Chemical modification of L-asparaginase with N, O-carboxymethyl chitosan and its effects on plasma half-life and other properties

E.coli L-asparaginase,an antitumor enzyme,was chemically modified with N,O-carboxymethyl chitosan to lower its artigenicity and increase its plasma half-life.The results showed that the modified L-asparaginase has almost the same apparent Km value as that of native enzyme.The modified L-asparaginase also showed a higher protease stability against trypsin and a-chymotrypsin.After being modified,the enzyme exhibited the complete loss of antigenicity towards antiasparaginase serum.In addition,the higher the molecular weight of modifying reagents,the better the effects on reduction of antigenicity.When tested in vivo,the plasma half-life of the modified enzyme (t1/2=40 h) was over 33 times longer than that of the native enzyme (t1/2=1.2 h).     

N,O-羧甲基壳聚糖修饰对L-天门冬酰胺酶血浆半衰期及其他性质影响

将具有抗癌作用的L-天门冬酰胺酶用N,O-羧甲基壳聚糖进行化学修饰,以降低酶的抗原性,提高其在体内的半衰期.实验结果表明,修饰酶的表观K_m值与自由酶相差不多,分别为2.6×10~(-4)mol/L和2.2×10~(-4)mol/L(20℃),而抗胰蛋白酶和糜蛋白酶水解能力有很大提高.酶经修饰后抗原性大大降低,且修饰剂分子量越高,降低抗原性的效果越好.自由酶在兔体内的半衰期为1.2h,而修饰酶为40h,提高了33倍.     

大孔载体对米曲霉氨基酰化酶的固定化研究

用合成的大孔丙烯酸甲酯-—二乙烯苯交联共聚物(聚丙烯酸甲酯)、丙烯酰胺-N,N′-亚甲基双丙烯酰胺交联共聚物(聚丙烯酰胺)及它们的功能基化产物作为固定化氨基酰化酶的载体。考察了载体性质对固定化氨基酰化酶效果的影响。比较了底物浓度、pH、磷酸缓冲液浓度、温度对氨基酰化酶溶液酶及固定化酶的影响。利用其中一种载体制成固定化酶柱,对DL-蛋氨酸进行了连续拆分,考察固定化酶的操作稳定性。     

Studies on Immobilization of Aminoacylase From Aspergillus Oryzae on Macroporous Carriers

Synthesized macroporous cross-linked copolymers of methyl acrylate-divinyl benzene (MA-DVB), acrylamide-N,N'-methylenebisacrylamide (AAM-BIS) and their functionalized products were used for immobilization of aminoacylase from Aspergillus oryzae. Effects of the carrier properties on the activity of immobilized aminoacylase were investigated and effects of substrate concentration, pH, phosphate buffer concentration and temperature on the immobilized aminoacylase were compared with those of the soluble aminoacylase. A column of immobilized aminoaoylase was prepared and used for continuous resolution of N-acyl-DL-methionine; the operational stability of immobilized enzyme was also investigated.     

壳聚糖微球固定化L-天门冬酰胺酶研究

以反相悬浮交联法合成了壳聚糖微球,并将其应用于固定化L-天门冬酰胺酶。探讨了不同合成条件对成球情况的影响,研究了固定化酶的性质。结果表明,壳聚糖载体可以较好地吸附L-天门冬酰胺酶。固定化酶的活力回收率较高,且稳定性得到提高。