Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran.
Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Biotechnol Prog. 2022 Sep;38(5):e3282. doi: 10.1002/btpr.3282. Epub 2022 Jul 1.
Electrospun nanofibers have gained much attention for enzyme immobilization due to their high surface-to-volume ratio. In this study, urease was immobilized on chitosan/poly(vinyl alcohol) (PVA) nanofibers by both adsorption and crosslinking methods. In order to obtain nanofibers with more desirable properties, solutions with different ratios of chitosan and PVA were electrospun and crosslinked using glutaraldehyde. Comparing SEM images of the nanofibers, before and after immersing them in phosphate buffer, it was shown that higher chitosan content leads to more stable fibers. So, the solution with the chitosan to PVA ratio of 40:60 was used for enzyme immobilization. Then, the effects of initial protein concentration, temperature, incubation time, and method of immobilization were investigated to reach the highest enzyme activity. Under similar immobilization conditions, covalently immobilized urease showed higher activity, compared to uncrosslinked immobilized enzyme. Besides, it retained 30% of its initial activity after 10 times usage. So, this method was chosen for further investigation. Not only the activity of the immobilized enzyme was much higher than the free enzyme in a wide range of pH and temperature, but also stability of the immobilized enzyme was improved. Immobilized urease was then used to remove thiourea which is a toxic compound. Findings indicated 60% hydrolysis of initial thiourea in 12 h. In conclusion, the findings showed that chitosan/PVA nanofibers are suitable candidates for the immobilization of urease.
静电纺丝纳米纤维因其高的比表面积与体积比而在酶固定化方面受到广泛关注。在这项研究中，通过吸附和交联两种方法将脲酶固定在壳聚糖/聚乙烯醇（PVA）纳米纤维上。为了获得具有更好性能的纳米纤维，用戊二醛对不同壳聚糖和 PVA 比例的溶液进行静电纺丝和交联。通过比较纳米纤维在浸入磷酸盐缓冲液前后的 SEM 图像，表明壳聚糖含量越高，纤维越稳定。因此，使用壳聚糖与 PVA 比例为 40:60 的溶液进行酶固定化。然后，研究了初始蛋白质浓度、温度、孵育时间和固定化方法对达到最高酶活性的影响。在相似的固定化条件下，与未交联固定化酶相比，共价固定化脲酶表现出更高的活性。此外，它在 10 次使用后保留了其初始活性的 30%。因此，选择了这种方法进行进一步研究。不仅固定化酶的活性在很宽的 pH 和温度范围内都高于游离酶，而且固定化酶的稳定性也得到了提高。然后将固定化脲酶用于去除有毒化合物硫脲。结果表明，在 12 小时内初始硫脲的水解率达到 60%。总之，研究结果表明，壳聚糖/PVA 纳米纤维是脲酶固定化的合适候选材料。
