Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Oligonucleotide-Based Photoaffinity Probes: Chemical Tools and Applications for Protein Labeling.

A variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non-covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid-protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.

Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

2D Atomic-Molecular Heterojunctions toward Brainoid Applications.

Brainoid computing using 2D atomic crystals and their heterostructures, by emulating the human brain's remarkable efficiency and minimal energy consumption in information processing, poses a formidable solution to the energy-efficiency and processing speed constraints inherent in the von Neumann architecture. However, conventional 2D material based heterostructures employed in brainoid devices are beset with limitations, performance uniformity, fabrication intricacies, and weak interfacial adhesion, which restrain their broader application. The introduction of novel 2D atomic-molecular heterojunctions (2DAMH), achieved through covalent functionalization of 2D materials with functional molecules, ushers in a new era for brain-like devices by providing both stability and tunability of functionalities. This review chiefly delves into the electronic attributes of 2DAMH derived from the synergy of polymer materials with 2D materials, emphasizing the most recent advancements in their utilization within memristive devices, particularly their potential in replicating the functionality of biological synapses. Despite ongoing challenges pertaining to precision in modification, scalability in production, and the refinement of underlying theories, the proliferation of innovative research is actively pursuing solutions. These endeavors illuminate the vast potential for incorporating 2DAMH within brain-inspired intelligent systems, highlighting the prospect of achieving a more efficient and energy-conserving computing paradigm.

Bifunctional enzyme provides absolute concentration robustness in multisite covalent modification networks.

Biochemical covalent modification networks exhibit a remarkable suite of steady state and dynamical properties such as multistationarity, oscillations, ultrasensitivity and absolute concentration robustness. This paper focuses on conditions required for a network of this type to have a species with absolute concentration robustness. We find that the robustness in a substrate is endowed by its interaction with a bifunctional enzyme, which is an enzyme that has different roles when isolated versus when bound as a substrate-enzyme complex. When isolated, the bifunctional enzyme promotes production of more molecules of the robust species while when bound, the same enzyme facilitates degradation of the robust species. These dual actions produce robustness in the large class of covalent modification networks. For each network of this type, we find the network conditions for the presence of robustness, the species that has robustness, and its robustness value. The unified approach of simultaneously analyzing a large class of networks for a single property, i.e. absolute concentration robustness, reveals the underlying mechanism of the action of bifunctional enzyme while simultaneously providing a precise mathematical description of bifunctionality.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Preparation of 4-Amino-3-hydrazino-1,2,4-triazol-5-thiol-Modified Graphene Oxide and Its Greatly Enhanced Selective Adsorption of Gallium in Aqueous Solution.

Efficient recovery of gallium (Ga) from vanadium slag processing residue (VSPR) solution is of great significance for environmental protection and resource utilization, but improving its selective adsorption against the coexisting Sc3+ and In3+ is still challenging. Herein, a novel adsorbent consisting of 4-amino-3-hydrazino-1,2,4-triazol-5-thiol (AHTZT)-modified graphene oxide (GO-AHTZT) was successfully synthesized that exhibits a higher adsorption selectivity for Ga3+ in VSPR solution with coexisting Sc3+ and In3+. Under optimal conditions, the adsorption capacity of GO-AHTZT for Ga3+ can reach 23.92 mg g-1, which is 4.9 and 12.6 times higher than that for Sc3+ (4.87 mg g-1) and In3+ (1.90 mg g-1) adsorption, indicating the excellent anti-interference ability of GO-AHTZT against Sc3+ and In3+. The process and mechanism of Ga3+ adsorption onto GO-AHTZT was also studied and discussed in detail. By measuring the adsorption process and by characterizing the adsorbent before and after adsorption, we demonstrate that the selective interaction between the Ga3+- and N-containing groups in AHTZT is the main reason for the improved adsorption selectivity. This work opens up an avenue for the design and synthesis of highly selective adsorbents for Ga3+ in complex VSPR solutions.

Multivariate Silicification-Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity-Stability Trade-Off.

The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long-term stability is highly desired but highly challenging. Herein, we propose the concept of the "silicazyme", where solid inorganic silica undergoes controlled hybridization with the fragile enzyme under moderate conditions at the single-enzyme level, thus enabling simultaneous structure augmentation, long-term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity-stability trade-off the structure flexibility/rigidity of the silicazyme was optimized by a component adjustment ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages, including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes with promise for catalysis, separations, and nanomedicine.

Reversible Postsynthetic Modification in a Metal-Organic Framework.

Postsynthetic modification (PSM) of metal-organic frameworks (MOFs) provides access to functional materials and advanced porous solid engineering. Herein, we report the reversible PSM of a multivariate isoreticular MOF by applying dynamic furan-maleimide Diels-Alder (DA) chemistry. The key step involves incorporating a furan group into the MOF via "click" PSM, which can then undergo repeated cycles of modification and de-modification with maleimides. The structural integrity, crystallinity, and porosity of the furan-appended MOF remained intact even after three consecutive PSM/de-modification cycles using three different functionalized maleimides.

Epigenetic meets metabolism: novel vulnerabilities to fight cancer.

Histones undergo a plethora of post-translational modifications (PTMs) that regulate nucleosome and chromatin dynamics and thus dictate cell fate. Several evidences suggest that the accumulation of epigenetic alterations is one of the key driving forces triggering aberrant cellular proliferation, invasion, metastasis and chemoresistance pathways. Recently a novel class of histone "non-enzymatic covalent modifications" (NECMs), correlating epigenome landscape and metabolic rewiring, have been described. These modifications are tightly related to cell metabolic fitness and are able to impair chromatin architecture. During metabolic reprogramming, the high metabolic flux induces the accumulation of metabolic intermediate and/or by-products able to react with histone tails altering epigenome homeostasis. The accumulation of histone NECMs is a damaging condition that cancer cells counteracts by overexpressing peculiar "eraser" enzymes capable of removing these modifications preserving histones architecture. In this review we explored the well-established NECMs, emphasizing the role of their corresponding eraser enzymes. Additionally, we provide a parterre of drugs aiming to target those eraser enzymes with the intent to propose novel routes of personalized medicine based on the identification of epi-biomarkers which might be selectively targeted for therapy. Video Abstract.

Photocatalysis-self-Fenton system over edge covalently modified g-C3N4 with high mineralization of persistent organic pollutants.

The Fenton process is a widely used to remedy organic wastewaters, but it has problems of adding H2O2, low utilization efficiency of H2O2 and low mineralization efficiency. Here, a new photocatalysis-self-Fenton process was exploited for the removal of persistent 4-chlorophenol (4-CP) pollutant through coupling the photocatalysis of 4-carboxyphenylboronic acid edge covalently modified g-C3N4 (CPBA-CN) with Fenton. In this process, H2O2 was in situ generated via photocatalysis over CPBA-CN, the photogenerated electrons assisted the accelerated regeneration of Fe2+ to improve the utilization efficiency of H2O2, and the photogenerated holes facilitated the enhancement of 4-CP mineralization. Under the conjugation of CPBA, the electronic structure of CN was optimized and the molecular dipole was enhanced, resulting in the deepening valence band position, accelerated electron-hole pair separation, and improved O2 adsorption-activation. Therefore, the incremental 4-CP degradation rate in the CPBA-CN photocatalysis-self-Fenton process was approaching 0.099 min-1, by a factor of 3.1 times compared with photocatalysis. The parallel mineralization efficiency increased to 74.6% that was 2.1 and 2.6 times than photocatalysis and Fenton, respectively. In addition, this system maintained an excellent stability in the recycle experiment and can be potentially applied in a wide range of pHs and under the coexistence of various ions. This study would provide new insights for improving Fenton process and promote further development of Fenton in organic wastewater purification.

Amyloid fibril reduction through covalently modified lysine in HEWL and insulin.

Proteins possess a variety of nucleophiles, which can carry out different reactions in the functioning cells. Proteins endogenously and synthetically can be modified through their nucleophilic sites. The roles of these chemical modifications have not been completely revealed. These modifications can alter the protein folding process. Protein folding directly affects the function of proteins. If an error in protein folding occurs, it may cause protein malfunction leading to several neurodegenerative disorders such as Alzheimer's and Parkinson's. In this study, Hen Egg White Lysozyme (HEWL) and bovine insulin, as model proteins for studying the amyloid formation, were covalently attached with 5(6)-thiophenolfluorescein. The amyloid formation of the covalently labeled lysozyme and insulin were compared with the native proteins. Interestingly, the results indicated that the covalent attachment of fluorescein slowed down the amyloid formation of HEWL and insulin significantly. The amyloid formation was examined using Thioflavin T (ThT) fluorescence assay, circular dichroism, FTIR, and gel electrophoresis. Tandem mass spectrometry was employed to identify the sites of covalent modifications in HEWL. It turned out that two surface lysine residues (K97 and K 116) in HEWL were modified. Computational studies, including docking and molecular simulations, revealed that 5(6)-thiophenolfluorescein makes several non-covalent interactions with HEWL residues, including Lys 97, leading to the reduction of the ß-sheet in the protein. Additionally, AFM analysis confirmed the amyloid fibril reduction of lysine-modified bovine insulin and HEWL. Altogether, our results expand mechanistic insights into preventing amyloid formation by providing an approach for reducing amyloid formation by modifying specific lysine residues in the proteins.

CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation.

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.

PES derivative PESA is a potent tool to globally profile cellular targets of PES.

PES (2-phenylethynesulfonamide, pifithrin-µ, PFTµ) is an electrophilic compound that exhibits anticancer properties, protects against chemotherapy-induced peripheral neuropathy in chemotherapy, and shows immunomodulatory, anti-inflammatory and anti-viral activities. PES generally shows higher cytotoxicity towards tumor cells than non-tumor cells. The mechanism of action of PES is unclear but may involve the covalent modification of proteins as PES has been found to be a covalent inhibitor of Hsp70. We developed a new PES derivative PESA with a terminal alkynyl group to perform click-reaction-assisted activity-based protein profiling (click-reaction ABPP) and used this to screen for cellular targets of PES. We found PES and its derivatives PES-Cl and PESA have comparable ability to undergo a Michael addition reaction with GSH and Hsp70, and showed similar cytotoxicity. By fluorescence imaging and proteomics studies we identified over 300 PESA-attached proteins in DOHH2 cells. Some proteins involved in cancer-related redox processes, such as peroxiredoxin 1 (PRDX1), showed higher frequency and abundance in mass spectrometry detection. Our results suggest that cytotoxicity of PES and its derivatives may be related to attack of protein thiols and cellular GSH resulting in breakdown of cellular redox homeostasis. This study provides a powerful new tool compound within the PES class of bioactive compounds and gives insight into the working mechanisms of PES and its derivatives.

Organic/Inorganic Species Synergistically Supported Unprecedented Vanadomolybdates.

Vanadomolybdates (VMos), comprised of Mo and V in high valences with O bridges, are one of the most important types of polyoxometalates (POMs), which have high activity due to their strong capabilities of gaining/losing electrons. Compared with other POMs, the preparation of VMos is difficult due to their relatively low structural stability, especially those with unclassical architectures. To overcome this shortcoming, in this study, triol ligands were applied to synthesize VMos through a beaker reaction in the presence of V2O5, Na2MoO4, and organic species in the aqueous solution. The single-crystal X-ray diffraction results indicate that two VMo clusters, Na4{V5Mo2O19[CH3C(CH2O)3]}∙13H2O and Na4{V5Mo2O19[CH3CH2C(CH2O)3]}∙13H2O, with a similar architecture, were synthesized, which were both stabilized by triol ligand and {MoO6} polyhedron. Both clusters are composed of five V ions and one Mo ion in a classical Lindqvist arrangement with an additional Mo ion, showing an unprecedented hepta-nuclear VMo structure. The counter Na+ cations assemble into one-dimensional channels, which facilitates the transport of protons and was further confirmed by proton conductivity experiments. The present results provide a new strategy to prepare and stabilize VMos, which is applicable for developing other compounds, especially those with untraditional architectures.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Probes for Photoaffinity Labelling of Kinases.

Protein kinases, one of the largest enzyme superfamilies, regulate many physiological and pathological processes. They are drug targets for multiple human diseases, including various cancer types. Probes for the photoaffinity labelling of kinases are important research tools for the study of members of this enzyme superfamily. In this review, we discuss the design principles of these probes, which are mainly derived from inhibitors targeting the ATP pocket. Overall, insights from crystal structures guide the placement of photoreactive groups and detection tags. This has resulted in a wide variety of probes, of which we provide a comprehensive overview. We also discuss several areas of application of these probes, including the identification of targets and off-targets of kinase inhibitors, mapping of their binding sites, the development of inhibitor screening assays, the imaging of kinases, and identification of protein binding partners.

Covalently modified enzymatic 3D-printed bioelectrode.

Three-dimensional (3D) printing has showed great potential for the construction of electrochemical sensor devices. However, reported 3D-printed biosensors are usually constructed by physical adsorption and needed immobilizing reagents on the surface of functional materials. To construct the 3D-printed biosensors, the simple modification of the 3D-printed device by non-expert is mandatory to take advantage of the remote, distributed 3D printing manufacturing. Here, a 3D-printed electrode was prepared by fused deposition modeling (FDM) 3D printing technique and activated by chemical and electrochemical methods. A glucose oxidase-based 3D-printed nanocarbon electrode was prepared by covalent linkage method to an enzyme on the surface of the 3D-printed electrode to enable biosensing. X-ray photoelectron spectroscopy and scanning electron microscopy were used to characterize the glucose oxidase-based biosensor. Direct electrochemistry glucose oxidase-based biosensor with higher stability was then chosen to detect the two biomarkers, hydrogen peroxide and glucose by chronoamperometry. The prepared glucose oxidase-based biosensor was further used for the detection of glucose in samples of apple cider. The covalently linked glucose oxidase 3D-printed nanocarbon electrode as a biosensor showed excellent stability. This work can open new doors for the covalent modification of 3D-printed electrodes in other electrochemistry fields such as biosensors, energy, and biocatalysis.

Nanocarriers for Biomedicine: From Lipid Formulations to Inorganic and Hybrid Nanoparticles.

Encapsulation of cargoes in nanocontainers is widely used in different fields to solve the problems of their solubility, homogeneity, stability, protection from unwanted chemical and biological destructive effects, and functional activity improvement. This approach is of special importance in biomedicine, since this makes it possible to reduce the limitations of drug delivery related to the toxicity and side effects of therapeutics, their low bioavailability and biocompatibility. This review highlights current progress in the use of lipid systems to deliver active substances to the human body. Various lipid compositions modified with amphiphilic open-chain and macrocyclic compounds, peptide molecules and alternative target ligands are discussed. Liposome modification also evolves by creating new hybrid structures consisting of organic and inorganic parts. Such nanohybrid platforms include cerasomes, which are considered as alternative nanocarriers allowing to reduce inherent limitations of lipid nanoparticles. Compositions based on mesoporous silica are beginning to acquire no less relevance due to their unique features, such as advanced porous properties, well-proven drug delivery efficiency and their versatility for creating highly efficient nanomaterials. The types of silica nanoparticles, their efficacy in biomedical applications and hybrid inorganic-polymer platforms are the subject of discussion in this review, with current challenges emphasized.

Covalent chemical modification of myofibrillar proteins to improve their gelation properties: A systematic review.

To improve the quality of meat products is a constant focus for both the meat industry and scientists. As major components in meat protein, the gelation properties of myofibrillar proteins (MPs) predominantly determine the sensory quality and product yield of the final product. Naturally or artificially occurring covalent modifications are known to largely affect MP functionality by changing the protein structure and forming aggregates, leading to both favorable and unfavorable outcomes. The review aims to summarize the mechanisms associated with several covalent modifications and the recent developments in enhancing MP gelation properties. Various extrinsic and intrinsic parameters controlling oxidation, phenolic-protein interactions, enzyme catalysis, glycation, and isoelectric solubilization/precipitation, and their effects on the characteristics of heat-induced MP gels are discussed. This article provides an improved understanding of the covalent modifications that occur mainly in the MP system and how they can be utilized to promote its gelation properties. Covalent modifications exhibited dose-dependent and dual-role manners for MP gelation properties. Mild oxidation, enzyme catalysis, and isoelectric solubilization/precipitation treatment would be beneficial to form more aligned and cross-linked three-dimensional networks for MP gels because of moderate protein aggregation. However, an excessive aggregate impedes the MP gelation behavior, leading to reduced gelation quality. Glycation effectively increased hydrophilicity of MPs and phenolic conjugation provides MPs with novel bioactivity. A proper utilization of such a process or even a rational combination of them allowed us to enhance the gelation properties of MP with assorted appreciated functionalities and further improve the quality of meat products.