文档介绍：Immobilized Creatine Kinase Continuous
Preparative Separation of Creatine
Phosphate

A Thesis Submitted to ChongqingUniversity
in Partial Fulfillment of the Requirement for the
Master’sDegree of Engineering
By
Yu Su

Supervised by Prof. Xiaohua Zhou
Specialty:Pharmaceutical Chemistry

College of Chemistry and Chemical Engineering of
Chongqing University, Chongqing, China
October, 2012
重庆大学硕士学位论文中文摘要

摘要

磷酸肌酸是医药上大量使用的原料药,用量不断增加,需求潜力巨大。磷酸
肌酸既可用化学法合成,也可用酶催化合成。化学法合成磷酸肌酸由于条件苛刻,
副反应多、分离纯化困难、产品纯度低、环境污染严重,不符合节能减排、可持
续发展的国策;因而,亟需研发一种既绿色、又具有竞争优势的制备磷酸肌酸的
方法。
以赖氨酸修饰的交联壳聚糖微球作为载体,以戊二醛作为交联剂,采用交换-
交联的方法固定化肌酸激酶,制备固定化肌酸激酶的最佳工艺条件为: pH 为 ,
戊二醛﹕载体氨基:酶(摩尔比)=﹕1﹕-04 下,常温交换平衡,室温交
联 3h,固定化酶活力为、活力回收 68.%55。
固定化肌酸激酶的主要酶学性质:Km=,为游离酶的倍,Vmax=
mol/min•mg 植酸酶,为游离酶的 %;最适 pH 范围 4~9,最适温度范围
15~65℃,60℃半衰期为 ,室温(25℃)重复使用半衰期 38 次,4℃贮存半
衰期 301 天。
以固定化肌酸激酶装配成柱式生物反应器连续制备磷酸肌酸,最佳工艺条件
为:温度 40℃,pH=,底物浓度为 40mmol/L,底物流速为时,连续
制备磷酸肌酸,操作 8h 后,磷酸肌酸生成率为 %,固定化肌酸激酶的残余活
力为 %,连续水解半衰期为。
以阴离子交换树脂交换水解生成的产物,以 ~2mol/L NaCl 为洗脱剂对产
物进行分离。以 NaCl,流速 1BV/h,基本将 ADP 洗脱下来。以 2mol/L
NaCl,流速为 2BV/h,基本将磷酸肌酸钠洗脱下来。磷酸肌酸钠洗脱液经结晶提
纯,其纯度达到 %,产品经红外、核磁共振和高效液相分析确证。

关键词:交联壳聚糖-赖氨酸树脂,肌酸激酶,磷酸肌酸,磷酸肌酸钠

I
重庆大学硕士学位论文英文摘要

ABSTRACT

Creatinephosphat used as raw materials of the pharmaceutical drug, is in huge
demand potential these days。Creatine phosphate can be synthesized by chemical
method as well as enzyme-catalyzed synthesis. Chemical synthesis of creatine
phosphate does ply with energy conservation and the sustainable development
of national policy due to harsh reaction conditions, side reactions, separation and
purification difficulties, low purity of the product and serious environmental
pollution。Therefore, it is urgent to develop a green and petitive method of
preparation of creatine phosphate.
The immobilized Creatine