Abstract

Nitrilase (NLase) was covalently immobilized on modified Relizyme supports or adsorbed on montmorillonite K-10 or entrapped in polyvinyl alcohol hydrogel. Although 80–90% of initial loaded protein was immobilized, however, unsatisfactory activity recoveries was obtained in both covalent immobilization and adsorption studies. Entrapping NLase in polyvinyl alcohol allowed us to obtain an immobilized NLase with 80% activity recovery and 100% initial protein recovery. The characterization studies showed that the free and entrapped NLase samples had both maximum activity at pH 7.5 and 30 °C. The free nitrilase completely lost its initial activity at 30 °C after 24 h pre-incubation. However, the entrapped NLase retained 60% of its initial activity under the same conditions. Acrylonitrile with a initial concentration of 0.1 M was all converted to acrylic acid by entrapped NLase in 30 min whereas the same yield was achieved by soluble enzyme in 60 min. The entrapped NLase retained 84% of its initial activity after 15 reuses for the hydrolysis of acrylonitrile. These results show that the entrapped NLase can be of great interest as a biocatalyst to synthesize acrylic acid under mild reaction conditions.

摘要

腈水解酶（NLase）共价固定在修饰的Relizyme载体上，或吸附在蒙脱石K-10上或包裹在聚乙烯醇水凝胶中。尽管固定了初始负载蛋白的80–90％，但是在共价固定和吸附研究中均获得了令人满意的活性回收率。将NLase捕获在聚乙烯醇中可使我们获得固定化的NLase，其活性回收率为80％，初始蛋白质回收率为100％。表征研究表明，游离和捕获的NLase样品在pH 7.5和30°C时均具有最大活性。预孵育24小时后，游离腈水解酶在30°C时完全失去了其初始活性。但是，在相同条件下，被捕获的NLase保留了其初始活性的60％。丙烯腈的初始浓度为0。包埋的NLase在30分钟内将1 M全部转化为丙烯酸，而可溶性酶在60分钟内获得了相同的收率。15次重复用于丙烯腈水解后，被捕获的NLase保留了其初始活性的84％。这些结果表明，被包埋的NLase作为在温和的反应条件下合成丙烯酸的生物催化剂可能是非常令人感兴趣的。