Due to their properties such as superparamagnetism, high surface area, large surface-to-volume ratio, easy separation under external magnetic fields, iron magnetic nanoparticles have attracted much attention in the past few decades. Various modification methods have been developed to produce biocompatible magnetic nanoparticles for protein immobilization. This review provides an updated and integrated focus on the fabrication and characterization of suitable magnetic iron nanoparticle-based nano-active materials for protein immobilization.

During the past few years, nanoparticles have been used for various applications including, but not limited to, protein immobilization, bioseparation, environmental treatment, biomedical and bioengineering usage, and food analysis. Among all types of nanoparticles, superparamagnetic iron oxide nanoparticles, especially Fe₃O₄, have attracted a great deal of attention due to their unique magnetic properties and the ability of being easily chemical modified for improved biocompatibility, dispersibility. This review covers recent advances in the fabrication of functional materials based on Fe₃O₄ nanoparticles together with their possibilities and limitations for application in different fields.

A series of stable mesoporous silica sieves (SBA-15) with different pore sizes (9.8, 7.2, and 5.5 nm) were synthesized using a hydrothermal method. The resulting mesoporous material was then utilized for protein immobilization using myoglobin (Mb) as the target protein. The effects of pore size and adsorption methods on the immobilization efficiency of Mb in a mesoporous material were studied. The SBA-15 with a pore size of 7.2 nm showed the best loading capacity, reaching 413.8 mg/g. The SBA-15 with a pore size of 9.8 nm showed the highest retained catalytic ability (92.36%). The immobilized enzyme was more stable than the free enzyme. After seven consecutive assay cycles, Mb adsorbed by SBA-15 (Mb/SBA-15) and Mb adsorbed by SBA-15 and crosslinked with glutaraldehyde (Mb/G/SBA-15) retained 36.41% and 62.37% of their initial activity, respectively.

The O-demethylation of lignin monomers, which has drawn substantial attention recently, is critical for the formation of phenols from aromatic ethers. The P450BM3 peroxygenase system was recently found to enable the O-demethylation of different aromatic ethers with the assistance of dual-functional small molecules (DFSM), but these prepared mutants only have either moderate O-demethylation activity or moderate selectivity, which hinders their further application. In this study, we improve the system by introducing different amino acids into the active site of P450BM3, and these amino acids with different side chains impacted the catalytic ability of enzymes due to their differences in size, polarity, and hydrophobicity. Among the prepared mutants, the combination of V78A/F87A/T268I/A264G and Im-C6-Phe efficiently catalyzed the O-demethylation of guaiacol (TON = 839) with 100% selectivity. Compared with NADPH-dependent systems, we offer an economical and practical bioconversion avenue.

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA’s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.

With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with k_cat of 1402 min⁻¹ and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (k_cat/K_m = 1388 mM⁻¹ min⁻¹) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.