Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.
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ABSTRACT:
SUBMITTER: Zhao Z
PROVIDER: S-EPMC4749968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin β. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 μg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 μg/mL of chymotrypsin, and there was no decrease in function at up to 5 μg/mL of meprin β.
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Project description:There is a considerable interest in the use of structurally stable and catalytically active enzymes, such as cytochrome C (Cyt C), in the pharmaceutical and fine chemical industries. However, harsh process conditions, such as temperature, pH, and presence of organic solvents, are the major barriers to the effective use of enzymes in biocatalysis. Herein, we demonstrate the suitability of bio-based ionic liquids (ILs) formed by the cholinium cation and dicarboxylate-based anions as potential media for enzymes, in which remarkable enhanced activity and improved stability of Cyt C against multiple stresses were obtained. Among the several bio-ILs studied, an exceptionally high catalytic activity (> 50-fold) of Cyt C was observed in aqueous solutions of cholinium glutarate ([Ch][Glu]; 1g/mL) as compared to the commonly used phosphate buffer solutions (pH 7.2), and > 25-fold as compared to aqueous solutions of cholinium dihydrogen phosphate ([Ch][Dhp]; 0.5g/mL) -the best known IL for long term stability of Cyt C. The catalytic activity of the enzyme in presence of bio-ILs was retained against several external stimulus, such as chemical denaturants (H2O2 and GuHCl), and temperatures up to 120 °C. The observed enzyme activity is in agreement with its structural stability, as confirmed by UV-Vis, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopies. Taking advantage of the multi-ionization states of di/tri-carboxylic acids, the pH was switched from acidic to basic by the addition of the corresponding carboxylic acid and choline hydroxide, respectively. The activity was found to be maximum at a 1:1 ratio of [Ch][carboxylate], with a pH in the range from 3 to 5.5. Moreover, it was found that the bio-ILs studied herein protect the enzyme against protease digestion and allow long-term storage (at least for 21 weeks) at room temperature. An attempt by molecular docking was also made to better understand the efficacy of the investigated bio-ILs towards the enhanced activity and long term stability of Cyt C. The results showed that dicarboxylates anions interact with the active site's amino acids of the enzyme through H-bonding and electrostatic interactions, which are responsible for the observed enhancement of the catalytic activity. Finally, it is demonstrated that Cyt C can be successfully recovered from the aqueous solution of bio-ILs and reused without compromising its yield, structural integrity and catalytic activity, thereby overcoming the major limitations in the use of IL-protein systems in biocatalysis.
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Project description:Enzymatic catalysis is of great importance to the chemical industry. However, we are still scratching the surface of the potential of biocatalysis due to the limited operating range of enzymes in harsh environments or their low recyclability. The role of Metal-Organic Frameworks (MOFs) as active supports to help overcome these limitations, mainly by immobilization and stabilization of enzymes, is rapidly expanding. Here we make use of mild heating and a non-polar medium during incubation to induce the translocation of a small enzyme like protease in the mesoporous MOF MIL-101(Al)-NH2. Our proteolytic tests demonstrate that protease@MIL-101(Al)-NH2 displays higher activity than the free enzyme under all the conditions explored and, more importantly, its usability can be extended to extreme conditions of pH and high temperatures. MOF immobilization is also effective in providing the biocomposite with long-term stability, recyclability and excellent compatibility with competing enzymes. This simple, one-step infiltration strategy might accelerate the discovery of new MOF-enzyme biocatalysts that meet the requirements for biotechnological applications.
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Project description:In the developing area of modern nanobiotechnology, the research is being focused on enhancement of catalytic performance in terms of efficiency and stability of enzymes to fulfill the industrial demand. In the context of this interdisciplinary era, we isolated and identified alkaline protease producer Bacillus aryabhattai P1 by polyphasic approach and then followed one variable at a time approach to optimize protease production from P1. The modified components of fermentation medium (g/L) were wheat bran 10, soybean flour 10, yeast extract 5, NaCl 10, KH2PO4 1, K2HPO4 1 and MgSO4·7H2O 0.2 (pH 9). The optimum alkaline protease production from P1 was recorded 75 ± 3 U/mg at 35 °C and pH 9 after 96 h of fermentation period. Molecular weight of partially purified P1 alkaline protease was 26 KDa as revealed by SDS-PAGE. Calcium based nanoceramic material was prepared by wet chemical precipitation method and doped in native P1 protease for catalytic activity enhancement. Catalytic activity of modified P1 protease was attained by nanoactivator mediated modulation was more by 5.58 fold at pH 10 and 30 °C temperature. The nanoceramic material named as nanoactivator, with grain size of 40-60 nm was suitable to redesign the active site of P1 protease. Such types of modified proteases can be used in different nanobiotechnological applications.
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Project description:Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.
| S-EPMC6109922 | biostudies-literature
Project description:Promoting the activity of biological enzymes under in vitro environment is a promising technique for bioelectrocatalytic reactions, such as the conversion of carbon dioxide (CO2 ) into valuable chemicals, which is a promising strategy to address the environmental issue of CO2 in the atmosphere; however, this technique remains challenging. Herein, a nanocage structure for enzyme confinement is synthesized to enable the in situ encapsulation of formate dehydrogenase (FDH) in a porous metal-organic framework, which acts as a coenzyme and boosts the hybrid synergistic catalysis using enzymes. This study reveals that the synthesized FDH@ZIF-8 nanocage-structured hybrid (CSH) catalyst exhibits an improved catalytic ability of the enzymes and increases the hydrophobicity of the electrode and its affinity to CO2 . Thus, CSH can trap CO2 and control its microenvironments. The CSH catalyst boosts the conversion rate of CO2 to formic acid (HCOOH) to 28 times higher than that when using pure FDH. The in situ attenuated total reflectance surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) spectra indicates that OCHO* is the key intermediate. Density functional theory (DFT) calculations show that CSH has extremely low overpotential and is particularly effective for producing formate. This protection architecture for enzymes considerably promotes their biological application under in vitro environments.
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.
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Project description:A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions.
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Project description:The possible relationship between the thermal stability and the catalytic power of enzymes is of great current interest. In particular, it has been suggested that thermophilic or hyperthermophilic (Tm) enzymes have lower catalytic power at a given temperature than the corresponding mesophilic (Ms) enzymes, because the thermophilic enzymes are less flexible (assuming that flexibility and catalysis are directly correlated). These suggestions presume that the reduced dynamics of the thermophilic enzymes is the reason for their reduced catalytic power. The present paper takes the specific case of dihydrofolate reductase (DHFR) and explores the validity of the above argument by simulation approaches. It is found that the Tm enzymes have restricted motions in the direction of the folding coordinate, but this is not relevant to the chemical process, since the motions along the reaction coordinate are perpendicular to the folding motions. Moreover, it is shown that the rate of the chemical reaction is determined by the activation barrier and the corresponding reorganization energy, rather than by dynamics or flexibility in the ground state. In fact, as far as flexibility is concerned, we conclude that the displacement along the reaction coordinate is larger in the Tm enzyme than in the Ms enzyme and that the general trend in enzyme catalysis is that the best catalyst involves less motion during the reaction than the less optimal catalyst. The relationship between thermal stability and catalysis appears to reflect the fact that to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall preorganization process. Thus, the optimized catalysts are less stable. This trend is clearly observed in the DHFR case.
| S-EPMC3174049 | biostudies-literature
Project description:L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.
| S-EPMC5670125 | biostudies-literature