CN106191151A - 一种生物转化联产d‑赖氨酸和5‑氨基戊酸的方法 - Google Patents
一种生物转化联产d‑赖氨酸和5‑氨基戊酸的方法 Download PDFInfo
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Abstract
一种生物转化联产D‑赖氨酸和5‑氨基戊酸的方法将L‑赖氨酸溶解,加入赖氨酸消旋酶全细胞,反应结束后去除赖氨酸消旋酶全细胞,得到D‑赖氨酸和L‑赖氨酸的混合水溶液,再加入表达L‑赖氨酸单加氧酶和5‑氨基酰胺水解酶的基因工程菌全细胞拆分DL‑赖氨酸,得到D‑赖氨酸和5‑氨基戊酸。本发明原料易得,生产成本低工艺简单,反应条件温和,D‑赖氨酸的高学纯度高达98%以上;而且L‑赖氨酸也转化为更有价值的5‑氨基戊酸,原料得到充分利用。
Description
技术领域
本发明涉及生物催化技术领域,特别涉及赖氨酸消旋酶快速消旋L-赖氨酸生成DL-赖氨酸,在L-赖氨酸单加氧酶和5-氨基酰胺水解酶催化作用下联合生产D-赖氨酸和5-氨基戊酸。
背景技术
D-赖氨酸为非蛋白质氨基酸,它具有有效的抗真菌性和抗菌性,是新药开发的重要药物中间体,在医药上具有重要的作用。D-Lys是合成促黄体生成素释放激素类似物的前体,也可以用于合成促性腺激素释放激素高活性类似物,口服和静脉给药均可降低肿瘤治疗中放射性肽的肾摄取。D-Lys比L-赖氨酸L-Lys更适合在癌症治疗中使用。D-赖氨酸的多聚体,能刺激人软骨细胞和脑星形胶质细胞的增值,也是一种良好的药物载体。
5-氨基戊酸是一种重要的C5平台化合物,在医药方面有着广泛的应用,例如可以作为合成5-羟基戊酸、戊二酸、1,5-戊二醇及其衍生物、戊二酸酐的重要前体,同时,它也可以用于合成尼龙-5,5(尸胺和戊二胺的共聚物)和尼龙-5(5-氨基戊酸均聚物)所以,5-氨基戊酸在纺织工业以及医药合成领域有着广泛的应用。
目前,国内外制备D-赖氨酸的方法主要是化学法,化学法是通过赖氨酸与手性酸生成非对映体盐,利用非对映体盐的溶解度差异而分离。通过化学方法来消旋制备DL-Lys的操作流程较为复杂,且成本较高,通常需要强酸、强碱或者高温的环境,然而,化学拆分DL-Lys的效率并不高,且产品的光学纯度较低。生物酶法转化因具有反应条件温和及催化效率高的优点,已成为拆分或合成手性化合物的重要途径。
发明内容
本发明所要解决的技术问题是提供一种绿色环保拆分DL-Lys的方法并且能够联产D-赖氨酸和5-氨基戊酸,与化学生产法相比,其反应简单,成本低廉,更适用于工业化生产。
一种生物法联产D-赖氨酸和1,5-戊二胺的方法,其生产方法包括以下步骤:
步骤一、表达赖氨酸消旋酶的基因工程菌和表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的构建:
根据已报导的Proteus mirabilis BCRC10725来源赖氨酸消旋酶lyr的氨基酸序列(NCBI登录号:WP_004243720.1)进行密码子优化后,全基因合成该序列,两端设计酶切位点NcoI和XhoI,亚克隆到载体pET28a上,获得重组质粒pET28a-lyr。将构建好的重组质粒pET28a-lyr用氯化钙法转化进入大肠杆菌表达宿主BL21(DE3),得到赖氨酸消旋酶表达菌种BL21(DE3)/pET28a-lyr。
L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的构建方法如下(同申请号201610500252X的专利中关于该菌株的构建方法):将davA(Gene ID: 1044092)片段与表达载体pET-22b相连,将davB (Gene ID: 1044093)片段与表达载体pRSF-dute相连,分别得到重组质粒pET-22b-DavA和pRSF-davB,将重组质粒pET-22b-DavA和pRSF-davB导入E.coliBL21的感受态细胞中,得到过表达了davBA的重组菌BL-22A-RB;另外将davA及davB片段与表达载体pET-22b相连,得到重组质粒pET22b-DavBA,将重组质粒pET22b-DavBA导入E.coliBL21的感受态细胞中,得到过表达了davBA的重组菌E.coliBL-22AB;将davA片段与表达载体pRSF-dute相连,得到重组质粒pRSF-DavA,将重组质粒pRSF-DavA导入重组菌E.coliBL-22AB的感受态细胞中,得到重组菌株E.coliBL-22AB-RA;最后,将davB片段与表达载体pACYC-dute相连,得到重组质粒pACYC-davB,将重组质粒pACYC-davB导入重组菌BL-22A-RB的感受态细胞中,得到重组菌株E.coli BL-22A-RB-YB。
步骤二、表达赖氨酸消旋酶的基因工程菌和表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的培养:
挑取表达赖氨酸消旋酶的基因工程菌和表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的单菌落,分别接种于LB培(酵母粉5g/L,蛋白胨10g/L,NaCl为5g/L)养基,置于37℃过夜培养;将所培养的赖氨酸消旋酶基因工程菌转接于LB培养基,接种量为LB培养基体积的1%,然后在37℃培养2-3小时,加入IPTG至终浓度1mmol/L,表达赖氨酸消旋酶的基因工程菌30℃继续培养12小时;表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌20℃继续培养12小时,揺瓶发酵结束后,分别离心收集表达赖氨酸消旋酶的基因工程菌和表达赖氨酸脱羧酶的基因工程菌,备用。
步骤三、以L-赖氨酸为原料,转化生产D-赖氨酸和5-氨基戊酸:
向磷酸钾缓冲溶液中加入赖氨酸消旋酶全细胞和L-赖氨酸盐酸盐,37 ℃、200 r /min振荡反应1h;然后12000rpm,离心5min,去除赖氨酸消旋酶细胞;向体系中补加表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌,并调节PH至7.0,继续37℃震荡反应15-40h,至高效液相色谱法检测L-赖氨酸完全被消耗,停止反应。
所述磷酸钾缓冲溶液的浓度为0.2 mol/L,pH为7.0。
加入的赖氨酸消旋酶全细胞OD600=10,表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌OD600=40。
有益效果是:
1、本发明针对D-赖氨酸和5-氨基戊酸的市场需求,克服了D-赖氨酸在生产中利用化学消旋的弊端,采用纯生物法消旋,在生物法拆分DL-赖氨酸过程中,将L-赖氨酸转化为附加价值更高的5-氨基戊酸,从而实现纯生物法联产D-赖氨酸和的5-氨基戊酸目的。
2、本发明前后两步催化反应都采用全细胞催化,反应结束后,反应体系中只剩下D-赖氨酸和5-氨基戊酸,L-赖氨酸被全部转化为5-氨基戊酸,使得D-赖氨酸的光学纯度能够达到98%以上。本方法的催化活性高,原料便宜,总的生产成本低,且转化率高,适用于大规模工业化生产。
附图说明
图1为本发明的联产D-赖氨酸和5-氨基戊酸的反应原理示意图。
具体实施方式
为更好的理解本发明的内容,下面结合具体实施例做进一步说明。
实施例1:D-赖氨酸的高效液相色谱检测方法
D-赖氨酸的检测条件为:Agilent 1200 高效液相色谱;色谱柱为Chirex ChiralColumns;流动相:1mM CuSO4。5H2O溶于水:异丙醇=95:5;流速:0.8ml/min;温度:25℃;波长:254nm。
实施例2:赖氨酸消旋酶、L-赖氨酸单加氧酶和5-氨基酰胺水解酶的诱导表达
表达赖氨酸消旋酶的基因工程菌的构建:根据已报导的Proteus mirabilisBCRC10725来源赖氨酸消旋酶lyr的氨基酸序列(NCBI登录号:WP_004243720.1)进行密码子优化后,全基因合成该序列,两端设计酶切位点NcoI和XhoI,亚克隆到载体pET28a上,获得重组质粒pET28a-lyr。将构建好的重组质粒pET28a-lyr用氯化钙法转化进入大肠杆菌表达宿主BL21(DE3),得到赖氨酸消旋酶表达菌种BL21(DE3)/pET28a-lyr。
表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌构建方法如下:(1)davB由金唯智合成,酶切位点为NdeI和XhoI,将davB片段经限制性内切酶NdeI和XhoI处理回收后,与经相同限制性内切酶处理的载体pACYCDuet-1相连,使用T4DNA连接酶25℃连接30min;
(2)将(1)中的连接液转入大肠杆菌Trans1-T1(由全式金生物技术有限公司提供)的感受态细胞中,涂布在带有氯霉素抗性的LB平板上,37℃过夜培养;
(3)挑取(2)中平板上生长的单菌落,转接到含有氯霉素抗性的LB培养基里,然后提取质粒,再利用限制性内切酶NdeI和XhoI进行酶切验证,最后得到了载体pACYCDuet-1-davB;
(4)将载体pACYCDuet-1-davB转入到E.coliBL-22A-RB的感受态细胞里,涂布在带有氨苄青霉素抗性、卡那霉素抗性和氯霉素抗性的LB平板,37℃过夜培养;
(5)挑取(4)中平板上生长的单菌落,转接到含有氨苄青霉素抗性、卡那霉素抗性和氯霉素抗性的LB培养基里培养8-10h后,用30%的甘油保存在-80℃冰箱里,得到了E.coliBL-22A-RB-YB菌株。
挑取表达赖氨酸消旋酶的基因工程菌和表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的单菌落,分别接种于LB培养基(酵母粉5g/L,蛋白胨10g/L,NaCl为5g/L),置于37℃过夜培养;将所培养的赖氨酸消旋酶基因工程菌转接于LB培养基,接种量为LB培养基体积的1%,然后在37℃培养2-3小时,加入IPTG至终浓度1mmol/L,表达赖氨酸消旋酶的基因工程菌30℃继续培养12小时,表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌20℃继续培养12小时,揺瓶发酵结束后,分别离心收集表达赖氨酸消旋酶的基因工程菌和表达赖氨酸脱羧酶的基因工程菌,备用。
实施例3:L-赖氨酸单加氧酶和5-氨基酰胺水解酶拆分DL-赖氨酸反应
在10mL 0.2 mol/L 磷酸钾缓冲溶液(PH 7.0)中,赖氨酸消旋酶全细胞OD600=10,1.2gL-赖氨酸盐酸盐,37 ℃、200 r /min振荡反应1h,反应结束后得到L-赖氨酸和D-赖氨酸的混旋物,然后12000rpm,离心5min,去除赖氨酸消旋酶细胞。
去除赖氨酸消旋酶后,向反应体系中补加表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌细胞OD600=20,调节PH至7.0,继续37℃震荡反应15h,至高效液相色谱法检测L-赖氨酸完全被消耗,停止反应。
结论:最终得到的D-赖氨酸的收率为48%,对映体过量值ee>98%,5-氨基戊酸的转化率为97%。
实施例4:不同表面活性剂对L-赖氨酸单加氧酶和5-氨基酰胺水解酶拆分DL-赖氨酸的影响
选取浓度为0.1g/L-0.7g/L等7个浓度梯度的Triton X-100。
在 0.2 mol/L 磷酸钾缓冲溶液(PH 7.0)中,赖氨酸消旋酶全细胞OD600=10,1.2gL-赖氨酸盐酸盐,37 ℃、200 r /min振荡反应1h,反应结束后得到L-赖氨酸和D-赖氨酸的混旋物,然后12000rpm,离心5min,去除赖氨酸消旋酶细胞。
去除赖氨酸消旋酶后,向反应体系中补加表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌细胞OD600=20,调节PH至7.0,继续37℃震荡反应40h,至高效液相色谱法检测L-赖氨酸完全被消耗,停止反应。
结论:当Triton X-100的浓度为0.5g/L时,反应速率有显著提高。
Claims (8)
1.一种生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:其生产方法包括以下步骤:
步骤一,构建表达赖氨酸消旋酶的基因工程菌、表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌;
步骤二,培养步骤一构建的基因工程菌;
步骤三,以L-赖氨酸为原料,利用步骤二培养的基因工程菌转化生产D-赖氨酸和5-氨基戊酸。
2.根据权利要求1所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:步骤一所述的构建方法具体为:根据已报导的Proteus mirabilis BCRC10725来源赖氨酸消旋酶lyr的氨基酸序列(NCBI登录号:WP_004243720.1)进行密码子优化后,全基因合成该序列,两端设计酶切位点NcoI和XhoI,亚克隆到载体pET28a上,获得重组质粒pET28a-lyr;将构建好的重组质粒pET28a-lyr用氯化钙法转化进入大肠杆菌表达宿主BL21(DE3),得到赖氨酸消旋酶表达菌种BL21(DE3)/pET28a-lyr。
3.根据权利要求1所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:步骤一所述表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的构建方法是:将davA片段与表达载体pET-22b相连,将davB片段与表达载体pRSF-dute相连,分别得到重组质粒pET-22b-DavA和pRSF-davB,将重组质粒pET-22b-DavA和pRSF-davB导入E.coli BL21的感受态细胞中,得到过表达了davBA的重组菌BL-22A-RB;另外将davA及davB片段与表达载体pET-22b相连,得到重组质粒pET22b-DavBA,将重组质粒pET22b-DavBA导入E.coli BL21的感受态细胞中,得到过表达了davBA的重组菌E.coliBL-22AB;将davA片段与表达载体pRSF-dute相连,得到重组质粒pRSF-DavA,将重组质粒pRSF-DavA导入重组菌E.coliBL-22AB的感受态细胞中,得到重组菌株E.coliBL-22AB-RA;最后,将davB片段与表达载体pACYC-dute相连,得到重组质粒pACYC-davB,将重组质粒pACYC-davB导入重组菌BL-22A-RB的感受态细胞中,得到重组菌株E.coli BL-22A-RB-YB。
4.根据权利要求1所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:步骤二所述的培养方法如下:挑取表达赖氨酸消旋酶的基因工程菌和表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌的单菌落,分别接种于LB培养基,置于37℃过夜培养;将所培养的赖氨酸消旋酶基因工程菌转接于LB培养基,接种量为LB培养基体积的1%,然后在37℃培养2-3小时,加入IPTG诱导,表达赖氨酸消旋酶的基因工程菌30℃继续培养12小时;表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌20℃继续培养12小时,揺瓶发酵结束后,分别离心收集表达赖氨酸消旋酶的基因工程菌和表达赖氨酸脱羧酶的基因工程菌。
5.根据权利要求1所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:步骤三所述的转化生产D-赖氨酸和5-氨基戊酸具体方法为:向磷酸钾缓冲溶液中加入赖氨酸消旋酶全细胞和L-赖氨酸盐酸盐,37 ℃、200 r /min振荡反应1h;然后12000rpm,离心5min,去除赖氨酸消旋酶细胞;向体系中补加表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌,并调节PH至7.0,继续37℃震荡反应15-40h,至高效液相色谱法检测L-赖氨酸完全被消耗,停止反应。
6.根据权利要求5所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:所述磷酸钾缓冲溶液的浓度为0.2 mol/L,pH为7.0。
7.根据权利要求5所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:加入的赖氨酸消旋酶全细胞OD600=10。
8.根据权利要求5所述的生物转化联产D-赖氨酸和5-氨基戊酸的方法,其特征在于:向体系中补加表达L-赖氨酸单加氧酶和5-氨基酰胺水解酶的基因工程菌OD600=40。
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