CN108384818A - 一种酶法转化制备d-组氨酸的方法 - Google Patents
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Abstract
本发明属于生物技术领域,具体涉及一种酶法转化制备D‑组氨酸的方法。该方法以L‑组氨酸为底物,加入具有氨基酸消旋酶活性的菌体细胞或粗酶液,经消旋反应得到DL‑组氨酸,去除氨基酸消旋酶后,再加入具有组氨酸脱羧酶活性的菌体细胞或粗酶液拆分DL‑组氨酸,得到D‑组氨酸和组胺,最终利用等电点结晶和离子交换树脂相结合的方法分离得到高品质的D‑组氨酸。该方法以L‑组氨酸为底物,双酶级联催化制备D‑组氨酸,同时副产高附加值的组胺,具有反应条件温和,绿色环保,操作简便,生产成本低,适合大规模工业化生产等优点。
Description
一、技术领域
本发明属于生物技术领域,具体涉及一种酶法转化制备D-组氨酸的方法。
二、背景技术
近些年来,D-氨基酸广泛用于医药化工领域,是合成新药如抗菌和抗病毒药物、食品添加剂如甜味剂等的重要中间体。其中D-组氨酸参与机体蛋白质的合成,是生物体内***形成的必要条件之一。此外,还参与PhScN、Facitidin、环(D-苯丙-D-组)等多肽的合成。D-组氨酸很难从自然资源中大量获取,目前文献报道的方法主要是化学法。即以L-组氨酸为原料,以酒石酸为拆分剂生成非对映体盐,利用溶解度差异达到拆分目的。该方法具有产率高,光学纯度好等优点,但拆分步骤繁琐,需要用到大量的有机溶剂和拆分剂,不利于大规模生产,也会对环境造成污染。
组胺是一种重要的生物活性物质,也是一种重要的医药中间体。例如,磷酸组胺可用于胃液分泌功能的检查,麻风病的辅助诊断;二盐酸组胺用于持续缓解和防止急性髓细胞样白血病成人患者首次缓解治疗后的复发。目前组胺的制备方法主要以L-组氨酸为原料,以富含电子的酮类为催化剂,在环己醇溶剂中高温脱羧。该方法反应条件苛刻,纯化难度大,产率低,污染大。
因此,研究既绿色环保又廉价高效的制备D-组氨酸和组胺的方法具有重要意义。
三、发明内容
本发明的目的在于避免现有技术制备D-组氨酸的不足,提供一种绿色环保、廉价高效的生产D-组氨酸的方法。
本发明可以通过以下技术方案来达到:
(1)将恶臭假单胞菌来源具有氨基酸消旋酶活性的菌株、产气肠杆菌来源具有组氨酸脱羧酶活性的菌株分别在培养基中培养,产生高活力的氨基酸消旋酶、组氨酸脱羧酶;
(2)以30~200g/L的L-组氨酸为底物,加入具有氨基酸消旋酶活性的菌体细胞或粗酶液,0.05~0.5g/L的磷酸吡哆醛,20~45℃,用氢氧化钠水溶液控制pH 7~10,酶法消旋,待反应液旋光为零,去除氨基酸消旋酶;反应液中再加入具有组氨酸脱羧酶活性的菌体细胞或粗酶液,盐酸控制pH 5~7,25~45℃酶促转化,得到D-组氨酸和组胺;
(3)利用等电点结晶和离子交换树脂相结合的方法分离转化产物,得到高纯度的D-组氨酸,同时得到高附加值的组胺。
上述步骤(1)所述培养基碳源采用葡萄糖、麦芽糖、蔗糖和/或乳糖,培养基中总碳源质量浓度为1~20g/L;氮源采用牛肉膏、酵母膏、玉米浆、蛋白胨和/或豆饼水解液,培养基中总氮源质量浓度为1~20g/L;
上述步骤(2)所述去除氨基酸消旋酶的方法有离心去除氨基酸消旋酶菌体细胞,和/或升温灭活氨基酸消旋酶。
本发明采用的工艺路线如下:
本发明与现有技术相比具有如下优点:
(1)相较于现有技术中需要使用大量有机溶剂而言,本发明反应过程以水为溶剂,符合绿色生产的要求,环境友好;
(2)本发明利用双酶级联反应催化制备D-组氨酸,具有催化效率高、立体选择性强、反应条件温和等特点,有效缩短反应周期;
(3)本发明以L-组氨酸为底物,先消旋再拆分制备D-组氨酸,工艺简单,适合工业化生产;
(4)本发明生产D-组氨酸的同时还获得高附加值的组胺,显著降低了综合生产成本,具有重要的应用价值和现实意义。
四、具体实施方式
以下实施例仅用于对本发明进行具体说明,但本发明的保护范围并非仅仅局限于以下实施例。
实施案例一
1.称取30g L-组氨酸溶解在1000mL水中,调pH 7.0,加入氨基酸消旋酶菌体细胞10g,磷酸吡哆醛0.05g,20℃搅拌反应4h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 5.0,再加入15g组氨酸脱羧酶全细胞,25℃搅拌反应,用2mol/L盐酸控制pH 5.0,反应5h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为14.9g/L,组胺浓度为10.6g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至150mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸9.65g,收率64.7%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐8.95g,收率51%。
实施案例二
1.称取50g L-组氨酸溶解在1000mL水中,调pH 8.0,加入氨基酸消旋酶菌体细胞15g,磷酸吡哆醛0.1g,30℃搅拌反应6h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 6.0,再加入15g组氨酸脱羧酶全细胞,35℃搅拌反应,用2mol/L盐酸控制pH 6.0,反应6h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为24.8g/L,组胺浓度为17.5g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至250mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸16g,收率64.5%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐15g,收率52%。
实施案例三
1.称取100g L-组氨酸溶解在1000mL水中,调pH 9.0,加入氨基酸消旋酶菌体细胞20g,磷酸吡哆醛0.3g,40℃搅拌反应10h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 7.0,再加入20g组氨酸脱羧酶全细胞,40℃搅拌反应,用2mol/L盐酸控制pH 7.0,反应12h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为49.5g/L,组胺浓度为35.1g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至400mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸35g,收率71%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐30.2g,收率52%。
实施案例四
1.称取100g L-组氨酸溶解在1000mL水中,调pH 10.0,加入氨基酸消旋酶菌体细胞20g,磷酸吡哆醛0.3g,45℃搅拌反应15h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 7.0,再加入20g组氨酸脱羧酶全细胞,45℃搅拌反应,用2mol/L盐酸控制pH 7.0,反应10h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为49.6g/L,组胺浓度为35.5g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至400mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸34g,收率68.5%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐29.9g,收率51%。
实施案例五
1.称取150g L-组氨酸溶解在1000mL水中,调pH 9.0,加入氨基酸消旋酶菌体细胞30g,磷酸吡哆醛0.5g,35℃搅拌反应20h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 7.0,再加入30g组氨酸脱羧酶全细胞,35℃搅拌反应,用2mol/L盐酸控制pH 7.0,反应20h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为74.2g/L,组胺浓度为52.7g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至600mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸51.7g,收率69.7%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐44.5g,收率51%。
实施案例六
1.称取200g L-组氨酸溶解在1000mL水中,调pH 10.0,加入氨基酸消旋酶菌体细胞40g,磷酸吡哆醛0.5g,35℃搅拌反应24h,检测反应液旋光为零,得到DL-组氨酸的混旋物;
2.6000r/min离心15min,去除氨基酸消旋酶全细胞,上清液加热至80℃维持10min,灭活残留的氨基酸消旋酶,冷却至室温后用6mol/L盐酸调pH 7.0,再加入40g组氨酸脱羧酶全细胞,35℃搅拌反应,用2mol/L盐酸控制pH 7.0,反应24h结束,手性HPLC检测反应体系中无L-组氨酸残留,D-组氨酸浓度为98.7g/L,组胺浓度为70.7g/L;
3.6000r/min离心15min,去除组氨酸脱羧酶全细胞,活性炭脱色,抽滤,滤液真空浓缩至800mL,冷却至室温结晶,析出白色固体,抽滤,无水乙醇洗涤,烘干,得D-组氨酸72g,收率73%,母液用离子交换树脂分离,低温真空干燥得到白色粉末组胺二盐酸盐58.6g,收率50%。
Claims (1)
1.一种酶法转化制备D-组氨酸的方法,其特征是由以下步骤构成:
(1)将恶臭假单胞菌来源具有氨基酸消旋酶活性的菌株、产气肠杆菌来源具有组氨酸脱羧酶活性的菌株分别在培养基中培养,产生高活力的氨基酸消旋酶、组氨酸脱羧酶;培养基碳源采用葡萄糖、麦芽糖、蔗糖和/或乳糖,培养基中总碳源质量浓度为1~20g/L;氮源采用牛肉膏、酵母膏、玉米浆、蛋白胨和/或豆饼水解液,培养基中总氮源质量浓度为1~20g/L;
(2)以30~200g/L的L-组氨酸为底物,加入具有氨基酸消旋酶活性的菌体细胞或粗酶液,0.05~0.5g/L的磷酸吡哆醛,20~45℃,用氢氧化钠水溶液控制pH 7~10,酶法消旋,待反应液旋光为零,去除氨基酸消旋酶;反应液中再加入具有组氨酸脱羧酶活性的菌体细胞或粗酶液,盐酸控制pH5~7,25~45℃酶促转化,得到D-组氨酸和组胺;
(3)利用等电点结晶和离子交换树脂相结合的方法分离转化产物,得到高纯度的D-组氨酸,同时得到高附加值的组胺。
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