Faculdade de Farmácia, Research Institute for Medicines and Pharmaceutical Sciences (i-Med-UL), University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
Appl Biochem Biotechnol. 2010 Apr;160(7):2129-47. doi: 10.1007/s12010-009-8733-6. Epub 2009 Aug 20.
A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA-alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 degrees C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)-alginate beads with three different sizes (1-3 mm), at three different alginate concentrations (0.2-1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 degrees C for the PVA-alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)-alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 degrees C. The Michaelis constant (K(Mapp)) and the maximum reaction velocity (V(maxapp)) were evaluated for both free (K(Mapp) = 0.233 mM; V(maxapp) = 0.13 mM min(-1)) and immobilized naringinase (K(Mapp) = 0.349 mM; V(maxapp) = 0.08 mM min(-1)). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 degrees C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.
一种合成聚合物，聚乙烯醇（PVA），一种廉价且对生物体无毒的合成聚合物，已被用于固定生物催化剂。在这项工作中，开发了具有热稳定性、机械稳定性和化学稳定性的 PVA-海藻酸盐珠，可在高温（<80°C）下使用。海藻酸盐与珠粒的联合处理以及用硫酸钠处理不仅防止了聚集，而且产生了凝胶强度高的珠粒，并防止了硼酸对酶的失活。从 Penicillium decumbens 中固定化的柚皮苷酶用三种不同大小（1-3mm）的 PVA（10%）-海藻酸盐珠、三种不同浓度的海藻酸盐（0.2-1.0%）进行固定化，并在珠内溶胀比、酶活性和固定化柚皮苷水解产率方面对这些特性进行了研究。固定化柚皮苷酶的最适 pH 和温度分别为 4.0 和 70°C。在 pH 4.0 和 70°C 下，2mm 的 PVA（10%）-海藻酸盐（1%）珠中的柚皮苷酶活性最高，达到 80%。游离柚皮苷酶的米氏常数（K（app））和最大反应速度（V（maxapp））分别为 0.233mM 和 0.13mM min（-1），固定化柚皮苷酶的 K（app）和 V（maxapp）分别为 0.349mM 和 0.08mM min（-1）。在 pH 4 的醋酸缓冲液中于 4°C 下连续进行了 8 次批次运行，固定化酶的保留活性为 70%。在 6 周后，将固定化生物催化剂储存在 pH 4 的醋酸缓冲液中 4°C 时，仍保留了初始活性的 90%。这些有希望的结果说明了这种固定化策略对所评估体系的潜力，并表明其应用可能有效地用于其他生物催化剂的包埋。
