Vmax Values Research Articles

Efficient degradation of aqueous dichloromethane by an enhanced microbial electrolysis cell: Degradation kinetics, microbial community and metabolic mechanisms

Dichloromethane (DCM) has been listed as a toxic and harmful water pollutant, and its removal needs attention. Microbial electrolysis cells (MECs) are viewed as a promising alternative for pollutant removal, which can be strengthened from two aspects: microbial inoculation and acclimation. In this study, the MEC for DCM degradation was inoculated with the active sludge enhanced by Methylobacterium rhodesianum H13 (strain H13) and then acclimated in the form of a microbial fuel cell (MFC). Both the introduction of strain H13 and the initiation in MFC form significantly promoted DCM degradation. The degradation kinetics were fitted by the Haldane model, with Vmax, Kh, Ki and vmax values of 103.2 mg/L/hr, 97.8 mg/L, 268.3 mg/L and 44.7 mg/L/hr/cm2, respectively. The cyclic voltammogram implies that DCM redox reactions became easier with the setup of MEC, and the electrochemical impedance spectrogram shows that the acclimated and enriched microbes reduced the charge transfer resistance from the electrode to the electrolyte. In the biofilm, the dominant genera shifted from Geobacter to Hyphomicrobium in acclimation stages. Moreover, Methylobacterium played an increasingly important role. DCM metabolism mainly occurred through the hydrolytic glutathione S-transferase pathway, given that the gene dcmA was identified rather than the dhlA and P450/MO. The exogenous electrons facilitated the reduction of GSSG, directly or indirectly accelerating the GSH-catalyzed dehalogenation. This study provides support for the construction of an efficient and stable MEC for DCM removal in water environment.

Metabolism characterization and chemical and plasma stability of casearin B and caseargrewiin F.

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory and antiulcerogenic activities. The clerodane diterpenes casearin B (cas B) and caseargrewiin F (casg F) are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of cas B and casg F were not previously investigated. We aimed to assess the stability of cas B and casg F in physiological conditions and their metabolism in human liver microsomes (HLM). The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of cas B and casg F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism is not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed log P in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with KM values of 61.4 μM and 66.4 μM and Vmax values of 327 and 648 nmol/min/mg of protein for cas B and casg F, respectively. Metabolism parameters in HLM were extrapolated to predict human hepatic clearance and suggest that casg F and cas B have a high hepatic extraction ratio. In conclusion, our data suggest that casg F and cas B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.

Nanoarchitectonic approaches for measuring the catalytic behavior of a membrane anchored enzyme. From Langmuir-Blodgett to a novel Langmuir-Schaefer based nanofilm building device

Self-organized lipid monolayers at the air-water interface (Langmuir films, LF) are commonly used for measuring the catalytic properties of membrane-bound enzymes. This methodology allows to provide a consistent flat topography molecular density, packing defects and thickness. The aim of the present work was to show the methodological advantages of using the horizontal transfer method (Langmuir-Schaefer) with respect to the vertical transfer method (Langmuir-Blodgett) when mounting a device to measure catalytic activity of membrane enzymes. Based on the results obtained we can conclude that it is possible to prepare stable Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) films from Bovine Erythrocyte Membranes (BEM) preserving the catalytic activity of its native Acetylcholinesterase (BEA). In comparison, the LS films showed Vmax values more similar to the enzyme present in the vesicles of natural membranes. In addition, it was much easier to produce large amounts of transferred areas with the horizontal transfer methodology. It was possible to decrease the time required to mount an assay with numerous activity points, such as building activity curves as a function of substrate concentration. The present results show that LSBEM provides a proof of concept for the development of biosensors based on transferred purified membrane for the screening of new products acting on an enzyme embedded on its natural milieu. In the case of BEA, the application of these enzymatic sensors could have medical interest, providing drug screening tools for the treatment of Alzheimer's disease.

The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sites.

Mammalian protein arginine methyltransferase 7 (PRMT7) has been shown to target substrates with motifs containing two arginine residues separated by one other residue (RXR motifs). In particular, the repression domain of human histone H2B (29-RKRSR-33) has been a key substrate in determining PRMT7 activity. We show that incubating human PRMT7 and [3H]-AdoMet with full-length Xenopus laevis histone H2B, containing the substitutions K30R and R31K (RKRSR to RRKSR), results in greatly reduced methylation activity. Using synthetic peptides, we have now focused on the enzymology behind this specificity. We show for the human and Xenopus peptide sequences 23-37 the difference in activity results from changes in the Vmax rather than the apparent binding affinity of the enzyme for the substrates. We then characterized six additional peptides containing a single arginine or a pair of arginine residues flanked by glycine and lysine residues. We have corroborated previous findings that peptides with an RXR motif have much higher activity than peptides that contain only one Arg residue. We show that these peptides have similar apparent km values but significant differences in their Vmax values. Finally, we have examined the effect of ionic strength on these peptides. We found the inclusion of salt had little effect on the Vmax value but a considerable increase in the apparent km value, suggesting that the inhibitory effect of ionic strength on PRMT7 activity occurs largely by decreasing apparent substrate-enzyme binding affinity. In summary, we find that even subtle substitutions in the RXR recognition motif can dramatically affect PRMT7 catalysis.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 μmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Biodegradation characteristics and mechanism of terbuthylazine by the newly isolated Agrobacterium rhizogenes strain AT13

Terbuthylazine (TBA) is an emerging environmental contaminant that poses moderate to high risk to non-target organisms. In this study, a newly TBA-degrading strain, Agrobacterium rhizogenes AT13, was isolated. This bacterium degraded 98.7% of TBA (100 mg/L) within 39 h. Based on the six detected metabolites, three novel pathways of strain AT13, including dealkylation, deamination–hydroxylation, and ring-opening reactions, were proposed. The risk assessment demonstrated that most degradation products might be substantially less harmful than TBA. Whole-genome sequencing and RT-qPCR analysis revealed that ttzA, which encodes S-adenosylhomocysteine deaminase (TtzA), is closely related to TBA degradation in AT13. Recombinant TtzA showed 75.3% degradation of 50 mg/L of TBA within 13 h and presented a Km value of 0.299 mmol/L and a Vmax value of 0.041 mmol/L/min. The molecular docking results indicated that the binding energy of TtzA to TBA was −32.9 kcal/mol and TtzA residue ASP161 formed two hydrogen bonds with TBA at distances of 2.23 and 1.80 Å. Moreover, AT13 efficiently degraded TBA in water and soil. Overall, this study provides a foundation for the characterization and mechanism of TBA biodegradation and may enhance our understanding of the TBA biodegradation by microbes.

The catalytic and kinetic characterization of Bacillus subtilis MK775302 milk clotting enzyme: comparison with calf rennet as a coagulant in white soft cheese manufacture

BackgroundCalf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purified Bacillus subtilis MK775302 MCE and to assess the role of enzyme in cheese manufacture. ResultsB. subtilis MK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculated Km and Vmax values were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purified B. subtilis MK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties. ConclusionsThe partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.

Laccase immobilized in adamantane-modified PEGMA-based microgels: Tuning enzyme activity by beta-cyclodextrin

Intelligent immobilized enzymes can be constructed by designing “smart” carrier materials. In this study, we successfully prepared adamantane-modified PEGMA-based microgel-immobilized laccases (AdM-Lac) by precipitation polymerization in a laccase solution. AdM-Lac-DMCD was obtained by mixing 2,6-di-O-methyl-beta-cyclodextrin (DMCD) and AdM-Lac. By comparison, DLS, AFM, and IR results indicate that AdM-Lac exhibits DMCD-induced volume phase transition behavior. NMR analysis confirms the successful formation of inclusion complexes of DMCDs and adamantyl groups on AdM-Lac. The catalytic tests show that the enzyme activity of AdM-Lac-DMCD at 20 °C (24.09 U/L) is ca.70% higher than AdM-Lac (14.20 U/L). The Vmax value of AdM-Lac-DMCD (1.22 µM min−1) is similar to AdM-Lac (1.24 µM min−1), Whereas the Km value (52 µM) decreases by ca. 69%, even close to free laccase (41 µM), and the Vmax/Km ratio (0.0235 min−1) increases by about 2-fold. These results imply that DMCD initiates the volume swelling of AdM-Lac, resulting in increased enzyme activity and improved kinetic properties. Due to AdM-Lac having better storage stability, we suggest that laccase is stored as AdM-Lac, and applied as AdM-Lac-DMCD. This study successfully achieved the regulation of enzyme activity through the addition of molecules, providing a novel strategy for the design of “smart” carrier materials.

Heterologous Expression and Biochemical Characterization of a Thermostable Endoglucanase (MtEG5-1) from Myceliophthora thermophila

Thermophilic endoglucanases have become of significant interest for effectively catalyzing the hydrolysis of cellulose. Myceliophthora thermophila is an ideal source of thermophilic enzymes. Interestingly, different hosts differently express the same enzymes. In this study, we successfully overexpressed endoglucanase (MtEG5-1) from M. thermophila in the methylotrophic yeast, Pichia pastoris GS115, via electroporation. We found that purified MtEG5-1 exhibited optimum activity levels at pH 5 and 70 °C, with 88% thermal stability after being incubated at 70 °C for 2 h. However, we observed that purified MtEG5-1 had a molecular weight of 55 kDa. The Km and Vmax values of purified MtEG5-1 were approximately 6.11 mg/mL and 91.74 μmol/min/mg at 70 °C (pH 5.0), respectively. Additionally, the optimum NaCl concentration of purified MtEG5-1 was found to be 6 g/L. Furthermore, we observed that the activity of purified MtEG5-1 was significantly enhanced by Mn2+ and was inhibited by K+. These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33. These properties of MtEG5-1 make it highly suitable for future academic research and industrial applications.

Immobilization of lipase on hydrophobic MOF synthesized simultaneously with oleic acid and application in hydrolysis of natural oils for improving unsaturated fatty acid production

The hydrolysis of natural oils (vegetable oils and fats) by lipase has significant applications in food and medicine. However, free lipases are usually sensitive to temperature, pH and chemical reagents in aqueous solutions, which hinders their widespread industrial application. Excitingly, immobilized lipases have been widely reported to overcome these problems. Herein, inspired by lipase interface activation, a hydrophobic Zr-MOF (UiO-66-NH2-OA) with oleic acid was synthesized for the first time in an emulsion consisting of oleic acid and water, and the Aspergillus oryzae lipase (AOL) was immobilized onto the UiO-66-NH2-OA through hydrophobic interaction and electrostatic interaction to obtain immobilized lipase (AOL/UiO-66-NH2-OA). 1H NMR and FT-IR data indicated that oleic acid was conjugated with the 2-amino-1,4-benzene dicarboxylate (BDC-NH2) by amidation reaction. As a result, the Vmax and Kcat values of AOL/UiO-66-NH2-OA were 179.61 μM﹒min−1 and 8.27 s−1, which were 8.56 and 12.92 times higher than those of the free enzyme, respectively, due to the interfacial activation. After treated at 70 °C for 120 min, the immobilized lipase maintained 52 % of its original activity, but free AOL only retained 15 %. Significantly, the yield of fatty acids by the immobilized lipase reached 98.3 % and still exceeded 82 % after seven times of recycling.

A HIGHLY PURIFIED L-GLUTAMINASE FROM IMMOBILIZED PSEUDOMONAS SP. RAS123 CULTURES WITH ANTITUMOR AND ANTIBACTERIAL ACTIVITIES

L-glutaminase (E.C.3.5.2.1) is an antineoplastic enzyme and in the present study, an extracellular L-glutaminase was produced from a marine local strain identified as Pseudomonas sp. RAS123. The enzyme was produced from free cultures and from cultures immobilized on and in different supports. Pseudomonas sp. RAS123 L-glutaminase produced from immobilized cultures was purified to homogeneity. The specific activity of the enzyme reached 698.655 U/mg protein, with Km and Vmax value of 3.2 mg/ml and 2000 U/ml, respectively. A single band with a molecular weight of about 32.0 kDa was produced by the purified enzyme on SDS-PAGE. Further findings indicated that the pure enzyme's maximum activity occurred at 50°C and pH 9. The enzyme was stable at 60°C for 60 min and in the pH range of 8.0 to 10.0, The effect of chemicals showed that Mn2+, Mg2+, Ni2+ and Fe2+activated the enzyme, while SDS (10% w/v) strongly inhibited the activity of the enzyme. The purified enzyme showed cytotoxic activity against HCT-116, HepG2, MCF-7, HeLa, and CCL-86 cell lines tested with IC50 values of 122, 175, 195, 306, and &gt; 500 µg/ml, respectively. Also, the antibacterial effect of the enzyme showed activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus mutants, Enterobacter cloacae and Escherichia coli. These findings demonstrate that L-glutaminase might be used in numerous biotechnological applications, particularly food and pharmaceutical processing.

Production, purification, and characterization of p-diphenol oxidase (PDO) enzyme from lignolytic fungal isolate Schizophyllum commune MF-O5.

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68kDa and showed stability at lower temperature (30°C) for 1h. The Km and Vmax values of the purified PDO recorded were 2.48mM and 6.20 U/min. Thermal stability results showed that at 30°C PDO had 119.17kJ/K/mol Ea value and 33.64min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1mM showing 12.04% activity. The enzyme showed stability against 10mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.

Amorphization-Modulated Metal Sulfides with Boosted Active Sites and Kinetics for Efficient Enzymatic Colorimetric Biodetection.

Colorimetric biosensing has become a popular sensing method for the portable detection of a variety of biomarkers. Artificial biocatalysts can replace traditional natural enzymes in the fields of enzymatic colorimetric biodetection; however, the exploration of new biocatalysts with efficient, stable, and specific biosensing reactions has remained challenging so far. Here, to enhance the active sites and overcome the sluggish kinetics of metal sulfides, the creation of an amorphous RuS2 (a-RuS2 ) biocatalytic system is reported, which can dramatically boost the peroxidase-mimetic activity of RuS2 for the enzymatic detection of diverse biomolecules. Due to the existence of abundant accessible active sites and mildly surface oxidation, the a-RuS2 biocatalyst displays a twofold Vmax value and much higher reaction kinetics/turnover number (1.63 × 10-2 s-1 ) compared to that of the crystallized RuS2 . Noticeably, the a-RuS2 -based biosensor shows an extremely low detection limit of H2 O2 (3.25× 10-6 m), l-cysteine (3.39× 10-6 m), and glucose (9.84× 10-6 m), respectively, thus showing superior detection sensitivity to many currently reported peroxidase-mimetic nanomaterials. This work offers a new path to create highly sensitive and specific colorimetric biosensors in detecting biomolecules and also provides valuable insights for engineering robust enzyme-like biocatalysts via amorphization-modulated design.

Isolation and Characterization of Extracellular Chitinase Produced by Chitinolytic Bacteria Isolated from Soil Samples

Chitin is one of the most common biopolymers found in both marine and terrestrial habitats. Chitinase enzyme has gained popularity due to its diverse biotechnological applications, particularly in agriculture for the biocontrol of phytopathogenic fungi and harmful insects. In this work bacteria producing chitinase enzyme were isolated from soil and water samples. Three bacterial isolates C2, C3, and C4 were found to be Gram-negative coccobacilli while bacterial isolate C9 was found to be Gram-positive cocci. The optimum pH and temperature for all the isolates were studied. The effect of pH, temperature, substrate variation, and incubation period on enzyme activity was studied. The activity of the chitinase enzyme for all the bacterial isolates increased with an increase in temperature up to 37°C. However, a further rise in reaction temperature caused a loss of chitinase activity. Michaelis-Menten plot and Line weaver Burk plot were constructed to calculate the Km and Vmax values. Partial Purification of the chitinase enzyme was carried out and the enzyme activity, protein content, specific activity, and purification fold were estimated. It was observed that the enzyme activity of the crude sample was less than the fractionated sample. The highest enzyme activity was found to be at a 40% fraction. Of the four isolates, C2 was the best in its enzyme activity. The future prospect is the production of chitinases at an industrial scale for various biotechnological applications. Chitinase-producing isolates have the ability to produce chitinase between temperatures 22ºC and 40ºC which is the field temperature for the cultivation of most the crop, so it may be applicable to field conditions against plant pathogenic fungi which is the major problem for agricultural food production. The management of seafood waste businesses may potentially benefit from the usage of this enzyme.

Influence of Mineral Fertilizers on the Dynamics of Biomass Aaccumulation and Plant Growth of Winter Rye

The dynamics of biomass and height of Secale cereale L. plants was studied in a long-term stationary field experiment on sod-podzolic light loamy soil. Systematic application of mineral fertilizers (option 2 – N65P50K50, option 3 – N100P75K75) had a significant impact on the height and biomass of plants at all tested stages of vegetation (eight time points within 50 days). To describe the dynamics of biomass and height of Secale cereale L. plants, sigmoid (logistic) and linear models are well suited, which made it possible to calculate such parameters as maximum biomass and height (M2, g : H2, cm), maximum speed (Vmax, g ⋅ day–1 (cm ⋅ day–1)) and average speed (b, g ⋅ day–1 (cm ⋅ day–1)). The Vmax values of the growth of raw plant biomass in variants 2 and 3 exceeded this indicator for the control variant by 1.7–2.0 times; Vmax of plant height growth varied within 4.2–4.6 cm ⋅ day–1, did not depend on the application of mineral fertilizers. The application of mineral fertilizers led to an increase in the average growth rate of the biomass of plants Secale cereale L. (b, g ⋅ day–1), in relation to the control, by more than 2 times.

Evaluation of Bacillus aryabhattai B8W22 peroxidase for phenol removal in waste water effluents

Environmental contamination by phenol has been reported in both aquatic and atmospheric environments. This study aimed to separate and purify the peroxidase enzyme from bacteria that degrade phenol from wastewater sources. An enrichment culture of MSM was used to screen 25 bacterial isolates from different water samples for peroxidase production, six of the isolates exhibited high levels of peroxidase enzyme activity. Qualitative analysis of peroxidase revealed that isolate No. 4 had the highest halo zones (Poly-R478: 14.79 ± 0.78 mm, Azure B: 8.81 ± 0.61 mm). The promising isolate was identified as Bacillus aryabhattai B8W22 by 16S rRNA gene sequencing with accession number OP458197. As carbon and nitrogen sources, mannitol and sodium nitrate were utilized to achieve maximum peroxidase production. A 30-h incubation period was used with pH 6.0, 30 °C, mannitol, and sodium nitrate, respectively, for maximal production of peroxidase. Purified peroxidase enzyme showed 0.012 U/mg specific activity, and SDS-PAGE analysis indicated a molecular weight of 66 kDa. The purified enzyme exhibits maximum activity and thermal stability at pH values of 4.0 and 8.0, respectively, with maximum activity at 30 °C and complete thermal stability at 40 °C. In the purified enzyme, the Km value was 6.942 mg/ml and the Vmax value was 4.132 mol/ml/hr, respectively. The results demonstrated that Bacillus aryabhattai B8W22 has promising potential for degrading phenols from various phenol-polluted wastewater sources.

Chitinase from Streptomyces mutabilis as an Effective Eco-friendly Biocontrol Agent.

Blood sucking parasites not only cause economic loss but also transmit numerous diseases. Dermanyssus gallinae, an obligatory blood feeding ectoparasite causes huge production loss to the poultry industry. Mosquitoes act as vector for transmitting several viral and parasitic diseases in humans. Acaricide resistance limits the control of these parasites. The present study was aimed to control the parasites using chitinase that have selective degradation of chitin, an important component in exoskeleton development. Chitinase was induced in Streptomyces mutabilis IMA8 with chitin extracted from Charybdis smithii. The enzyme showed more than 50% activity at 30-50°C and the optimum activity at 45°C. The enzyme activity of chitinase was highest at pH 7.0. The kinetic parameters Km and Vmax values of chitinase were determined by non-linear regression using Michaelis-Menten equation and its derivative Hanes-Wolf plot. The larvicidal effect of different concentrations of chitinase was evaluated against all instar larvae (I-IV) and pupae of An. stephensi and Ae. aegypti after 24h of exposure. The percentage of mortality was directly proportional to the chitinase concentration. Bioassay for miticidal activity showed that chitinase had excellent miticidal activity (LC50 = 24.2ppm) against D. gallinae. The present study suggested the usage of Streptomyces mutabilis for preparation of chitinase in mosquito and mite control.

Molecular cloning, expression analysis and functional characterization of chicken cytochrome P450 27A1: A novel mitochondrial vitamin D3 25-hydroxylase

Vitamin D3 is hydroxylated by cytochrome P450 (CYP) before exerting biological effects. The chicken CYP involved in vitamin D3 25-hydroxylation has yet to be cloned, and little is known about its functional characteristics, tissue distribution, and cellular expression. We identified a novel, full-length CYP27A1 gene cloned from chicken hepatocyte cDNA that encodes a putative protein of 518 amino acids. Swiss modeling revealed that chicken CYP27A1 has a classic open-fold form. Multisequence homology alignment determined that CYP27A1 contains conserved motifs for substrate recognition and binding. Quantitative real-time PCR analysis in 2-mo-old Partridge Shank broilers demonstrated that CYP27A1 mRNA levels were highest in the liver, followed by the thigh muscles, the breast muscles, and kidneys. The transcripts of CYP27A1 in breast muscles were significantly higher in males than in females. A subcellular localization analysis demonstrated that CYP27A1 was mainly expressed in the mitochondria. In vitro enzyme assays suggested that recombinant CYP27A1 hydroxylates vitamin D3 at the C-25 position to form 25-hydroxyvitamin D3 (25(OH)D3). The Km and Vmax values for CYP27A1-dependent vitamin D3 25-hydroxylation were estimated to be 4.929 μM and 0.389 mol min-1 mg-1 protein, respectively. In summary, these results suggest that CYP27A1 encodes a mitochondrial CYP that plays an important physiologic role in the 25-hydroxylation of vitamin D3 in chickens, providing novel insights into vitamin D3 metabolism in this species.

Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from Thermococcus radiotolerans DSM-15228

Backgroundβ-glucosidase (EC 3.2.1.21) catalyzing the β-glycosidic linkages in polysaccharides is a ubiquitous enzyme with great importance in biofuel and other industries. Enzyme inhibition by glucose has been considered as the major hurdle in the practical applications of this enzyme. Therefore, there has been a continuous search for novel β-glucosidase with high glucose tolerance and stability at industrial temperature. In the present study, recombinant of β-glucosidase from Thermococcus radiotolerans has been produced and characterized. ResultsThe enzyme was overexpressed in Escherichia coli strain BL21 (DE3) codon plus RIPL and purified by selective heat denaturation, ethanol precipitation and anion exchange chromatography. Purified enzyme displayed a band on SDS-PAGE with a molecular weight of 50 kDa. Optimum enzyme activity was found at pH 5, and 85°C, it retained more than 46% activity when incubated at 100°C for 5 min and exhibited 80% activity in the presence of 800 mM glucose. Km and Vmax values of the purified enzyme were found as 16.3 mM of pNPG and 25.8 µ moles per min. Molecular docking studies have shown a strong binding affinity of pNPG with the enzyme active site consisting of Glu365, Asn266, and Trp295 as major the active site amino acids. MD simulation analysis has shown a significantly high stability of enzyme active site and high potential of enzyme substrate complex formation. ConclusionsThe novel characteristics such as relatively low Km value, extremely high-temperature stability and tolerance of high glucose concentration advocate the enzyme as a potential candidate for the industrial applications.How to cite: Albalawi H, Altayeb HN, Iftikhar S, et al. Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from T. radiotolerans DSM-15228. Electron J Biotechnol 2023; 64. https://doi.org/10.1016/j.ejbt.2023.03.002.

The signal conversion strategy using the lytic transglycosylase Cag4-double nanoporous gold co-catalysis for the rapid screening of drugs against Helicobacter pylori infection

The cag pathogenicity island (cagPAI) is the main virulence factor of gastric carcinoma induced by Helicobacter pylori (H. pylori). The lytic transglycosylase Cag4 is an important component that assists in the translocation of the bacterial oncoprotein CagA and maintains the peptidoglycan cycle. The allosteric regulation of Cag4 has been preliminarily demonstrated to inhibit H. pylori infection. Unfortunately, a rapid screening technology for allosteric regulators of Cag4 has not been established. In this study, a novel Cag4-double nanoporous gold (NPG) biosensor based on enzyme-inorganic co-catalysis was constructed using the heterologously expressed H. pylori 26695 Cag4 as the biological recognition element for screening Cag4 allosteric regulators. The results showed that chitosan or carboxymethyl chitosan was a mixed Cag4 inhibitor combining non-competition with uncompetition. The inhibition constants were Ki' Chitosan = 0.88909 mg/mL and Ki' Carboxymethyl chitosan = 1.13480 mg/mL, respectively. Surprisingly, D-(+)-cellobiose showed the activation effect of Cag4 on E. coli MG1655 cell wall lysis by decreasing the Ka value by 29.7% and increasing the Vmax value by 71.3%. In addition, molecular docking revealed the importance of the polarity of the C2 substituent group with glucose as the main structure in the Cag4 allosteric regulator. This study provides a fast and useful platform for screening potential new drugs based on the Cag4 allosteric regulator.