Hyperthermophilic xylanase and thermophilicity analysis by molecular dynamic simulation with quantum mechanics

Molecular dynamics simulations, at different scales, have been exploited for investigating complex mechanisms ruling biologically inspired systems. Nonetheless, with recent advances and unprecedented achievements, the analysis of molecular dynamics simulations requires customized workflows. In 2018, we developed Morphoscanner to retrieve structural relations within self-assembling peptide systems. In particular, we conceived Morphoscanner for tracking the emergence of β-structured domains in self-assembling peptide systems. Here, we introduce Morphoscanner2.0. Morphoscanner2.0 is an object-oriented library for structural and temporal analysis of atomistic and coarse-grained molecular dynamics (CG-MD) simulations written in Python. The library leverages MDAnalysis, PyTorch and NetworkX to perform the pattern recognition of secondary structure patterns, and interfaces with Pandas, Numpy and Matplotlib to make the results accessible to the user. We used Morphoscanner2.0 on both simulation trajectories and protein structures. Because of its dependencies on the MDAnalysis package, Morphoscanner2.0 can read several file formats generated by widely-used molecular simulation packages such as NAMD, Gromacs, OpenMM. Morphoscanner2.0 also includes a routine for tracking the alpha-helix domain formation.

Xylanase is efficient for xylan degradation and widely applied in industries. We found a GH11 family xylanase (Xyn11A) with high thermostability and catalytic activity from compost metatranscriptome. This xylanase has the optimal reaction temperature at 80°C with the activity of 2907.3 U/mg. The X-ray crystallographic structure shows a typical "right hand" architecture, which is the characteristics of the GH11 family enzymes. Comparing it with the mesophilic XYN II, a well-studied GH11 xylanase from Trichoderma reesei, Xyn11A is more compact with more H-bonds. Our mutagenic results show that the electrostatic interactions in the thumb and palm region of Xyn11A could result in its high thermostability and activity. Introducing a disulfide bond at the N-terminus further increased its optimal reaction temperature to 90°C with augmented activity. KEY POINTS: • A hyperthermophilic xylanase with high activity was discovered using the metatranscriptomic method. • The mechanisms of thermophilicity and high activity were revealed using X-ray crystallography, mutagenesis, and molecular dynamics simulations. • The thermostability and activity were further improved by introducing a disulfide bond.

Nowadays, oleogels have gained attentions as promising fat substitutes. Lipid-based compounds, mono- and diacylglycerols, fatty acids (FAs), fatty alcohols, waxes, sterols, ceramides, and phospholipids exhibit excellent oil-gelling potential due to their structural similarity to triacylglycerols. This review addresses how oleogels composition affects their nutritional functionality and further applications in food industry. The properties of oleogels structured by lipid-based compounds depend on structurant (concentration, type, ratio), solvent oil (type, FA profile, minor polar components), and processing conditions (cooling rate, shear force). Synergistic effects between FAs and fatty alcohols have been confirmed, and a thermodynamics-based screening model has been developed to assist identification of potential oil-structuring combinations, though further validation is needed. Oleogel offers health benefits contributed by its structure, oleogelator, and solvent oil type, including replacing harmful fats, delaying lipid release, inhibiting lipid-lipase interactions, and delivering bioactives, but long-term safety studies, especially for FAs and fatty alcohols, are required. Thus, modulating gel processing conditions and the composition of structurants and solvent oils can tailor the properties of oleogels, enhancing their nutritional function and broadening potential applications in food industry with desired product quality. Oleogels have been successfully applied in bakery, dairy, meats, spreads, margarine, confectionary, frying medium, and delivery systems, with complete fat substitution strategies developed for cheese, meat, spreads, and margarines. Further research could explore their nutritional roles and applications in functional foods, particularly considering the regulatory limitations and the influence of long-term consumption on human metabolisms.

The mammalian tetrahydrobiopterin (BH4)-dependent phenylalanine hydroxylases (PAH), involved in important metabolic pathways of phenylalanine, belong to non-heme iron-containing aromatic acid hydroxylases’ enzyme (AAH) family. AAHs utilize BH4 as protein co-factor and thus promote hydroxylation reactions of their substrates. Any alterations in BH4 -mediated AAH’s pathway or mutations in these enzymes are responsible for various disorders, and thus highlights the importance of mutational analysis to assess the effect on their biosynthetic pathways. Our present studies are aimed at single-site mutations in PAH that lead to thermodynamic stability change upon folding and further validation of designed non-reduced BH2 designed co-factors. We have presented single-site mutational analysis of PAH where single-site mutations have been identified from known literature. Further, in silico studies with the PAH, in silico mutant PAH, and crystallized known mutant A313T forms, involved QM/MM and Molecular Dynamics (MD) simulations analysis. The modified co-factor A showed high affinity with PAH and all mutant PAH with high G-score of −14.851. The best pose high affinity co-factor A subjected to QM/MM optimization which leads to square-pyramidal coordination of non-heme active site. The structural and energetic information obtained from the production phase of 20 ns MD simulation of co-factor-metalloprotein complex results helped to understand the binding mode and involvement of three molecules throughout the reaction pathways’ catalysis of PAH. The free energies of binding (dG) of A were found to be −68.181 kcal/mol and −72.249 for 1DMW and 1TDW for A313T mutant. Binding of Co-factor A do not perturb the coordination environment of iron at the active site which resides in 2-Histdine and 1-Glutamate triad, and may enhance the percentage response towards co-factor-mediated therapy.

Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene, Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated Tm values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.

"Everything that living things do can be understood in terms of jigglings and wigglings of atoms." Richard Feynman's remarks in the early 1960's summarize what is today widely accepted, namely, that biological processes can be described by the dynamics of biomolecules. Molecular dynamics (MD) simulation, in this regard, is the main methodology employed in structural biology to explore the dynamical behavior of macromolecules at a microscopic level. Aided by MD, researchers have been able, for instance, to resolve atomic structures of multi-protein complexes from cryo-EM densities, thus unveiling the atomistic details of enzymatic mechanisms and characterize the binding of small molecules to proteins. To achieve all this, the capabilities of MD packages are constantly evolving, providing a multitude of complex simulation and analysis techniques, e.g., enhanced sampling and free energy calculations. Although applicable to a wide variety of research problems, a broader usage of MD is hindered by a steep initial learning curve imposed by nearly every MD software. To reduce this initial barrier and make the methodology more accessible to the general community of biomolecular researchers, we developed an intuitive tool named QwikMD (1), which assists the users in the preparation, execution, and analysis of biomolecular MD simulations. Among many other features, QwikMD automatically checks the initial structure for structural inconsistencies, facilitates structure manipulations such as point mutations and partial deletions, simplifies the protein insertion in lipid membranes and enables the visualization and analysis of MD simulations on the fly. The user-friendly graphical interface of QwiKMD allows the preparation of MD simulations in a point-and-click fashion, offering the user multiple MD protocols, such as unbiased MD simulations, Steered MD, MD Flexible Fitting (MDFF), and, most recently, hybrid QM/MM simulations. The latter exploits the recently developed VMD and NAMD interface to common quantum mechanics software packages. QwikMD facilitates performing MD simulations for nearly any user, novice or expert. While assisting the user, QwikMD ensures reproducibility of the results by recording all parameters and steps into two log files, one in a script-like format and another in a "methods section" format. QwikMD also serves as a learning tool, providing the theoretical background of the different MD protocols and options in many "info buttons". J. V Ribeiro et al., QwikMD — Integrative Molecular Dynamics Toolkit for Novices and Experts. Sci. Rep. 6, 26536 (2016).

In the present study, 300 plant derived secondary metabolites (100 each of alkaloid, flavonoid, and terpenoid), have been screened for their anti-cancerous activity through inhibition of selected key enzymatic targets, namely cyclooxygenases (COXs), topoisomerases (Topos), and aromatase by molecular docking approach. Furthermore, the stability of the complexes of top hits, from each class of secondary metabolites, with their respective enzymatic targets was analyzed using molecular dynamics (MD) simulation analyses and binding free energy calculations. Analysis of the results of the docking in light of the pharmacokinetically screened 18 alkaloids, 26 flavonoids, and 9 terpenoids, revealed that the flavonoid, curcumin, was the most potent inhibitor for all the selected enzymatic targets. The stability of the complexes of COX-1, COX-2, Topo I, Topo IIβ and aromatase with the most potent inhibitor curcumin and those of the respective drugs, namely ibuprofen, aspirin, topotecan, etoposide, and exemestane were also analyzed through MD simulation analyses which revealed better stability of curcumin complexes than those of respective drugs. Binding energy calculations of the complexes of the curcumin with all the targets, except those of Topos, exhibited lower binding energies for the curcumin complexes than those of respective drugs which corroborated with the results of molecular docking analyses. Thus, the present study affirms the versatile and multipronged nature of curcumin, the traditionally used herbal medicine, as anti-cancer molecule directed against these enzymatic targets.

This study investigates the biochemical properties of two xylanases, ZgXyn10A and CaXyn10B, which are members of the glycoside hydrolase family 10 (GH10) and originate from the marine Bacteroidetes species Zobellia galactanivorans and Cellulophaga algicola, respectively. Utilizing an auto-induction expression system in Escherichia coli, high-purity recombinant forms of these enzymes were successfully produced. Biochemical assays revealed that ZgXyn10A and CaXyn10B exhibit optimal activities at 40 °C and 30 °C, respectively, and demonstrate a high sensitivity to temperature fluctuations. Unlike conventional low-temperature enzymes, these xylanases retain only a fraction of their maximal activity at lower temperatures. To gain deeper insights into the structural and functional properties of these marine xylanases, two thermostable GH10 xylanases, TmxB and CoXyn10A, which share comparable amino acid sequence identity with ZgXyn10A and CaXyn10B, were selected for structural comparison. All four marine xylanases share a nearly similar three-dimensional structural topology. Molecular dynamics simulation indicated a striking difference in structural fluctuations between the low-temperature and thermostable xylanases, as evidenced by the distinct root mean square deviation values. Moreover, root mean square fluctuation analysis specifically identified the β3-α3 and β7-α7 loop regions within the substrate-binding cleft as crucial determinants of the temperature characteristics of these GH10 xylanases. Our findings establish loop dynamics as a key evolutionary driver in the thermal adaptation of GH10 xylanases and propose a loop engineering strategy for the development of industrial biocatalysts with tailored temperature responses, particularly for lignocellulosic biomass processing under moderate thermal conditions.

Hydrocolloids: Structure, preparation method, and application in food industry

Enzymes are readily inactivated in harsh micro-environment due to changes in pH, temperature, and ionic strength. Developing suitable and feasible techniques for stabilizing enzymes in food sector is critical for preventing them from degradation. This review provides an overview on chitosan (CS)-based enzymes encapsulation techniques, enzyme release mechanisms, and their applications in food industry. The challenges and future prospects of CS-based enzymes encapsulation were also discussed. CS-based encapsulation techniques including ionotropic gelation, emulsification, spray drying, layer-by-layer self-assembly, hydrogels, and films have been studied to improve the encapsulation efficacy (EE), heat, acid and base stability of enzymes for their applications in food, agricultural, and medical industries. The smart delivery design, new delivery system development, and in vivo releasing mechanisms of enzymes using CS-based encapsulation techniques have also been evaluated in laboratory level studies. The CS-based encapsulation techniques in commercial products should be further improved for broadening their application fields. In conclusion, CS-based encapsulation techniques may provide a promising approach to improve EE and bioavailability of enzymes applied in food industry.HighlightsEnzymes play a critical role in food industries but susceptible to inactivation.Chitosan-based materials could be used to maintain the enzyme activity.Releasing mechanisms of enzymes from encapsulators were outlined.Applications of encapsulated enzymes in food fields was discussed.

Microbial transglutaminase (MTG) has numerous industrial applications in the food and pharmaceutical sectors. Unfortunately, the thermostability of MTG is too low to tolerate the desired conditions used in many of these commercial processes. In a previous study, we used protein engineering to improve the thermostability of MTG. Specifically, we generated a T7C/E58C mutant of MTG from Streptomyces mobaraensis that displayed enhanced resistance to thermal inactivation. In this study, a rational structure-based approach was adopted to introduce a disulfide bridge to further increase the thermostability of MTG. In all, four new mutants, each containing a novel disulfide bond, were engineered. Of these four mutants, D3C/G283C showed the most promising thermostability with a significantly higher ∆T50 (defined as the temperature of incubation at which 50% of the initial activity remains) of + 9°C by comparison to wild-type MTG. Indeed, D3C/G283C combined enhanced thermostability with a 2.1-fold increased half-life at 65°C compared with the wild-type enzyme. By structure-based rational design, we were able to create an MTG variant which might be useful for expanding the scope of application in food. KEY POINTS: • Microbial transglutaminase (MTG) is an enzyme used in many food applications • The applicability of MTG to various industrial processes other than the food sector is being investigated • Improvement of thermostability was confirmed for the disulfide bridge mutant D3C/G283C.

Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification.

This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of 2-4.6. The influence of dynamical fluctuations of the protein structure is investigated for Bacillus agaradhaerens and Aspergillus kawachii xylanase using molecular dynamics (MD) simulations to provide snapshots from which average values can be computed. This decreases the respective predicted pH optima from 6.2-6.7 and 4.8 to 5.3 +/- 0.3 and 4.0 +/- 0.2, respectively, which are in better agreement with the observed values of 5.6 and 2, respectively. The change is primarily due to structural fluctuations of an Arg residue near the catalytic nucleophile, which lowers its pKa value compared to using the X-ray structure. The MD simulations and some X-ray structures indicate that this Arg residue can form a hydrogen bond to the catalytic base, and it is hypothesized that this hydrogen bond is stabilized by an additional hydrogen bond to another Glu residue present only in acidic xylanases. Formation of such a hydrogen bond is predicted to lower the pH optimum of A. kawachii xylanase to 2.9 +/- 0.3, which is in reasonable agreement with the observed value of 2. The predicted pH of optimal stability is in excellent agreement with the pH value at which the melting temperature (Tm) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and Tm, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pKa values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pKa values significantly lower than standard values.

The structure of xylan, which has a 1,4-linked β-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and β-xylosidase was also found to significantly accelerate the conversion of XOS into xylose.

Xylanases have important industrial applications but are most extensively utilized in the pulp and paper industry as a pre-bleaching agent. We characterized a xylanase from Bacillus amyloliquefaciens strain SK-3 and studied it for kraft pulp bleaching. The purified enzyme had a molecular weight of ~50 kDa with optimal activity at pH 9.0 and 50 °C. The enzyme showed good activity retention (85%) after 2 h incubation at 50 °C and pH 9.0. This enzyme obeyed Michaelis–Menten kinetics with regard to beechwood xylan with Km and Vmax values of 5.6 mg/ml, 433 μM/min/mg proteins, respectively. The enzyme activity was stimulated by Mn2+, Ca2+ and Fe2+ metal ions. Further, it also showed good tolerance to phenolics (2 mM) in the presence of syringic acid (no loss), cinnamic acid (97%), benzoic acid (94%) and phenol (97%) activity retention. The thermostability of xylanase was increased by 6.5-fold in presence of sorbitol (0.75 M). Further, pulp treated with 20U/g of xylanase (20IU/g) alone and with sorbitol (0.75M) reduced kappa number by 18.3 and 23.8%, respectively after 3 h reaction. In summary, presence of xylanase shows good pulp-bleaching activity, good tolerance to phenolics, lignin and metal ions and is amenable to thermostability improvement by addition of polyols. The SEM image showed significant changes on the surface of xylanase-treated pulp fiber as a result of xylan hydrolysis.