Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes

The use of protein-inorganic hybrid nanoflowers for the immobilization of enzymes has received a significant degree of attention owing to their capability to retain high enzymatic activity and stability. However, the relative lack of reusability due to the weakness of the flower-like structure has limited their practical applications. Herein, we have developed a simple but efficient method to synthesize highly robust enzyme-inorganic hybrid nanoflowers,which relies on further crosslinking of the enzyme molecules entrapped in the hybrid nanoflowers by treatment with glutaraldehyde (GA). By employing lipase from Candida rugosa as a model enzyme with copper phosphate during 3 days incubation followed by the additional GA treatment for only 1 h, we could successfully synthesize GA-treated lipase nanoflowers having similar flower-like morphology and hydrolytic activity (ca. 95% compared with the free lipase) as conventionally synthesized lipase nanoflowers without GA treatment. Importantly, the conventional lipase nanoflowers seemed not to be reusable because they lost most of their activity (90%) after recycling 4 times, whereas GA-treated lipase nanoflowers exhibited higher retention of their initial activity (over 70%) after 4 reuses, which was also accompanied by an efficient maintenance of their flower-like morphology. Based on our results, we expect that this simple GA-mediated strategy to synthesize enzyme-inorganic hybrid nanoflowers can be readily extended to other enzymes for various biotechnological applications.