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.
Mesoporous molecular sieve SBA-15 was successfully modified with 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS). The functionalized SBA-15 were characterized by small-angle X-ray (SAXRD), thermogravimetric analysis (TG), N2 adsorption, and Fourier transformed infrared spectrum (FT-IR). APTES functionalized SBA-15 (named SBA-15-A) and GPTMS functionalized SBA-15 (named SBA-15-G) were used to immobilize myoglobin (Mb). The loading amounts of Mb by SBA-15-A and SBA-15-G were 511.2 and 547.8 mg/g, respectively, whereas only 359.6 mg/g was achieved by SBA-15. Mb/SBA-15-G and Mb/SBA-15-A demonstrated better reusability than SBA-15, retaining 84.6% and 82.7% of the initial activity after repeated use seven times. The Mb/SBA-15-A and Mb/SBA-15-G also exhibited improved thermal stability and storage stability.
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