Mekras C I, George M H, Barrie J A
Department of Chemistry, Imperial College of Science, Technology and Medicine, London, UK.
Int J Biol Macromol. 1989 Apr;11(2):113-8. doi: 10.1016/0141-8130(89)90052-4.
A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.
开展了一项关于将α-胰凝乳蛋白酶（α-CT）固定在先前已充分表征的接枝有丙烯酸的合成聚氨酯P（U-g-AA）上的研究。P（U-g-AA）先前是在60℃的溶液中，以2,2'-偶氮二异丁腈（AIBN）作为自由基引发剂，丙烯酸作为单体，在不饱和聚氨酯存在的情况下制备的。评估了天然酶和固定在P（U-g-AA）上的酶的一些动力学参数。使用Lineweaver-Burk图（双倒数）发现，米氏常数（Km），固定化酶的为（4.0±0.9）×10⁻³M，游离酶的为（3.0±0.2）×10⁻³M。α-胰凝乳蛋白酶固定在接枝聚氨酯微胶粒/聚集体上，活性保留约45%。固定化的α-CT也能将该活性保持至少6周。发现固定化酶在43℃时具有最大稳定性，而游离酶在36℃时具有最大稳定性，并且最适pH从pH 6.6变为pH 8.2。研究了该酶的长期操作稳定性，这一点很重要，因为该酶可能被物理捕获在胶束环境中。使用对硝基苯乙酸作为底物，在pH 7.5的0.01M磷酸盐缓冲液中进行酶的测定。未观察到在合成的未接枝和接枝聚氨酯存在下α-CT受到抑制。
