Vmax Values Research Articles

Heterologous expression, purification, and characterization of thermo- and alkali-tolerant laccase-like multicopper oxidase from Bacillus mojavensis TH309 and determination of its antibiotic removal potential.

Laccases or laccase-like multicopper oxidases have great potential in bioremediation to oxidase phenolic or non-phenolic substrates. However, their inability to maintain stability in harsh environmental conditions and against non-substrate compounds is one of the main reasons for their limited use. The gene (mco) encoding multicopper oxidase from Bacillus mojavensis TH309 were cloned into pET14b( +), expressed in Escherichia coli, and purified as histidine tagged enzyme (BmLMCO). The molecular weight of the enzyme was about 60kDa. The enzyme exhibited laccase-like activity toward 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The highest enzyme activity was recorded at 80°C and pH 8. BmLMCO showed a half-life of ~ 305, 99, 50, 46, 36, and 20min at 40, 50, 60, 70, 80, and 90°C, respectively. It retained more than 60% of its activity after pre-incubation in the range of pH 5-12 for 60min. The enzyme activity significantly increased in the presence of 1mM of Cu2+. Moreover, BmLMCO tolerated various chemicals and showed excellent compatibility with organic solvents. The Michaelis constant (Km) and the maximum velocity (Vmax) values of BmLMCO were 0.98mM and 93.45µmol/min, respectively, with 2,6-DMP as the substrate. BmLMCO reduced the antibacterial activity of cefprozil, gentamycin, and erythromycin by 72.3 ± 1.5%, 79.6 ± 6.4%, and 19.7 ± 4.1%, respectively. This is the first revealing shows the recombinant production of laccase-like multicopper oxidase from any B. mojavensis strains, its biochemical properties, and potential for use in bioremediation.

Label-Free Detection of Epinephrine Using Flower-like Biomimetic CuS Antioxidant Nanozymes.

A biosensor comprising crystalline CuS nanoparticles (NPs) was synthesized via a one-step simple coprecipitation route without involvement of a surfactant. The powder X-ray diffraction method has been used to evaluate the crystalline nature and different phases consist of the formation of CuS NPs. Mainly hexagonal unit cells consist of the formation of CuS NP unit cells. Most of the surfaces are covered with rhombohedral microparticles with a smooth exterior and surface clustering, examined by SEM images, and the shape of NPs was spherical, having an average size of 23 nm, as confirmed by TEM analysis. This study has focused on the peroxidase-mimicking activity, superoxide dismutase (SOD)-mimicking activity, and chemosensor-based colorimetric determination and detection of epinephrine (EP) neurotransmitters with excellent selectivity. The CuS NPs catalyzed the oxidation of the oxidase substrate 3, 3-5, 5 tetramethyl benzidine (TMB) with the help of supplementary H2O2 that followed Michaelis-Menten kinetics with excellent Km and Vmax values calculated by the Lineweaver-Burk plot. Taking advantage of the drop in absorbance upon introduction of EP for the CuS NPs-TMB/H2O2 system, a colorimetric route has been developed for selective and real-time detection of EP. The sensitivity of the new colorimetric probe was vibrant, having a linear range of 0-16 μM, and achieved a low limit of detection of 457 nM. Moreover, the present nanosystem exhibited appreciable SOD-mimicking activity which could effectively remove O2•- from commercial cigarette smoke, along with it acting as a potential radical scavenger as well. The new nanosystem effectively scavenged •OH, O2.-, and metal chelation which were investigated calorimetrically.

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6β-Hydroxylation Catalyzed by CYP3A Subfamilies.

The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6β-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6β-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. 6β-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. Lower concentrations of DHEA and ANF increased cortisol 6β-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6β-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6β-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 μM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 μM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6β-hydroxylation mediated by CYP3A4 with similar inhibition constants. The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Kinetic Studies of Enzymatic Hydrolysis Pre-Treatment of Waste Bread Under Preliminary Conditions

This study investigate the process conditions of enzyme concentration (2.0, 4.0, 6.0% w/v), temperature (30, 40, 50 °C), and reaction time (0, 60, 90, 120, 180, 240 min) of enzymatic hydrolysis for sugar recovery from waste bread. The kinetics of the enzyme (α-amylase) was studied to analyse the chemical reaction and the activity of the α-amylase towards the starch present in the waste bread. Enzymatic hydrolysis with different substrate concentration (5, 10, 15, 20 g) was conducted to analyse the kinetics of the enzyme with increasing amount of substrate concentration. The highest reducing sugar yield from the waste bread was 119.73 mg/mL at 15g of substrate concentration, 6.0% w/v enzyme concentration, temperature 50°C and 180 mins reaction time. From this value, the percentage recovery of the reducing sugar was 71.84%. The Michaelis-Menten equation was applied to study the kinetics of enzymatic hydrolysis of waste bread. The Vmax value was 3443.234 µmol/L/min and the Km value was 6.0g.

Structural and functional insights of a cold-adaptive β-glucosidase with very high glucose tolerance from Microbacterium sp. CIAB417

A gene glu1 (WP_243232135.1) coding for β-glucosidase from the genome of Microbacterium sp. CIAB417 was characterized for its cold adaptive nature and tolerance to high levels of glucose and ethanol. The phylogenetic analysis suggested the close association of glu1 with a similar gene from a mesophilic bacterium Microbacterium indicum. The purified recombinant GLU1 displayed its optimal activity and stability at pH 5 and temperature 30ᴼC. Additionally, the presence of L3 loop in GLU1 suggested its cold adaptive nature. The glucose tolerant Gate keeper residues (Leu 174 & Trp 169) with a distance of ∼ 6.953 Å between them was also predicted in GLU1. The GLU1 enzyme showed ≥ 95% and ≥ 40% relative activity in the presence of 5 M glucose and 20% ethanol. The Vmax, Km, and Kcat values of GLU1 for cellobiose substrate were observed to be 45.22 U/mg, 3.5 mM, and 41.0157 s-1, respectively. The GLU1 was found to be highly efficient in hydrolysis of celloologosaccharides (C2-C5), lactose and safranal picrocrocin into glucose. Hence, cold adaptive GLU1 with very high glucose and ethanol tolerance could be very useful in bio-refinery, dairy, and flavor industries.

Silver(I) and Copper(II) 1,10-Phenanthroline-5,6-dione Complexes as Promising Antivirulence Strategy against Leishmania: Focus on Gp63 (Leishmanolysin).

Leishmaniasis, caused by protozoa of the genus Leishmania, encompasses a group of neglected diseases with diverse clinical and epidemiological manifestations that can be fatal if not adequately and promptly managed/treated. The current chemotherapy options for this disease are expensive, require invasive administration and often lead to severe side effects. In this regard, our research group has previously reported the potent anti-Leishmania activity of two coordination compounds (complexes) derived from 1,10-phenanthroline-5,6-dione (phendione): [Cu(phendione)3].(ClO4)2.4H2O and [Ag(phendione)2].ClO4. The present study aimed to evaluate the effects of these complexes on leishmanolysin (gp63), a virulence factor produced by all Leishmania species that plays multiple functions and is recognized as a potential target for antiparasitic drugs. The results showed that both Ag-phendione (-74.82 kcal/mol) and Cu-phendione (-68.16 kcal/mol) were capable of interacting with the amino acids comprising the active site of the gp63 protein, exhibiting more favorable interaction energies compared to phendione alone (-39.75 kcal/mol) or 1,10-phenanthroline (-45.83 kcal/mol; a classical gp63 inhibitor) as judged by molecular docking assay. The analysis of kinetic parameters using the fluorogenic substrate Z-Phe-Arg-AMC indicated Vmax and apparent Km values of 0.064 µM/s and 14.18 µM, respectively, for the released gp63. The effects of both complexes on gp63 proteolytic activity were consistent with the in silico assay, where Ag-phendione exhibited the highest gp63 inhibition capacity against gp63, with an IC50 value of 2.16 µM and the lowest inhibitory constant value (Ki = 5.13 µM), followed by Cu-phendione (IC50 = 163 µM and Ki = 27.05 µM). Notably, pretreatment of live L. amazonensis promastigotes with the complexes resulted in a significant reduction in the expression of gp63 protein, including the isoforms located on the parasite cell surface. Both complexes markedly decreased the in vitro association indexes between L. amazonensis promastigotes and THP-1 human macrophages; however, this effect was reversed by the addition of soluble gp63 molecules to the interaction medium. Collectively, our findings highlight the potential use of these potent complexes in antivirulence therapy against Leishmania, offering new insights for the development of effective treatments for leishmaniasis.

Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis

Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry.

Kinetic behaviour of pancreatic lipase inhibition by wine

This study determined the in vitro inhibitory effects of Mukuzani (dry red) wine and its ethyl acetate/aqueous fractions against pancreatic lipase (PL). Also, it characterises the kinetic behaviour of such inhibition. To current knowledge, no studies have been conducted to describe the kinetic behaviour of PL inhibition by wine or the combined effect on the PL activity of some individual phenolic compounds present in wine. Additionally, the influence of different winemaking methods on PL activity is unknown. Lipase activity was determined by the titrimetric assay method. The kinetic analysis was determined by using the graphical method via Lineweaver-Burk plots. The mode of inhibition (MOI) was determined by looking at the interception pattern and crossing linear lines for the reciprocal data of PL activity with and without inhibitors versus olive oil concentration. The mode of inhibition identified for the wine sample was mixed-mode inhibition. In this type of inhibition, the value of Km (244.329±10.214 μmol·mL-1) was higher, and Vmax (80875.4±3489.754 μmol·mL-1·hour-1) was lower compared to the value of Km (170.901±7.544 μmol·mL-1) and Vmax (88735±4036.741 μmol·mL-1·hour-1) for the non-inhibited PL. This inhibitor decreased the PL activity rate and reduced the affinity between the substrate (olive oil) and enzyme (PL). Selected wine showed predominantly competitive inhibition. The value of inhibition constant Ki1 was equal to 40.556±1.932, and Ki2 was equal to 179.361±8.678 μmol·mL-1, meaning that binding affinity to the pancreatic lipase was higher than to the enzyme-substrate complex. The liquid/liquid extraction method was used to fractionate phenolic compounds of wine into aqueous and ethyl acetate fractions. Fractionated phenolic compounds of wine un-competitively inhibited PL activity. They were considered mutually nonexclusive inhibitors, while a synergistic relationship was found among them. The kinetic parameters of apparent Vmax and Km decreased. Due to the uncompetitive inhibition, the same factor decreased these kinetic parameters. The results obtained herein allow one to conclude that red wine made with the Kakhetian winemaking method can inhibit pancreatic lipase in vitro and play a role in body weight management.

Galactosyl trehalose synthesis by recombinant Lactobacillus fermentum β-galactosidase

A gene encoding a β-Galactosidase (LacLM) from the Lactobacillus fermentum 9–4 was isolated, cloned and expressed in Escherichia coli. The purified recombinant LacLM consisted of two subunits with approximately 35 and 72 kDa, respectively, and displayed the maximum activity toward o-nitrophenol-β-D-galactopyranoside (oNPG) at 45 ℃ and pH 7.0. The Vmax and Km values of LacLM toward oNPG were determined to be 36.64 µmol/(mL·min) and 1.02 mM, respectively, corresponding to a kcat value of 327.79 s−1 and kcat/Km value of 322.80 s−1·mM−1. After optimizing the conditions of glycosylation reaction, the maximum galactooligosaccharides conversion rate of 33.16 % was achieved after 12 h of reaction at pH 7.0 and 30 ℃ with 50 % (W/V) lactose as substrate and 10 U/mL of LacLM. At the same conditions, LacLM successfully synthesized galactosyl trehaloses including β-Gal(1→6)-α-Glc-(1→1)-α-Glc using 25 % (w/v) lactose and 25 % (w/v) trehalose as substrates. This is the first report that β-galactosidase derived from lactic acid bacteria can be used for enzymatic production of galactosyl trehalose.

Identification of Cytochrome P450 Enzymes Responsible for Oxidative Metabolism of Synthetic Cannabinoid (1-Hexyl-1H-Indol-3-yl)-1-naphthalenyl-methanone (JWH-019)

(1-Hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-019) is one of the second-generation synthetic cannabinoids which as a group have been associated with severe adverse reactions in humans. Although metabolic activation can be involved in the mechanism of action, the metabolic pathway of JWH-019 has not been fully investigated. In the present study, we aimed to identify the enzymes involved in the metabolism of JWH-019. JWH-019 was incubated with human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s or CYPs). An animal study was also conducted to determine the contribution of the metabolic reaction to the onset of action. Using an ultra-performance liquid chromatography system connected to a single-quadrupole mass detector, we identified 6-OH JWH-019 as the main oxidative metabolite in HLMs supplemented with NADPH. JWH-019 was extensively metabolized to 6-OH JWH-019 in HLMs with the KM and Vmax values of 31.5 µM and 432.0 pmol/min/mg. The relative activity factor method estimated that CYP1A2 is the primary contributor to the metabolic reaction in the human liver. The animal study revealed that JWH-019 had a slower onset of action compared to natural and other synthetic cannabinoids. CYP1A2 mediates the metabolic activation of JWH-019, contributing to the slower onset of its pharmacological action.

DEVELOPMENT OF ENTEROBACTER CLOACAE IP8 MUTANT STRAIN PRODUCING THERMOSTABLE CELLULASE

Mutagenesis of Enterobacter cloacae IP8 for enhanced cellulase production was carried out using ultraviolet (UV) irradiation and ethyl methanesulphonate (EMS) treatment. The mutant strain exhibited cellulolytic activity which was 2.18-fold higher than that of the wildtype strain. The optimal conditions for cellulase production were an incubation period of 28 h, a temperature of 45 oC, and pH 7.0, using CMC and peptone as carbon and nitrogen sources, respectively. The cellulases from both strains were purified by using ammonium sulfate precipitation, CM Sephadex C-50, and Biogel P-100 column chromatography. The specific activity of the purified cellulase from the mutant strain was 29.47 U/mg while that of wildtype cellulase was 21.5 U/mg. Biochemical characterization of the purified enzyme revealed the optimum pH and temperature of 8.0 and 65 oC, respectively, for the cellulase from the mutant strain, and 7.0 and 60 oC for the wild-type cellulase. The mutant cellulase was thermally stable up to 70 oC retaining 86.5% of its original activity after 180 h. Metal ions Na+ and Ca2+ remarkably enhanced the activity of the cellulase from both strains while Al3+ and the chelating agent, EDTA, strongly inhibited the activity. Mutagenesis of E. cloacae IP8 using combined UV and EMS treatment led to the development of mutant strain with enhanced capacity for the production of cellulase exhibiting novel properties such as thermostability, alkalinity, low Km, and high Vmax values. Therefore, the enzyme from the mutant strain of E. cloacae IP8 has the potential for broad industrial applications.

(1E)-1-(2-pyrazinyl)ethanone thiosemicarbazone (PT) as a tyrosinase inhibitor with anti-browning activity: Spectroscopy, DFT and molecular docking studies

In this study, (1E)-1-(2-Pyrazinyl)ethanone thiosemicarbazone (PT) was synthesized and characterized using Fourier-transform infrared (FTIR), ultraviolet-visible (UV–Vis), proton nuclear magnetic resonance (1HNMR ) and carbon-13 nuclear magnetic resonance (13CNMR ) spectroscopy. The vibrational frequencies were analysed using the Vibrational Energy Distribution Analysis (VEDA) program. The chemical properties of PT, including electron density distribution and intramolecular interactions, were characterized using in-silico methods based on Mulliken atomic charges, molecular electrostatic potential (MEP), quantum theory of atoms in molecules and reduced density gradient noncovalent interaction approaches. PT exhibits a significant inhibitory effect on enzymatic browning (6 and 24 h) and tyrosinase enzymes (IC50 = 7.75 μM). Kinetic analysis shows that PT is a mixed-type inhibitor, with respective Km and Vmax values of 0.632 mM and 0.0044 μM/s, and that it forms a reversible enzyme-inhibitor interaction. The PT-tyrosinase interaction was experimentally evaluated based on 1D, second derivatives of 1D, 2D and 3D IR spectroscopy. Furthermore, analysis of absorption, distribution, metabolism, excretions and toxicity properties revealed good physicochemical properties of PT according to the drug scores and Lipinski's Rule of Five. Lastly, the mechanisms of PT against tyrosinase were visualized and supported by means of a molecular docking approach.

Selective and sensitive colorimetric sensing of iodine ions based on porous MoS2 particles with excellent haloperoxidase-like activity

Nanozymes continue to attract great interest due to their high enzyme activities, good biocompatibility, chemical stability and adjustable activity, which have been widely used in biosensing, disease therapy and antibacterial treatment. Herein we report porous MoS2 particles to have both excellent bromoperoxidase-like and iodoperoxidase-like activity. The MoS2 particles exhibited the highest Vmax value of 83 µM/s (H2O2 as substrate), remarkably superior to haloperoxidase and other haloperoxidase-like reported so far. The underlying mechanism of the iodoperoxidase mimics is oxygen vacancies. The iodoperoxidase-like can catalyze the oxidization of iodide ions (I−) by H2O2 to iodic acid (HIO3), which further enables to readily oxidize colorless 3,3’,5,5’-tetramethylbenzidine to green ox-TMB. Significantly, among the halogen anions (Cl−, Br− and I−), only I− was found to cause this oxidation reaction to occur. Thereafter, a fast, selective and cost-effective colorimetric detection of I− ion was developed with the linear range of 0.5–70 μM and detection limit of 292 nM (3σ/S). Further, the as-developed assay was applicable to detect seawater and human urines samples with satisfactory recoveries and accuracy. Thus the colorimetric assay based on MoS2 particles with haloperoxidase-like activity show great promise for I− sensing in environmental analysis and biochemical analysis.

Bioconversion of chitin into chitin oligosaccharides using a novel chitinase with high chitin-binding capacity

Chitin is the second largest renewable biomass resource in nature, it can be enzymatically degraded into high-value chitin oligosaccharides (CHOSs) by chitinases. In this study, a chitinase (ChiC8–1) was purified and biochemically characterized, its structure was analyzed by molecular modeling. ChiC8–1 had a molecular mass of approximately 96 kDa, exhibited its optimal activity at pH 6.0 and 50 °C. The Km and Vmax values of ChiC8–1 towards colloidal chitin were 10.17 mgmL−1 and 13.32 U/mg, respectively. Notably, ChiC8–1 showed high chitin-binding capacity, which may be related to the two chitin binding domains in the N-terminal. Based on the unique properties of ChiC8–1, a modified affinity chromatography method, which combines protein purification with chitin hydrolysis process, was developed to purify ChiC8–1 while hydrolyzing chitin. In this way, 9.36 ± 0.18 g CHOSs powder was directly obtained by hydrolyzing 10 g colloidal chitin with crude enzyme solution. The CHOSs were composed of 14.77–2.83 % GlcNAc and 85.23–97.17 % (GlcNAc)2 at different enzyme-substrate ratio. This process simplifies the tedious purification and separation steps, and may enable its potential application in the field of green production of chitin oligosaccharides.

Polyamidoamine Dendrimers Functionalized with ZnO-Chitosan Nanoparticles as an Efficient Surface for L-asparaginase Immobilization.

In this study, the third-generation polyamidoamine dendrimer was functionalized with a 5-amino-1H-tetrazole heterocycle to load the synthesis enzyme and its surface groups. Then, chitosan was attached to the dendrimer by a suitable linker, and finally, zinc oxide nanoparticles were inserted into dendrimer cavities to increase loading. FTIR, FESEM, TEM, and DLS analysis showed that this new dendrimer has specific branches, and ZnO nanoparticles were spread between the branches and connected with the branches and chitosan biopolymer. Also proved the presence of stabilized L-asparaginase enzyme and ZnO nanoparticles in the designed system. Furthermore, the extent of L-asparaginase enzyme loading and release was investigated in the laboratory with a dialysis bag. Examining the toxicity of the new third-generation polyamidoamine (PAMAM) dendrimeric nanocarrier based on chitosan-zinc oxide biopolymer (PAMAM-G3@ZnO-Cs nanocarrier) on the Jurkat cell line (human acute lymphoblastic leukemia) at pH 7.4 showed that this nanocarrier effectively encapsulates the drug L-asparaginase and slowly releases it and also preventing the growth of cancer cells. The activity of the loaded enzyme in the nanocarrier and the free enzyme was calculated. During the investigations, it was found that the enzyme attached to the nanocarrier is more stable than the free enzyme at optimal pH and temperature and at high temperatures, acidic and basic pHs. Vmax and Km values were lower for loaded enzymes. The synthesized PAMAM-G3@ZnO-Cs nanocarrier can be a promising candidate in the pharmaceutical industry and medical science for cancer treatment due to its biocompatibility, non-toxicity, stability, and slow release of L-asparaginase.

Exploring the Potential of ω3 Derivatives as Tyrosinase Inhibitors: A Comprehensive Study Combining Experimental, Computational, and Biological Approaches

AbstractIn this work, the inhibitory ability and mechanism of ω3‐nicotinic acid (ω3‐NA) and ω3‐Picolinic acid (ω3‐PA) complexes on the activity of mushroom tyrosinase (MT) were scrutinized for progressing a novel MT inhibitor. The complexes were synthesized. It was shown to have a considerable inhibition on the MT activity and Ki value of ω3‐NA and ω3‐PA on MT equal to 5.2 and 5.1 mM, respectively. ω3‐NA and ω3‐PA inhibited MT with Vmax values in the range of 0.134 mM and 0.14 mM, respectively. The outputs obtained from fluorescence quenching specified that ω3‐NA and ω3‐PA could interact with MT. Especially, the decrease in fluorescence intensity was due to the formation of a ligand‐enzyme complex which was mostly motivated by hydrogen bonding and hydrophobic forces. The presence of ω3‐NA and ω3‐PA altered the structure of MT and reduced the α‐helix of the enzyme. Molecular docking investigation along with molecular dynamics simulation exhibited that ligands‐MT formation is directed by hydrogen binding with Trp136, His263, and Val299 residues. The results highlight that ω3‐NA and ω3‐PA can be considered as possible inhibitors in treating hyperpigmentation via MT enzyme inhibition.

Glutaraldehyde-pea protein grafted polysaccharide matrices for functioning as covalent immobilizers

Three polysaccharide matrices (κ-Carrageenan (Carr), gellan gum, and agar) were grafted via glutaraldehyde (GA) and pea protein (PP). The grafted matrices covalently immobilized β-d-galactosidase (β-GL). Nonetheless, grafted Carr acquired the topmost amount of immobilized β-GL (iβ-GL). Thus, its grafting process was honed via Box-Behnken design and was further characterized via FTIR, EDX, and SEM. The optimal GA-PP-Carr grafting comprised processing Carr beads with 10% PP dispersion of pH 1 and 25% GA solution. The optimal GA-PP-Carr beads acquired 11.44 Ug−1 iβ-GL with 45.49% immobilization efficiency. Both free and GA-PP-Carr iβ-GLs manifested their topmost activity at the selfsame temperature and pH. Nonetheless, the β-GL Km and Vmax values were reduced following immobilization. The GA-PP-Carr iβ-GL manifested good operational stability. Moreover, its storage stability was incremented where 91.74% activity was offered after 35 storage days. The GA-PP-Carr iβ-GL was utilized to degrade lactose in whey permeate with 81.90% lactose degradation efficiency.

Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 μmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 μmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 μmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 μmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 μmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Effect of Long-Term and Short-Term Droughts on the Hydrolytic Enzymes in Haplic Luvisol

The transformation of organic matter in the soil is largely determined by hydrolytic enzymes. Under the conditions of climate change, understanding the mechanisms of microbial response is of particular importance for predicting the carbon cycle. Until now, the effect of drought duration and frequency on soil hydrolytic enzymes has been little studied. A multifactorial field manipulation experiment was carried out, simulating in the presence of plants and without them: two short-term droughts, a long-term drought, and an optimal level of soil moisture. The maximum reaction rate Vmax, Michaelis constant Km, and catalytic efficiency Ka of five groups of enzymes involved in the carbon cycle (cellobiase, glucosidase, xylanase), phosphorus (phosphatase), and nitrogen (chitinase) were determined. In phosphatases, glucosidases, and xylanases, Vmax decreased during short-term drought. During prolonged drought, the Vmax value of phosphatases, cellobiohydrolases, and xylanases decreased and increased in chitinases, while remaining unchanged in glucosidases. Both long-term and short-term droughts led to an increase in Km and a decrease in catalytic efficiency (Ka) for almost all enzymes. Short-term droughts were not a “weakened version” of a long-term drought, but had their own specifics – a decrease in Km in glucosidases, which led to an increase in Ka. Long-term drought was characterized by an increase in Vmax of chitinases and spatial variability of Vmax of phosphatases and glucosidases. The influence of the presence of plants was secondary and affected only during short droughts. The reversibility of the effect of drought on Vmax, Km, Ka decreased in the series first short-term drought second short-term drought long-term drought due to an increase in the total duration of the stress impact.

Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 μM and 528 pmol/min/mg protein toward serotonin and 201 μM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.