**1. Introduction**

Enzymes are proteins (or ribonucleic acids) that act as catalysts to quicken chemical reactions by decreasing the activation energy. They are environmentally friendly and do not alter or ge<sup>t</sup> consumed during chemical reactions. Enzymes as biocatalysts have recently become an intensive area of research. Currently, enzymes are used in many industrial applications, including food, drugs, and water remediation [1–7], and have numerous benefits, including a high catalytic efficiency, a high selectivity, and biodegradability. Despite all of these benefits, the use of enzymes in industrial applications has some limitations, such as a low operational stability, difficult recovery, low reproducibility, and a high cost. Immobilization of the enzyme on an insoluble solid support has been found to be a useful

**Citation:** Al-Maqdi, K.A.; Bilal, M.; Alzamly, A.; Iqbal, H.M.N.; Shah, I.; Ashraf, S.S. Enzyme-Loaded Flower-Shaped Nanomaterials: A Versatile Platform with Biosensing, Biocatalytic, and Environmental Promise. *Nanomaterials* **2021**, *11*, 1460. https://doi.org/10.3390/ nano11061460

Academic Editor: Daniela Lannazzo

Received: 29 March 2021 Accepted: 28 May 2021 Published: 31 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

way to overcome some of these limitations. The solid support must be inert, insoluble, nontoxic, environmentally safe, easily accessible, affordable, highly resistant to decay and microbial attack, and have an affinity to the enzyme used. Enzyme immobilization has many advantages, including functional stability, stability against extreme reaction conditions (changes in the pH and temperature), reusability, easy separation and recovery of enzymes, and increased catalytic performance. Many researchers have shown that immobilized enzymes are more stable than free enzymes. However, in some instances, immobilization can limit the enzyme performance and lower its catalytic activity. The reasons for this are the blocking of the active site on the enzyme and the conformational changes that happen to the enzyme after immobilization, as well as the limitations of mass transfer [8–14]. Therefore, there is a need to develop new and unique methods and materials to overcome these shortcomings caused by traditional immobilization methods. There is currently growing interest in the use of nanoscale materials, such as nanoparticles and nanocrystals, for enzyme immobilization [15–19].

More recently, Jun Ge et al. developed a new method of immobilizing enzymes on solid supports, known as organic–inorganic hybrid nanoflowers or hybrid nanoflowers, (hNFs), which are flower-like hybrid nanomaterials produced between a metal node and a protein through coordination interactions [20]. This review will focus on enzyme immobilization using organic–inorganic hybrid nanoflowers (hNFs), and will cover their synthesis, advantages, different types of nanoflowers, and applications.

#### **2. Organic–Inorganic Hybrid Nanoflowers**

Organic–inorganic hybrid nanoflowers were accidentally discovered in 2012. hNFs were first detected when 0.8 mM CuSO4 was added to phosphate-buffered saline (PBS) with 0.1 mgml−<sup>1</sup> bovine serum albumin (BSA). The reaction pH was 7.4 at 25 ◦C. After 3 days, a blue precipitate was formed at the bottom of the reaction tube, resembling a flower structure. The formation of the organic–inorganic hNFs was confirmed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) [20]. The formation mechanism (self-assembly) of organic–inorganic hybrid nanoflowers occurs through the following three stages (Figure 1): nucleation, growth, and completion. In the first step, the nucleation of primary crystals is formed from protein molecules and Cu2+ ions. This occurs through binding to the amide group onto the protein backbone. The second step is the growth of seed particles of the Cu2+ binding site, which leads to the production of nano petals. This continues with protein nanoparticles and primary crystals. In the last step, the formation of organic–inorganic hybrid nanoflowers is completed. Figure 2 shows the SEM images of the organic–inorganic hybrid nanoflower formation after 2 h, 12 h, and 3 days [20].

Additional research on organic–inorganic hybrid nanoflowers shows that the presence of a protein (enzyme) is essential for the formation of nanoflowers, and without an enzyme, only a crystal structure is formed. A research group formed hNFs between *Burkholderia cepacia* lipase (BCL) and calcium phosphate, and a part of their results (Figure 3) shows that the morphology transforms from sheet stacking to flower-like after the addition of lipase [21]. Another research group used α-chymotrypsin (ChT) and calcium phosphate to form hNFs, testing the effect of different enzyme concentrations on the formation of hNFs (Figure 4). It is clear from the SEM image that for samples a1 and a2, where ChT was not added, there is no formation of nanoflowers, and only the presence of large crystals are observed. However, when 0.05 mg/mL ChT was added, small buds were observed (b1 and b2), and as the enzyme concentration increased, nanoflower formation was more evident [22]. Lin et al. immobilized trypsin on hNFs and studied its application as a reactor for highly efficient protein digestion. One of the experiments they ran shows that for hNFs to be formed, the trypsin enzyme must be added, and without trypsin, large crystals are formed (Figure 5). The figure also shows that with an increasing trypsin concentration, a flower-like structure appears [23]. Researchers in another study produced hNFs from soybean peroxidase (SBP) and Cu2+. Their outcomes showed the same result as previous

work, where the absence of a protein (SBP) produced a crystal-like structure, but not nanoflowers (Figure 6) [24]. Moreover, their research shows that the presence of proteins in the formation of hybrid nanoflowers is crucial.

Currently the majority of enzyme immobilization techniques use preexisting carriers. In such a case, the enzyme can be immobilized by physical adsorption or attachment through covalent bonds between the carrier and the enzyme. These methods usually consist of the following two stages: one is the synthesis of the carriers, and the second is the immobilization process of the enzyme on the carrier. These two steps can cause a reduction in the reaction efficiency. Moreover, they require a lengthier process, which results in a higher cost. One factor that distinguishes hNFs from other immobilization techniques is that it is a one-step reaction, i.e., the carrier synthesis and enzyme immobilization processes occur in one step. This leads to a simpler procedure, and thus a lower cost [25].

**Figure 1.** Schematic illustration of the hybrid nanoflower (hNF) formation process.

**Figure 2.** Formation of BSA-Cu3(PO4)2.3H2O nanoflowers. The SEM images at different times: (**a**)2h, (**b**) 12 h, and (**c**) 3 days. Reprinted from [20] with permission from Springer Nature. Copyright © 2021, Nature Publishing Group. License Number: 5031780508604.

**Figure 3.** SEM image (**a**) without the addition of lipase enzyme, (**b**) with the addition of lipase enzyme. Reprinted from [21] with permission from the Royal Society of Chemistry. Copyright © The Royal Society of Chemistry. License Number: 1105113-1.

**Figure 4.** SEM images of different ChT concentrations on the formation of nanoflowers: (**a1**,**a2**) 0.0 mg/mL, (**b1**,**b2**) 0.05 mg/mL, (**c1**,**c2**) 0.1 mg/mL, and (**d1**,**d2**) 0.5 mg/mL. Reprinted from [22] with permission from the Royal Society of Chemistry. Copyright © The Royal Society of Chemistry. License Number: 1105121-1.

**Figure 5.** SEM image of different trypsin concentrations on the formation of nanoflowers: (**A1**,**A2**) 0.0 mg/mL, (**B1**,**B2**) 0.02 mg/mL, (**C1**,**C2**) 1.0 mg/mL, and (**D1**,**D2**) 5.0 mg/mL. Reprinted from [23] with permission from the Royal Society of Chemistry. Copyright © The Royal Society of Chemistry. License Number: 1105124-1.

**Figure 6.** SEM image of different soybean peroxidase (SBP) concentrations on the formation of nanoflowers: (**A**) 0.0 mg/mL, (**B**) 0.5 mg/mL, (**C**) 1.0 mg/mL, and (**D**) 2.0 mg. Reprinted from [24] with permission from Elsevier. Copyright © 2021 Elsevier B.V. License Number: 5031790214453.

#### **3. Advantages or (Properties) of Hybrid Nanoflowers**

## *3.1. Catalytic Activity*

Several enzymes, including carbonic anhydrase, lipase, trypsin, laccase, and different types of peroxidases, have been used for the formation of hNFs through immobilization techniques [23,24,26–30]. A significant advantage of hNF formation over other immobilization methods is the increase in the immobilized enzyme's catalytic activity [24]. Studies have shown that immobilization only improves the stability, not the catalytic activity of the enzyme (with rare exceptions). This could be as a result of the mass transfer limitations and conformational changes in the enzyme [31,32]. For hNFs, the enhancement of the catalytic activity of the immobilized enzyme possibly arises from several reasons, namely: (i) the high surface area of hNFs, (ii) less mass transfer limitation, (iii) cooperative effect of the nanoscale-entrapped enzyme, and (iv) favorable enzyme conformation in hNFs [31,33,34].

A study showed that the enzymatic activities of soybean peroxidase in hNFs formed from different concentrations of crude SBP (0.5, 1, and 2 mg/mL) were 787, 1857, and 2500 U/mg, respectively. These results showed a ~137%, ~325%, and ~446% increase in activity compared with free crude SBP, respectively, which has an activity of 572 U/mg [24]. Another study produced hNFs and magnetic hNFs with laccase enzyme and copper(II) sulfate pentahydrate (CuSO4.5H2O). Laccase showed a higher activity in both hNFs than free laccase. The laccase hNF activity was 3.3 times greater than that of free laccase, and the laccase magnetic hNF activity was 2.7 times greater than that of free laccase. The reduction in activity in the magnetic hNFs was attributed to the shielding of active sites on laccase by the magnetic nanoparticles on the surface of the hNFs [34]. Another research group working with lipase enzymes found that lipase/Zn3(PO4)2 hNFs had a higher enzyme activity (855 ± 13 U/g) than free lipase (328 ± 6 U/g). The increase in the enzyme activity was 147% of the pure enzyme [35]. Yin et al. synthesized α-chymotrypsin (ChT) hNFs and studied their use as immobilized ChT reactors for successful protein digestion. They determined the ChT activity in hNFs to be 3410 U/mg compared with free ChT, which

has an activity of 1123 U/mg. The results show that the enhancement in the ChT activity was approximately 266% higher [22]. Lin et al., in another study, synthesized hNFs using horseradish peroxidase enzyme (HRP) and copper phosphate (Cu3(PO4).3H2O) to use as a colorimetric platform for the visual identification of phenol and hydrogen peroxide. The results obtained showed a considerable improvement in the activity of the embedded HRP enzyme in the nanoflowers. The free HRP activity was 2970.5 U/mg, whereas the embedded HRP activity was 15,040.5 U/mg. This led to a 506% increase in the inactivity of the HRP-embedded nanoflowers [36]. All of these studies solidified that organic–inorganic hybrid nanoflowers significantly boost the catalytical activity of the embedded enzyme, which can be ascribed to the four previously-mentioned reasons.
