*3.2. Stability*

## 3.2.1. Thermal Stability

Another advantage of hNFs is the stability they provide to the enzyme. Regarding thermal stability, a research group, Yu et al., studied the temperature effect on hNFs formed using calcium phosphate and six different enzymes, namely: papain, bromelain, trypsin, lipase from porcine pancreas (PPL), lipase from *Thermomyces lanuginosus* (TLL), and lipase B from *Candida antarctica* (CALB). The results were tested in different temperature ranges of 50, 60, and 70 ◦C, and showed that all enzyme-hNFs were more thermally stable than their corresponding free enzymes. For example, after heating at 70 ◦C for 6 h, the residual activity of the enzymes in hNFs was as follows: TLL-hNFs (78.3%), PPL-hNFs (72.9%), and CALB-hNFs (84.3%), counting for a 4.57, 2.61, and 2.35 times higher activity than the corresponding free enzymes, respectively. The authors attributed this stability to the strong interaction between the Ca2+ ions and the functional groups on the enzymes in the hNFs and the rigidity in the inorganic hNFs that enclosed the enzyme and stopped the peptide chains from unfolding, thus improving the thermal stability of the enzyme [37]. Another study performed on magnetic hNFs embedded with the laccase enzyme showed that hNFs had a significantly better thermal stability than free laccase. One example is that at 55 ◦C (incubation time of 1 h), the magnetic laccase-hNFs retained 80% of their activity, which was more than that of free laccase. The results showed that a temperature of 35 ◦C had no effect on the activity, and a temperature of 85 ◦C made laccase inactive for both laccase-hNFs and free laccase [34].

## 3.2.2. Storage Stability

The storage stability of enzymes in hNFs is another essential influencing factor that has been intensively studied. The study mentioned above also examined the storage stability of the laccase enzyme in magnetic hNFs. The findings showed that, at room temperature, free laccase lost 77% of its activity after 30 days and 90% after 60 days. On the other hand, laccase-magnetic hNFs sustained 60% of their activity after 30 days and 45% after 60 days. At 4 ◦C and higher, over 60 days, both laccase-magnetic hNFs and free laccase maintained high activities. The authors suggested that the loss of activity for the free laccase was a result of conformational changes in the enzyme. Additionally, the morphology of the magnetic hNFs was examined using SEM over a 60-day storage period, and there was no visible difference in the nanoflower size and hierarchical structure. In addition, leaching of hNFs at room temperature and at 4 ◦C over 60 days of storage was studied by examining the protein content in the supernatant of hNFs. The results showed that there was nearly no detectable protein in the supernatant at the two different temperatures, which indicates the stability of the formed hNF complex [34].

Another study synthesized hNFs using copper ions and urease enzyme at different pH values, and examined the enzyme activity after 30 days at 4 ◦C and room temperature. Both experiments showed that hNFs had a better storage stability over time than the free enzyme. At 4 ◦C, the hNFs produced at different pH values of 6, 7.4, 8, and 9 lost 22.55%, 3.7%, 10%, and 15% of their initial activities, respectively. On the other hand, free urease lost 73.55% of its initial activity. At room temperature, the hNFs lost 35%, 9.28%, 13.22%, and

22.34% of their initial activities at a pH of 6, 7.4, 8, and 9, respectively, whereas free urease lost 90.25% of its initial activity. These findings not only show that enzyme-embedded hNFs are more stable than their corresponding free enzyme, but also indicate that hNFs synthesized at pH 7.4 have the best storage stability [38].

Nadar et al. prepared hNFs from glucoamylase enzymes and copper ions, and studied their storage stability over 25 days with five-day intervals at 30 ◦C. Their results showed that the free glucoamylase activity slowly decreased to 68% of its original activity, whereas the hNFs were able to maintain 91% of their original activity [39]. Similarly, Patel et al. found that laccase-hNFs and cross-linked-laccase-hNFs were more stable than free laccase when stored at 4 ◦C for 60 days. The laccase residual activity was 53.3%, 91.5%, and 3.8% for laccase-hNFs, cross-linked-laccase-hNFs, and free laccase, respectively. Hence, laccase-hNFs and cross-linked-laccase-hNFs had 14 and 24 times, respectively, the free enzyme's residual activity [30].
