CN101775415B - 全细胞生物催化合成磷酸胆碱的方法 - Google Patents
全细胞生物催化合成磷酸胆碱的方法 Download PDFInfo
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Abstract
本发明公开了一种全细胞生物催化合成磷酸胆碱的方法,以氯化胆碱和磷酸根离子为底物,以葡萄糖为能量供体,加入小分子化学效应物质,利用有透性的微生物全细胞生物催化合成磷酸胆碱。本发明采用全细胞生物催化合成磷酸胆碱,克服了化学合成法成本高、转化率低、溶剂毒性大等缺陷。全细胞催化稳定性好,采用小分子化学效应物质调控代谢流量从而提高能量原位再生和偶联效率的方法,产品浓度大幅度提高,转化率也有提高。
Description
技术领域
本发明属于生物催化技术领域,具体涉及全细胞生物催化合成磷酸胆碱的方法。
背景技术
磷酸胆碱是真核细胞卵磷脂生物合成的重要中间体,在动物及酵母菌中存在的胆碱激酶的催化下,由胆碱及ATP缩合形成。它是磷酸单酯中与CRP(C反应蛋白)结合力最强的,在钙离子存在下可以增强机体对细菌、真菌的防御力(European Journal of InternetMedicine,2002,13:412-422)。磷酸胆碱可以用于制备光电化学电池产生光电压。辉光放电修饰的磷酸胆碱双层分子生物化学特性与人的血红细胞抗血栓表面相似,极大地抑制了血小板的表面连接(Journal of American Chemical Society,2005,127:14473-14478)。磷酸胆碱钠盐和钙盐有阻止各种已知的膜解构物分解作用保护血小板、红细胞和溶酶体膜的作用。磷酸胆碱钙盐和钠盐能够增强肝素和血清β-脂蛋白间的相互作用。它的镁盐和钙盐可用于肝胆损伤的治疗(Clinica Terapeutica,1984,110(3):251-257)。它是工业制备CDP-胆碱、L-α-甘油磷酸胆碱等药物的原料。
磷酸胆碱已经有许多种化学合成方法:卤化胆碱的磷酸化包括使用三氯氧磷、五氧化二磷、五氧化二磷和无水磷酸、乙基偏磷酸盐、二苯氯磷酸等有机磷磷酸化试剂和通过三氯氧磷、乙基偏磷酸盐磷酸化氯乙醇制备磷酸氯乙醇再与三甲胺结合形成磷酸胆碱。但是,这些方法存在分离困难,得率低的缺陷。1944年Baer和McArthur报道了利用二苯氯磷酸作为磷酸化试剂合成磷酸胆碱尽管得率较高,分离产物纯度高,但是需要使用大量的金盐和铂盐催化二苯基磷酸胆碱的裂解,成本昂贵(The Journal of BiologicalChemistry,1944,154:451-460)。1947年Baer设计出两步法制备磷酸胆碱:首先由氯化胆碱与二苯氯磷酸在吡啶中反应生成二苯磷酸胆碱和吡啶盐酸盐,然后再与氢氧化钡反应直接生成磷酸胆碱钡盐和苯酚。该方法得率达到60-63%,用酒精沉淀所得粗品纯度达到96-97%(Journal of American Chemical Society,1947,69:1253-1254)。由于这些方法需要使用极度污染环境的有机磷酸化试剂和对人体有害的有机溶剂作反应介质,无论从环境的角度或者成本的角度,都是不经济的。目前,已经有很多原来用化学法合成的生物活性物质改由生物法合成,例如:cAMP,各种氨基酸、核苷等。因此,使用生物合成法取代化学合成法制备磷酸胆碱是技术发展的趋势。生物法的优势在于采用无机磷酸盐代替有机磷试剂和以水为反应介质极大地减少污染,而且反应条件温和。目前国内还没有生物催化合成磷酸胆碱的相关报道。
发明内容
本发明所要解决的技术问题是提供一种低成本、有利于环境的磷酸胆碱绿色制备方法。
为解决上述技术问题,本发明的思路是:
胆碱磷酸化过程中需要大量ATP。由于高效的ATP再生系统使得ATP浓度或者是ATP/ADP维持在较高的水平,这样强烈地抑制了磷酸果糖激酶的活性。磷酸果糖激酶是ATP再生体系的关键酶,它的活性直接影响到ATP再生体系的效率。另外体系中氧化还原平衡(NADH/NAD)也制约着ATP再生系统的效率。ATP既是胆碱激酶的底物之一,也是胆碱激酶的激活因子。同时,在微生物全细胞催化中,ATP可以通过腺苷酸环化酶的作用下合成cAMP,而后者可以激活蛋白激酶A和蛋白激酶C。胆碱激酶在被蛋白激酶A和蛋白激酶C磷酸化后,活性可以分别提高到1.9倍。文献报道,在啤酒酵母中,葡萄糖信号能激活胞内cAMP水平和蛋白激酶A,这个信号途径被称为cAMP-protein kinase(PKA)途径(Journal of Bioscience and Bioengineering,2007,104(4):245-250)。综上所述,利用酿酒酵母进行全细胞催化合成磷酸胆碱的关键是提高ATP再生的效率。
本发明的关键在于:
1、全细胞催化。
由于细胞具有维持其生命活动的完整的多酶系统,各种酶又保持着原有活细胞所处的状态和特定位置,因此用微生物细胞直接作为酶源进行酶催化反应,能够迅速有效地完成多步酶催化反应。在本发明中利用静息的微生物细胞,采用全细胞催化技术,同时添加促渗透剂对酵母进行细胞渗透性增强处理,从而既保持了细胞酶系的完整性,又有利于底物的利用,并使得产物能够从胞内释放出来,从而可以降低后续分离的难度。
本发明是建立在全细胞催化的基础上的,其特点在于克服了化学合成法成本较高,条件苛刻,转化率低,使用大量易挥发的有毒试剂的缺陷。制备过程更加环保,能耗更低。
2、利用微生物细胞合成磷酸胆碱的自身代谢途径。
在微生物细胞中生物合成磷酸胆碱需要EMP途径酶系(己糖激酶、磷酸葡萄糖异构酶、磷酸果糖激酶、醛缩酶、磷酸丙糖异构酶、3-磷酸甘油醛脱氢酶、磷酸甘油酸激酶、磷酸甘油酸变位酶、烯醇化酶、丙酮酸激酶、丙酮酸脱羧酶、乙醇脱氢酶)和胆碱激酶进行催化反应,由于细胞具有维持其生命活动的完整的多酶系统,各种酶又保持着原有生活细胞所处的状态和特定位置,反应所需要的能量和辅酶因子不需要外界供给,直接由细胞产生,因此能够迅速有效地完成多步酶催化反应,在大规模生产方面,有转化效率高、成本低,以及污染小的优点。
3、小分子小效应物的加入。
通过锌离子和半胱氨酸的加入,激活了胆碱激酶,使得磷酸胆碱能够快速有效地积累。
本发明的具体技术方案如下:
一种全细胞生物催化合成磷酸胆碱的方法,以氯化胆碱和磷酸根离子为底物,以葡萄糖为能量供体,加入小分子化学效应物质,利用有透性的微生物细胞生物催化合成磷酸胆碱。
其中,底物中,氯化胆碱的起始反应浓度为10~500mmol/L,优选20~300mmol/L,磷酸根离子可选自正磷酸、焦磷酸、三聚磷酸等多磷酸,磷酸二氢钾、磷酸二氢钠、磷酸氢二钠等无机磷酸盐,磷酸根离子的起始反应浓度为0.1~2mol/L,优选0.2~0.5mol/L。
其中,葡萄糖的起始反应浓度为0.1~1mol/L,优选0.2~0.5mol/L。
其中,所述的小分子化学效应物质为锌离子和半胱氨酸的组合物,其中,锌离子可选自硫酸锌、硝酸锌、氯化锌等无机盐,锌离子的起始反应浓度为1~200mmol/L,优选2~50mmol/L,半胱氨酸起始反应浓度为1~50mmol/L,优选2~30mmol/L。
其中,所述的微生物细胞是指能够利用氯化胆碱合成磷酸胆碱的微生物,包括气杆菌属、埃希氏菌属、赛氏杆菌属、微球菌属的细菌;酵母属、假丝酵母属、毕赤酵母属、球拟酵母属、德巴利酵母属、接合酵母属、克鲁维酵母属、汉逊酵母属和酒香酵母属的酵母;优选产氨短杆菌、枯草芽孢杆菌、酿酒酵母或白球拟酵母。微生物细胞的使用量为按湿菌体100~800g/L,优选200~600g/L。
其中,所述的有透性的微生物细胞是指通过化学、物理或生物方法处理过的细胞膜的通透性改变过的微生物细胞,具体方法包括表面活性剂法、有机溶剂法、冻融法、超声波处理法、风干法、冷冻干燥法或溶菌酶法。
表面活性剂法中使用的表面活性剂为非离子型表面活性剂聚环氧乙烷胺或曲拉通X-100、阳离子型表面活性剂十六烷基三甲胺·溴化物,或者阴离子表面活性剂月桂酰·肌氨酸盐,使用量为0.1~50g/L,优选1~20g/L,即表面活性剂法处理微生物细胞时,将表面活性剂直接加入反应液,对于总体积为1L的反应液,加入0.1~50g,优选加入1~20g。
有机溶剂法中使用的有机溶剂为二甲苯、甲苯、脂肪醇、丙酮或乙酸乙酯,使用量为0.1~50mL/L,优选1~20mL/L,即有机溶剂法处理生产菌株时,将有机溶剂直接加入反应液,对于总体积为1L的反应液,加入0.1~50mL,优选加入1~20mL。
上述微生物的利用形式可以是微生物细胞的干燥物、经过发酵培养分离离心得到的细胞、细胞的冻干物、市售酵母粉、风干酵母或废酵母泥。
上述磷酸胆碱的生成反应在水溶液中进行,pH4.0~10.0,20℃~40℃条件下反应4~30小时,优选在pH6.0~10.0,22℃~35℃的条件下反应。
本发明的制备胞二磷胆碱的主要代谢途径如图1所示。
本发明的有益效果为:
本发明采用全细胞生物催化合成磷酸胆碱,克服了化学合成法成本较高,条件苛刻,转化率低,使用大量易挥发的有毒试剂的缺陷。本发明成本低廉,操作简单,易于实现工业化,制备过程更加环保,能耗更低,为磷酸胆碱的绿色制备提供了一种新的思路和方法。
附图说明
图1是本发明全细胞生物催化合成磷酸胆碱的代谢途径。其中,ATP为三磷酸腺苷,ADP为二磷酸腺苷,NADH为还原型烟酰胺腺嘌呤二核苷酸,NAD为氧化性烟酰胺腺嘌呤二核苷酸,PPi为焦磷酸,Pi为磷酸根离子。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:
酵母培养基:葡萄糖40g/L,尿素2.0g/L,磷酸二氢钾1.5g/L,七水合硫酸锌4.0×10-3g/L,七水合硫酸亚铁3.0×10-3g/L,四水合氯化锰0.3×10-3g/L,无水氯化钙1.0×10-3g/L,生物素0.05×10-3g/L。
酵母接种量10%,于30℃下120rpm摇床培养24小时,离心4000rpm,20分钟。取酵母泥,-7℃保藏备用。
实施例2:
在容量15L的反应槽中调制由氯化胆碱100mmol、葡萄糖1mol、硫酸锌10mmol、实施例1方法培养的酿酒酵母泥经反复冻融5次1000g、半胱氨酸10mol、磷酸二氢钠1mol和水组成的反应液10L,用氢氧化钠调pH为5.0,温度为20℃,反应4小时后结束反应,用高氯酸沉淀,用HPLC对产物进行定量分析,磷酸胆碱含量为5.6mmol/L,得率为56%。
实施例3:
在容量15L的反应槽中调制由氯化胆碱5000mmol、葡萄糖10mol、氯化锌2mol、实施例1培养的产氨短杆菌湿菌体经超声波处理30min后8000g、半胱氨酸0.5mol、磷酸二氢钾20mol和水组成的反应液10L,用氢氧化钠调pH为10.0,温度为40℃,反应30小时后结束反应,用高氯酸沉淀,用HPLC对产物进行定量分析,磷酸胆碱含量为251.3mmol/L,得率为50.3%。
实施例4:
在容量15L的反应槽中调制由氯化胆碱200mmol、葡萄糖8mol、氯化锌200mmol、白球拟酵母湿菌体2400g、半胱氨酸50mmol、磷酸二氢钠2mol、月桂酰·肌氨酸盐50g和水组成的反应液10L,用氢氧化钠调pH为8.0,温度为30℃,反应24小时后结束反应,用高氯酸沉淀,用HPLC对产物进行定量分析,磷酸胆碱含量为13.2mmol/L,得率为66.0%。
实施例5:
在容量15L的反应槽中调制由氯化胆碱300mmol、葡萄糖5mol、硝酸锌20mmol、枯草芽孢杆菌湿菌体2500g、半胱氨酸100mmol、磷酸二氢钾3mol、十六烷基三甲胺·溴化铵10g、丙酮10mL和水组成的反应液10L,用氢氧化钠调pH为6.0,温度为35℃,反应14小时后结束反应,用高氯酸沉淀,用HPLC对产物进行定量分析,磷酸胆碱含量为22.5mmol/L,得率为75%。
Claims (10)
1.一种全细胞生物催化合成磷酸胆碱的方法,其特征在于以氯化胆碱和磷酸根离子为底物,以葡萄糖为能量供体,加入小分子化学效应物质,利用有透性的微生物细胞生物催化合成磷酸胆碱;
其中,所述的有透性的微生物细胞是指通过化学、物理或生物方法处理过的细胞膜的通透性改变过的微生物细胞;
所述的微生物细胞是指能够利用氯化胆碱合成磷酸胆碱的微生物;
所述的小分子化学效应物质为锌离子和半胱氨酸的组合物。
2.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征在于底物中,氯化胆碱的起始反应浓度为10~500mmol/L,磷酸根离子的起始反应浓度为0.1~2mol/L。
3.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征在于葡萄糖的起始反应浓度为0.1~1mol/L。
4.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征在于锌离子的起始反应浓度为1~200mmol/L,半胱氨酸起始反应浓度为1~50mmol/L。
5.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征在于微生物细胞的使用量为按湿菌体100~800g/L。
6.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征是所述的化学、物理或生物方法是表面活性剂法、有机溶剂法、冻融法、超声波处理法、风干法或冷冻干燥法。
7.根据权利要求6所述的全细胞生物催化合成磷酸胆碱的方法,其特征是表面活性剂法中使用的表面活性剂为非离子型表面活性剂、阳离子型表面活性剂或者阴离子表面活性剂,使用量为0.1~50g/L。
8.根据权利要求6所述的全细胞生物催化合成磷酸胆碱的方法,其特征是有机溶剂法中使用的有机溶剂为二甲苯、甲苯、脂肪醇、丙酮或乙酸乙酯,使用量为0.1~50mL/L。
9.根据权利要求1至7中任意一项所述的全细胞生物催化合成磷酸胆碱的方法,其特征在于磷酸胆碱的生成反应在水溶液中进行,pH4.0~10.0,20℃~40℃条件下反应4~30小时。
10.根据权利要求1所述的全细胞生物催化合成磷酸胆碱的方法,其特征是,所述的微生物细胞是气杆菌属、埃希氏菌属、赛氏杆菌属或微球菌属的细菌;酵母属、假丝酵母属、毕赤酵母属、球拟酵母属、德巴利酵母属、接合酵母属、克鲁维酵母属、汉逊酵母属或酒香酵母属的酵母。
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