BACKGROUND: Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified.
RESULTS: A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate.
CONCLUSIONS: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0-8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH 2 -glutaraldehyde quantity, and stabilization time (0-180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH 2 -glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH 2 -glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH 2 -glutaraldehyde quantity is more significant (p < 0.05). In terms of beta-galactosidase enzymatic activity, the study results highlight that among the seven variables considered, only the glutaraldehyde molarity, activation time, and the interplay of activation time and the quantity of MWCNT-NH 2 -glutaraldehyde can exert a statistically significant positive impact on the enzyme\'s activity (p < 0.05). The combination of activation time and buffer solution molarity, as well as the interactive effect of buffer pH and MWCNT-NH2-glutaraldehyde, can lead to a significant improvement in the stabilization efficiency of the protease of carbon nanotubes. The analysis of variance results demonstrated that the efficiency of covalently immobilizing β-galactosidase from Aspergillus oryzae on amino-functionalized multi-walled carbon nanotubes is influenced by the molarity of glutaraldehyde, buffer pH, stabilization time, and the interplay of activation time + buffer pH, buffer pH + activation time, activation time + buffer molarity, and glutaraldehyde molarity + MWCNT-NH 2 -glutaraldehyde (p < 0.05). Through the optimization and selection of optimal formulations, the obtained results indicate enzyme activities and stabilization efficiencies of 64.09 % ± 72.63 % and 65.96 % ± 71.77 % for protease and beta-galactosidase, respectively. Moreover, increasing the enzyme stabilization time resulted in a reduction of enzyme activity. Furthermore, an increase in pH, temperature, and the duration of milk storage passing through the enzyme-immobilized carbon nanotubes led to a decrease in enzyme stabilization efficiency, and lactose hydrolysis declined progressively over 8-h. Hence, the covalent immobilization of β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis onto amino-functionalized multi-walled carbon nanotubes is anticipated to be achievable for milk applications.

This study evaluated Streptomyces rochei strain NAM-19 solid-state fermentation of agricultural wastes to produce alkaline protease. Alkaline protease production increased with flaxseed, rice bran, and cheese whey fermentation reaching 147 U/mL at 48 h. Statistical optimization of alkaline protease production was performed using the central composite design (CDD). Results of CDD and the optimization plot showed that 4.59 g/L flaxseed, 4.31 g/L rice bran, 4.17 mL cheese whey, and a vegetative inoculum size of 7.0% increased alkaline protease production by 27.2% reaching 186 U/mL. Using the 20-70% ammonium sulfate fractionation method, the optimally produced enzyme was partially purified to fivefold. The partially purified alkaline protease was then covalently immobilized on a biopolymer carrier, glutaraldehyde-polyethylene-imine-κ-carrageenan (GA-PEI-Carr), with 90% immobilization efficiency. Characterizations revealed that immobilization improved thermostability, reusability, optimum temperature, and sensitivity towards metal ions of the free enzyme. The optimal temperature for free and immobilized enzymes was 40 and 50 °C, respectively. Both enzymes had the same optimum pH of 10. Immobilization increased Km from 19.73 to 26.52 mM and Vmax from 56.7 to 62.5 mmol min-1L-1. The immobilized enzyme retained 35% of its initial activity at 70 °C, while the free enzyme retained only 5%. The immobilized enzyme kept 80% of its initial activity at the 20th cycle. After 7 weeks of storage, the free enzyme lost all its initial activity, whereas the immobilized enzyme retained 50%. The free and immobilized enzymes were able to hydrolyze gelatin, and azo-casein demonstrating different relative activity, 85, 80, 90 and 95%, respectively, compared to casein (100%).

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.

Microorganisms have long captivated researchers for their potential to produce enzymes with diverse industrial applications. Efficient production of proteases from new strains is crucial as these enzymes play a vital role in breaking down protein bonds, enabling their use in industrial applications. Therefore, a novel Exiguobacterium indicum 1.2.3 was isolated (Istanbul, Turkiye) and characterized in this study. This strain produced alkaline serine protease, which works in lower temperatures (20-40 °C) with casein as a specific substrate. The protease was utterly stable for 3 h at 30 °C. The enzyme was also highly stable in the pH range of 8-11. The optimum activity was obtained at pH 10. The crude enzyme activity was enhanced by various metal ions and retained 147%, 125%, 124%, and 117% of its activity within 1 mM Ca2+, Mn2+, Cu2+, and Mg2+, respectively. The crude enzyme was inactive with phenylmethylsulfonyl fluoride, indicating a serine residue on the active side. The enzyme exhibited a significant proteolytic effect in the presence of surfactants and oxidizing agents. The addition of Tween 80, Triton X-100, and sodium perborate improved enzymatic activity up to 135%, 109%, and 105%, respectively. According to the washing results, the crude enzyme effectively removed the blood on different types of standard pre-stained textiles at 30 °C. In conclusion, Exiguobacterium indicum 1.2.3 is a promising candidate for protease production, with its diverse applications spanning various industrial sectors, particularly detergents.

Alkaline protease is widely used in the food, detergent, and pharmaceutical industries because of its comparatively great hydrolysis ability and alkali tolerance. To improve the ability of the recombinant Bacillus licheniformis to produce alkaline protease, single-factor experiments and response surface methodology (RSM) were utilized to determine and develop optimal culture conditions. The results showed that three factors (corn starch content, soybean meal content, and initial medium pH) had significant effects on alkaline protease production (P < 0.05), as determined through the Plackett‒Burman design. The maximum enzyme activity was observed with an optimal medium composition by central composite design (CCD): corn starch, 92.3 g/L; soybean meal, 35.8 g/L; and initial medium pH, 9.58. Under these optimum conditions, the alkaline protease activity of strain BL10::aprE was 15,435.1 U/mL, 82% higher than that in the initial fermentation medium. To further investigate the application of the optimum fermentation medium, the overexpressed strain BL10::aprE/pHYaprE was cultured using the optimized medium to achieve an enzyme activity of 39,233.6 U/mL. The present study achieved the highest enzyme activity of alkaline protease by B. licheniformis at the shake-flask fermentation level, which has important application value for large-scale production.

The present study aimed to investigate the functional properties of soybean protein isolate (SPI) treated with alkaline protease and high-speed shearing homogenization. Alkaline protease-hydrolyzed SPIs that were characterized by varying degrees of hydrolysis between 0 and 6% were treated with high-speed shearing homogenization to obtain different micro-particulate proteins. The results showed that this combined treatment could significantly reduce the particle size of SPI by markedly degrading the structure of both the 7S and 11S subunits, thereby resulting in a significantly reduced content of β-sheet and β-turn structures. The surface hydrophobicity increased considerably for samples with hydrolysis below the threshold of 2% and then declined gradually above this threshold. Furthermore, the combination of hydrolysis and homogenization significantly improved the emulsion stability of SPI hydrolysates. It also significantly improved the foaming properties of SPI. These results demonstrated that alkaline protease hydrolysis combined with high-speed shearing homogenization represents a promising approach for improving the functional and structural properties of SPI.

Here, we reported the process for the production of Pd/CuO/ZnO nanocomposite utilizing alkaline protease from Phalaris minor seed extract, which is a unique, effective biogenic approach. Alkaline protease performed a crucial part in the reduction, capping and stabilization of Pd/CuO/ZnO nanocomposites. A series of physicochemical techniques were used to inquire the formation, size, shape and crystalline nature of Pd/CuO/ZnO nanocomposites. The notable performance of the synthesized nanocomposite as a photocatalyst and an antibacterial disinfectant was astonishing. The Pd/CuO/ZnO nanocrystals showed considerable photocatalytic activity by eliminating 99 % of the methylene blue (MB) in <30 min of exposure. After three test cycles, the nanocatalyst demonstrated exceptional reliability as a photocatalyst. The nanocomposite was also discovered to be an effective antibacterial agent, with zones of inhibitory activity for Staphylococcus aureus and Escherichia coli bacteria of 30(±0.2), 27(±0.3), 22(±0.2), and 21(±0.3) mm, respectively, in both light and dark conditions. Moreover, the Pd/CuO/ZnO nanocomposites showed strong antioxidant activity by efficiently scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The photocatalytic, antibacterial and antioxidative performance of Pd, CuO, ZnO, and CuO/ZnO were also assessed for the sake of comparison. This work shows that biogenic nanocomposites may be employed as a feasible alternative photocatalyst for the decomposition of dyes in waste water as well as a sustainable antibacterial agent.

We studied the influences of hydrolysis time on the structure, functional properties, and emulsion stability of insoluble soybean meal hydrolysate aggregates (ISMHAs). We assume that the ISMHAs produced by soybean meal can be used as emulsifiers to prepare stable emulsions. The molecular weights of these ISMHAs were below 53 kDa. After hydrolysis, a decrease in α-helices and an increase in random coils indicated that the soybean meal proteins were unfolding. Moreover, the fluorescence intensity, UV absorption, and surface hydrophobicity of ISMHAs increased. These results would contribute to their antioxidant activity and functional properties. Additionally, the 90-min ISMHA sample exhibited the highest ABTS+• scavenging activity (80.02 ± 4.55 %), foaming stability (52.92 ± 8.06 %), and emulsifying properties (emulsifying activity index of 97.09 m2/g; emulsifying stability index of 371.47 min). The 90-min ISMHA emulsion exhibited the smallest particle size and excellent storage stability. Soybean meal peptide by-product emulsifier has potential for sustainable application.

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5\'-TAAAT……AAAAT…….AACAT…TAAAA-3\') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.