固定化肌酸激酶柱连续合成磷酸肌酸工艺  

Process of Continuously Synthesizing Creatine Phosphate by Immobilized Creatine Kinase Column

作　　者：杨志雄[1] 周小华[1] 赵春花[1] 蒋小琴[1] 王江[1] 

机构地区：[1]重庆大学化学化工学院,重庆400044

出　　处：《药物生物技术》2015年第3期213-217,共5页Pharmaceutical Biotechnology

摘　　要：为了得到高纯度磷酸肌酸产品和优化磷酸肌酸生产工艺,利用接枝赖氨酸壳聚糖微球固定化肌酸激酶与离子交换树脂耦合技术,研究连续合成并分离磷酸肌酸的工艺条件。首先制备固定化肌酸激酶,结果表明：肌酸激酶固定化的条件及其固定化酶的酶学性质为：p H 5.0,戊二醛-氨基酸载体-酶为1.36∶1∶1.6E-4,吸附温度为25℃,交联时间为2 h,所得固定化酶活力为21.6 U/mg,Km=37.18 mmol/L,活力回收为68.55%;其次考察p H,温度,底物浓度、体积流量等因素的影响。结果表明：在p H 6.0、温度为40℃、ATP浓度为10 mmol/L、肌酸浓度为60 mmol/L、体积流量为4.0 m L/min的条件下,利用固定化肌酸激酶连续合成磷酸肌酸8 h,以717阴离子交换树脂交换合成物,以0.05~2 mol/L的Na Cl溶液洗脱,分别以0.05 mol/L Na Cl溶液洗脱ADP,以2 mol/L的Na Cl溶液洗脱磷酸肌酸钠,获得纯度达到90.2%结晶体,产物经红外光谱、核磁共振氢谱分析确证为磷酸肌酸。利用此耦合技术能连续合成分离得到高纯度的磷酸肌酸,提供一种连续生产磷酸肌酸的优化工艺,具有广阔的发展前景。To obtain a high purity product and to optimize the process conditions of phosphocreatine,making the most of the coupling technique of immobilized creatine kinase of chitosan microspheres modified by L-lysine and ion exchange resin. The first step was preparation of immobilized creatine kinase. The results indicated that optimum conditions for preparation and characterization of immobilized creatine kinase were as follows： the p H was 5. 0,Glutaraldehyde; Amino carrier; Enzyme was 1. 36 ∶ 1 ∶ 1. 6E- 4; adsorption temperature was 25 °C,crosslinking time was 2 h,the immobilized enzyme vitality was 21. 6 U / mg,the Kmwas 37. 18 mmol / L,and the vitality recovery was 68. 55%. And then the effects of the process conditions of synthesizing and separating creatine phosphate continuously were studied,and the effects of temperature,p H,concentration of substrate,and volume flow were investigated.The results indicated that optimum conditions for synthesis and separation of creatine phosphate through coupling techuique were as follows： the p H was 6. 0,the temperature was 40 ° C,the volume flow was 4. 0 m L / min,and the concentration of ATP and creatine were 10 mmol / L and 60 mmol / L respectively. It takes 8 hours to synthesize creatine phosphate by immobilized creatine kinase.Exchanging the compound product with 717 anion exchange resin and eluting it by 0. 05 ~ 2 mol / L Na Cl solution. ADP could be eluted with 0. 05 mol / L Na Cl and phosphate sodium could also be eluted with 2 mol / L Na Cl. Finally the crystal product of creatine phosphate was obtained,and the purity could reach 90. 2%,and the crystals were identified as creatine phosphate by infrared spectrum and HNMR. In conclusion,a high purity phosphocreatine product could be obtained by the coupling technique of immobilized creatine kinase of chitosan microspheres modified by L-lysine and ion exchange resin,also providing a kind of optimized process about producing phosphocreatine continuously. It has a broad prospect on synthesis of phosphocreatine.

关 键 词：磷酸肌酸 固定化 肌酸激酶 离子交换树脂 耦合技术 合成 

分 类 号：R284[医药卫生—中药学] TQ460.6[医药卫生—中医学]