后进行PCR扩增反应。PCR反应体系为50μL体系。体系中各组分如表5所示。
表5 PCD和DHPR基因的PCR扩增反应体系
PCR反应条件及过程与前述苯丙氨酸羟化酶基因的PCR扩增反应相同。
反应结束后,取2μL反应产物进行0.8%琼脂糖凝胶电泳,观察结果并纯化回收约672bp处和363bp处的PCR扩增产物(即目的基因DHPR和PCD产物)。目的基因产物的DNA的纯化回收过程与前述苯丙氨酸羟化酶目的基因产物的纯化回收相同。
(2)采用NcoⅠ和BamHⅠ两种限制性内切酶分别对纯化回收的二氢生物蝶呤还原酶的目的基因产物和pETDuet-1载体进行双酶切。其中,目的基因产物的酶切反应体系为50μL,体系中各组分如表6所示:
表6 DHPR目的基因产物的酶切反应体系
载体的酶切反应体系为50μL,体系中各组分如表7所示:
表7 pETDuet-1载体的酶切反应体系
双酶切反应体系于37℃反应2h,琼脂糖凝胶电泳鉴定酶切产物。酶切产物采用OMEGA公司的胶回收纯化试剂盒(货号:D2500-02)进行纯化回收。
(3)通过琼脂糖电泳确定酶切后的目的基因产物与载体的相对浓度,调整两者的倍数使载体与目的基因产物在连接反应体系中的摩尔比为1:1~3:1。采用DNA连接试剂盒进行目的基因产物和载体的连接,连接反应体系如表8所示:
表8载体与目的基因产物的连接反应体系
连接体系于16℃反应6h或过夜,得到连接产物。
(4)将连接产物转化大肠杆菌BL21,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为表达二氢生物蝶呤还原酶的工程菌株。具体操作与前述苯丙氨酸羟化酶的工程菌株构建过程(转化、筛选以及阳性克隆的验证)完全相同。
(5)从步骤(4)获得的工程菌株(可事先进行扩大培养)中提取可表达二氢生物蝶呤还原酶基因的pETDuet-1载体,采用NdeⅠ和XhoⅠ两种限制性内切酶分别对载体和喋呤-4α-甲醇胺脱水酶的目的基因产物进行双酶切,将酶切后的目的基因产物和载体进行连接。双酶切反应、酶切产物的纯化回收以及目的基因和载体的连接与前述苯丙氨酸羟化酶表达载体的构建过程中的对应步骤操作完全相同。
(6)将连接产物转化大肠杆菌BL21,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为共表达二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶的工程菌株。具体操作与前述苯丙氨酸羟化酶的工程菌株构建过程(转化、筛选以及阳性克隆验证)完全相同。
2.纯酶液的制备
(1)将苯丙氨酸羟化酶的工程菌株和共表达喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的工程菌株接种于LB培养基中,于37℃、220rpm振荡发酵培养2.5~3h,当OD值达到0.6~0.8后,加入终浓度为0.1mM的IPTG,16℃、120rpm诱导表达16~18h。
(2)于4℃,8000rpm离心收集表达后的菌体,用Lysis Buffer(20mM Tirs-HCl、20mM咪唑、500mM NaCl,pH7.4)悬浮菌体细胞,混匀后加入200μL溶菌酶、0.3μL 0.1%的TritonX-100以及240μL苯甲基磺酰氟(PMSF),混匀后倒入烧杯中冰浴20min。
(3)冰浴后的细胞悬液用超声波细胞破碎仪进行破碎处理,处理时间为20min(超声2.5s,间隔3.0s)。
(4)将细胞破碎液于4℃、12000r/min离心30min,将离心后的上清液加载到经Lysis Buffer平衡后的镍树脂亲和层析柱(Ni-NTA superflow)中结合1h,使混合的样品流经纯化柱,用Wash Buffer(20mM Tirs-HCl、20mM咪唑、500mM NaCl,pH7.4)洗去杂质蛋白,再用Elution Buffer(20mM Tirs-HCl、500mM咪唑、500mM NaCl,pH7.4)溶出目的蛋白,最后用pH7.5的HEPES-NaOH缓冲液将Elution Buffer透析去除,得到置换后的苯丙氨酸羟化酶的纯酶液,以及二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶的混合酶液。
酶液的酶浓度由BCA蛋白浓度测定试剂盒测定(北京索莱宝,货号:PC0020)。
实施例2
固定化多酶体系的制备。
(1)将大孔载体置于磷酸钾缓冲液中,于25℃、150rpm振荡1h,振荡处理重复2~3次,之后用超纯水清洗树脂1~2次。所述磷酸钾缓冲液由0.1mol/L的K2HPO4和0.1mol/L的KH2PO4按体积比15~16:1混合配制而成。
(2)将苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶加入磷酸钠缓冲液中,之后再加入适量经步骤1处理后的大孔载体,于25℃、150rpm振荡反应12h。
本步骤中苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶是以酶液(酶液浓度为5~8mg/ml)的形式逐滴加入磷酸钠缓冲液(2mol/L,pH6.4),苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的加入量按质量比为:1~3:1~2:1~2,滴加过程中可对反应容器辅以振荡;大孔载体与酶的加量比例为:每20~35mg酶对应加入1g大孔载体。
(3)反应产物用pH7.5的HEPES-NaOH缓冲液清洗3次,洗去未固定到载体上的游离酶,得到固定化多酶体系。
上述固定化多酶体系制备过程中用到的大孔载体可以是大孔吸附树脂、环氧树脂、氨基树脂、硅藻土、大孔冻凝胶中的任意一种,本实施例选用大孔吸附树脂。
实施例3
利用实施例2制备的固定化多酶体系制备5-羟基色氨酸。具体包括如下步骤:
(1)向浓度为100mM的色氨酸溶液中加入微量的四氢生物蝶呤;
(2)继续向色氨酸溶液中加入固定化多酶体系,之后于30℃下反应1.5~2.5h;该步骤中,固定化多酶体系的加入量参照如下比例:每50mL色氨酸溶液中加入1g固定化多酶体系。
(3)将反应后的溶液过滤去除固定化多酶体系,即获得含有5-羟基色氨酸的粗溶液产品。
进一步地,上述固定化多酶体系制备5-羟基色氨酸的方法还包括步骤(4):将所述步骤(3)中过滤得到的固定化多酶体系用pH7.5的HEPES-NaOH缓冲液洗涤,洗涤后的固定化多酶体系继续放置在同一缓冲液中,于4℃保存备用。
重复上述步骤可完成多批次的催化反应。本实施例总共进行了15个批次的催化反应。其中,前9个批次的转化率始终保持在80%以上,表明酶活性稳定,无明显衰减(图1所示)。
以上各实施例中用到的HEPES-NaOH的配置方法:将4.766g HEPES、8.775g NaCl、0.771g DTT加入800ml ddH2O中,待溶质充分溶解后用NaOH调pH值至7.5,然后将溶液定容至1L,通过抽滤使溶液过0.22μm水系过滤膜,滤液即为pH7.5的HEPES-NaOH。
实施例4
5-羟基色氨酸产物的HPLC鉴定:将实施例3催化反应完成后的反应液稀释至合适浓度后加入到液相小瓶中进行定量分析,测定条件如下:
色谱仪:Agilent1260;
检测器:Agilent 1260Infinity II可变波长检测器;
进样:Agilent自动进样器;进样量20μL;
流动相:70%甲醇;30%0.05%三氯乙酸;流速0.6ml/min;
检测结果如图2所示,底物色氨酸和产物5-羟基色氨酸在柱子中均得到很好的保留和分离。
Claims (10)
1.一种用于生产5-羟基色氨酸的体外多酶体系,其特征在于包括:苯丙氨酸羟化酶和构成辅因子循环系统的四氢生物蝶呤的二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶。
2.根据权利要求1所述的体外多酶体系,其特征在于:所述苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的质量比为:1~3:1~2:1~2。
3.根据权利要求1或2所述的体外多酶体系,其特征在于:所述体外多酶体系为固定在大孔载体上的固定化多酶体系。
4.根据权利要求3所述的固定化多酶体系,其特征在于通过如下步骤制备获得:
1)将大孔载体置于磷酸钾缓冲液中,于25℃、150rpm振荡1h,振荡处理重复2~3次,之后用超纯水清洗大孔载体1~2次;
2)将苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶加入磷酸钠缓冲液中,之后再加入适量经步骤1)处理后的大孔载体,于25℃、150rpm振荡反应12h;
3)反应产物用pH7.5的HEPES-NaOH缓冲液清洗至少3次后得到固定化多酶体系。
5.根据权利要求4所述的固定化多酶体系,其特征在于:所述步骤2)中,苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶是以浓度为5~8mg/ml的酶液的形式逐滴加入2mol/L,pH6.4的磷酸钠缓冲液中,苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的加入量按质量比为:1~3:1~2:1~2,滴加过程中对反应容器辅以振荡;大孔载体与酶的加量比例为:每20~35mg酶对应加入1g大孔载体。
6.根据权利要求5所述的固定化多酶体系,其特征在于:所述大孔载体为大孔吸附树脂、环氧树脂、氨基树脂、硅藻土或大孔冻凝胶中的任意一种。
7.一种利用权利要求3~6中任意一项所述的固定化多酶体系制备5-羟基色氨酸的方法,其特征在于包括如下步骤:
1)向浓度为100mM的色氨酸溶液中加入微量的四氢生物蝶呤;
2)继续向色氨酸溶液中加入固定化多酶体系,之后于30℃下反应1.5~2.5h;该步骤中,固定化多酶体系的加入量参照如下比例:每50mL色氨酸溶液中加入1g固定化多酶体系;
3)将反应后的溶液过滤去除固定化多酶体系,即获得含有5-羟基色氨酸的粗溶液产品。
8.根据权利要求7所述的制备5-羟基色氨酸的方法,其特征在于还包括步骤4):将所述步骤3)中过滤得到的固定化多酶体系用pH7.5的HEPES-NaOH缓冲液洗涤,洗涤后的固定化多酶体系放置在pH7.5的HEPES-NaOH缓冲液中,于4℃保存备用。
9.一种用于共表达权利要求1~6中任意一项所述多酶体系中的喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的工程菌株,该工程菌株通过如下步骤构建获得:
1)分别以喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的核苷酸序列为模板设计引物并进行PCR扩增反应;
2)反应结束后,取少量反应产物进行琼脂糖凝胶电泳,观察结果并纯化回收喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的目的基因产物;
3)采用NcoⅠ和BamHⅠ两种限制性内切酶分别对纯化回收的二氢生物蝶呤还原酶的目的基因产物和pETDuet-1载体进行双酶切;
4)对双酶切产物进行琼脂糖凝胶电泳的鉴定、纯化回收并确定二氢生物蝶呤还原酶的目的基因产物与pETDuet-1载体的相对浓度;
5)将酶切后的二氢生物蝶呤还原酶的目的基因产物与pETDuet-1载体进行连接;连接前调整两者的浓度比例,使pETDuet-1载体与二氢生物蝶呤还原酶的目的基因产物在连接反应体系中的摩尔比为1:1~3:1;
6)将连接产物转化大肠杆菌,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为表达二氢生物蝶呤还原酶的工程菌株;
7)对表达二氢生物蝶呤还原酶的工程菌株进行扩大培养后收集菌细胞,从中提取可表达二氢生物蝶呤还原酶基因的pETDuet-1载体,采用NdeⅠ和XhoⅠ两种限制性内切酶分别对载体和喋呤-4α-甲醇胺脱水酶的目的基因产物进行双酶切;
8)将步骤7)双酶切后的目的基因产物和载体进行连接;将连接产物转化大肠杆菌,转化后的宿主菌通过抗生素平板进行筛选,对筛选获得的阳性克隆进行测序验证,验证无误的阳性克隆即为共表达二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶的工程菌株,其菌细胞内包含可同时表达二氢生物蝶呤还原酶和喋呤-4α-甲醇胺脱水酶基因的载体。
10.用于在PCR反应中扩增权利要求1~6中任意一项所述的多酶体系中苯丙氨酸羟化酶、喋呤-4α-甲醇胺脱水酶和二氢生物蝶呤还原酶的基因序列的引物组合,其特征在于包括:
用于扩增苯丙氨酸羟化酶基因序列的上游引物PAH-NdeI和下游引物PAH-EcoRI,核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;
用于扩增喋呤-4α-甲醇胺脱水酶基因序列的上游引物PCD-NdeⅠ和下游引物PCD-XhoⅠ,核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示;
用于扩增二氢生物蝶呤还原酶基因序列的上游引物DHPR-NcoⅠ和下游引物DHPR-BamHⅠ,核苷酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。
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Citations (4)
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CN102286563A (zh) * | 2011-07-19 | 2011-12-21 | 南开大学 | 一种采用固定化酶制备l-鸟氨酸的方法 |
CN106414755A (zh) * | 2014-05-16 | 2017-02-15 | 乔治亚大学研究基金公司 | 用于产生5‑羟基色氨酸的微生物途径 |
CN109777813A (zh) * | 2019-01-25 | 2019-05-21 | 浙江工业大学 | 一种转氨酶-plp共固定化酶及其制备与应用 |
CN114685629A (zh) * | 2020-12-28 | 2022-07-01 | 湖南引航生物科技有限公司 | 一种提高5-羟基色氨酸微生物转化浓度的方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102286563A (zh) * | 2011-07-19 | 2011-12-21 | 南开大学 | 一种采用固定化酶制备l-鸟氨酸的方法 |
CN106414755A (zh) * | 2014-05-16 | 2017-02-15 | 乔治亚大学研究基金公司 | 用于产生5‑羟基色氨酸的微生物途径 |
CN109777813A (zh) * | 2019-01-25 | 2019-05-21 | 浙江工业大学 | 一种转氨酶-plp共固定化酶及其制备与应用 |
CN114685629A (zh) * | 2020-12-28 | 2022-07-01 | 湖南引航生物科技有限公司 | 一种提高5-羟基色氨酸微生物转化浓度的方法 |
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